Hamourabi medical college

University of Babylon

Membranes and receptors

module

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 MEMBRANES AND RECEPTORS MODULE

AIMS OF THE MODULE

The aims of this module are that students should
 understand membrane structure and function and be able to relate this to cell behavior;
 understand how the movement of ions and molecules across membranes may contribute to pH and
cell volume regulation and electrical excitability and nerve impulse conduction,
 appreciate how chemical messengers, such as hormones and neurotransmitters, influence the
activity of cells and organs by interacting with receptors;
 understand in principle how drugs might modify the action of such chemical messengers.

PRE-REQUISITES
At the beginning of this module the students should be able to
 describe basic cell structure and function, including the pathway for protein secretion
 outline the non-covalent forces governing the structure and interactions of biomolecules
 discuss protein structure and function, including the properties of enzymes
 discuss factors influencing the association and dissociation of protein-ligand complexes

SUMMARY OF INTENDED LEARNING OUTCOMES

On completion of this module, students should be able to:
 describe the main features of the fluid mosaic model of biological membrane structure and discuss
the features of membrane asymmetry and cytoskeletal interactions.
 describe how membrane transporter mechanisms and ion channels contribute to the maintenance of
ionic gradients across membranes, the transport of solutes through membranes and the regulation of
intracellular pH and cell volume
 describe the ionic basis of the membrane potential
 describe the properties of voltage-gated ion channels, the general features of electrical excitability
of membranes, and the permeability changes associated with the action potential
 discuss factors affecting impulse conduction velocity in nerves
 describe and compare biological communication processes involving hormones, local mediators and
neurotransmitters
 outline the variety of receptor mechanisms which influence the behaviour of cells

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 outline the variety of effector mechanisms involved in cellular signalling pathways, including the
concepts of transducing proteins, second messengers and signal pathway cascades
 define the concept of receptor specificity, and define the terms agonist and antagonist. Distinguish
competitive and non-competitive antagonism
 describe the anatomical and pharmacological divisions of the autonomic nervous system. Outline
the steps of neurotransmission at cholinergic and adrenergic synapses in the autonomic nervous
system and the mammalian neuromuscular junction
 summarise whole-body considerations of drugs reaching their sites of therapeutic action, including
principles of drug bioavailability, inactivation and elimination and describe the adaptive changes
which can occur in receptor populations when exposed to agonists and antagonists
 describe the principles of drug action using the autonomic nervous system as an example drug
target.

What we should know about this module

 Module Handbook

 Assignment book

 Lectures

 Study sessions

 Tutorials

 Tutor sessions and private study

 Formative assessment

 Presentations and assignments

 Timetable

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 MODULE HANDBOOK

Sections of the module handbook will be distributed before the module started. The
handbook will consist of a synopsis of each lecture together with exercises for use in tutorials
and study sessions. This handbook will be available for collection from the Medical Education
Unit as follow:

Section Sessions
Membranes and membrane 1 -9
transport Membrane
excitability
Receptors and membrane turnover
Signal transduction
Module presentationin assignments
biological membranes 6 - 7
Drugs, receptors and the Autonomic Nervous 10 -12
System

STUDY GROUPS
For all activities (tutorials, study sessions, presentation sessions) you will work in your normal
study groups of students.

LECTURES
All lectures will be held in the Hamourabi College of Medicine. Between some

lectures and/or work sessions there may not be a timetabled break – please
get from one to the other as quickly as possible.

STUDY SESSIONS
In these sessions the student will work in his normal study groups using study sheet
materials provided.
Please you should come to these sessions with answers to as many of the questions on the study
sheet as he can. It is recommended that he bring personal copies of relevant texts to study

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sessions, particularly in the earlier sessions. The tutors will be available to help him in these
sessions.
It is module policy that where work in study sessions is consolidated either by a tutor-led
discussion or by a subsequent tutorial session, it is the students’ responsibility to ensure that they
have obtained from the discussion the answers they need against questions set in the
study sheets.

TUTORIAL SESSIONS
These, tutor led, sessions are to provide an opportunity to consolidate material worked on
previously, either in study sessions or during private study. The students are requested to
come to these sessions with completed study sheet exercises and prepared to contribute to the
discussion. Answer sheets WILL NOT be provided after tutorial sessions, so the student must
make sure that he has sufficient notes against each study sheet question to aid future reference
and revision.

ASSIGNMENT PRESENTATION SESSIONS

There are 2 assignment presentation sessions in the module in Sessions 6 &7. In each session,
each Study Group will be responsible for a presentation on the topic allocated in a given
table.
Groups of students should prepare the presentations together and one or more group members
may make the presentation required. In the second session, different students should make the
presentation. Study Groups should decide amongst themselves who should make the
presentations. Presentations should be approximately 8 minutes in length. The following 7-
8 minutes will be available for students to ask questions of the presenting group and to
be used by the tutor to develop discussion of the topic from the presentation. Assignment
handbook material to guide this preparation will be provided. All students should make notes
during presentations against the guideline questions in the accompanying handbook

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materials, particularly for the topics not covered during preparation. In this way, each student
should leave the session with a complete set of answers to the questions on the assignment
worksheet. Answer sheets WILL NOT be provided for these sessions. Most aspects will be
covered again at other points in the module. It is very important that study groups work together in
researching and preparing the material for each assignment presentation. In addition, it is
important that each oral presentation is practiced within the group. It should be noted that it
is the responsibility of the whole group, not just the presenter(s), to answer questions about
the presentation asked by either other students or the tutor.

ASSIGNMENT HANDBOOK
The presentation assignments are designed to cover important material in this course, much of which
will complement work in other sessions, particularly those centered on the Autonomic Nervous
System later in the module. The format of the assignments will develop student skills in
researching and distilling new information and give him valuable experience in preparing and
delivering specific scientific information at an appropriate level through oral presentation.

FORMATIVE ASSESSMENT
It will be held in Session 9. The session will consist of a paper sat under examination
conditions but followed by a debriefing session with the module leader. Students will be able to
bring study materials and textbooks for consultation to maximize the usefulness of this
session. The formats of sub-questions in the formative assessment and the time available to answer
will be similar to those to be used in the End of Semester Assessments (ESAs).

AFTERNOON SESSIONS - VOLUNTARY CLINIC SESSIONS

A module tutor will be available in a specified time for consultation. These sessions are
voluntary. Tutors will be pleased to provide help individually or in groups. Please make use of them.
Additional voluntary tutorials on any material from the module may be arranged if students request
this. Any afternoon session of this type will be additional to the module content and will
not be obligatory.

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Organization of membrane and receptors sessions and lectures

Topic Session/Lecture Lecturer

The membrane bilayer 1.1 Biochemist

Proteins of cell membrane 1.2 Biochemist

Role of membranes as permeability barrier 2.1 Biochemist

ATP-dependent ion pumps and ion 2.2 Biochemist

exchangers

The resting membrane potential 3.1 Physiologist

Changing membrane potential 3.2 Physiologist

Action potential and its properties 4.1 Physiologist

Conduction of the nerve impulse 4.2 Neurophysiologist

Electrical excitability 5.1 Neurophysiologist

Control of intracellular calcium 5.2 Biochemist

concentration
Receptors in cell signalling 6.1 Dr Israa Al-Humairi

Principles of receptor mediated endocytosis 6.2 Dr Israa Al-Humairi

Receptors effector signallig via G proteins 7.1 Dr Israa Al-Humairi

Effector mechanisms in intracellular 7.2 Dr Israa Al-Humairi

signalling

Drugs and receptors/pharmacokinetics 8.1 Pharmacologist

Drugs And Receptors
 8.2 Pharmacologist

Formative assessment and data handling 9.0 Dr Israa Al-Humairi

Autonomic nervous system 10 &11 Pharmacologist

pharmacokinetics

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BOOKS
You are recommended to buy a copy of:
 Page, C.P., Hofmann, B., Curtis, M., & Walker, M.. Integrated Pharmacology, With Student
Consult Online Access, 3rd Edition, Mosby, 2006, ISBN 0323040802, £51.34 (An integrated
medical pharmacology text)
Or
 Rang, H.P., Dale, M.M., Ritter, J.M., and Flower, R. Rang & Dale's Pharmacology: With Student
Consult Online Access, 6th Edition, Churchill Livingstone, 2007, ISBN 0443069115, £45.99 (A
pharmacology text based on different extracellular signalling molecules)
You should also have access to at least one of the recommended Physiology textbooks.
 Koeppen, B.M. & Stanton, B.A. Berne & Levy: Principles of Physiology, 6th Edition, Wolfe
Publications, 2006, ISBN 9780323073622, £56.79.
 Widmaier, E.P., Raff, H. & Strang, H. Vander‟s Human Physiology: the mechanisms of body
function, 11th Edition, McGraw-Hill, 2005, ISBN 9780077350017, £58.20
At the very least, Study Groups should possess between them one Pharmacology and one Physiology
textbook. It is recommended that you bring personal copies of relevant texts with you to module
study sessions.
You are also recommended to consider purchase a copy of the following:

 Norman, R.I. & Lodwick, D. Flesh and Bones of Medical Cell Biology, Elsevier, April 2007,
ISBN-13: 978-0-7234-3367-5. ISBN-10: 0-7234-3367-4, £19.99
In addition to its relevance to the Membranes and Receptors module, the subject material in this book
is applicable to a number of other modules in the Phase I course. This book is an updated version of
 Norman, R.I. & Lodwick, D. Medical Cell Biology Made Memorable, Churchill Livingstone, 1999,
ISBN 0443058156, £25.99
 Barritt, G.J., Communication within Animal cells, Oxford Science, 1992, ISBN 0198547269
 Bray, J.J., Cragg, P.A., Macknight, A.D.C., Mills, R.G. & Taylor, D.W. (Eds), Lecture Notes on
Human Physiology, 4th Edition, Blackwell Scientific Publications, 1999, ISBN 0865427755
 Ganong, W.F., Review of Medical Physiology, 23rd Edition, McGraw-Hill, 2009,
ISBN9780071605670
 Golan, D.E., Tashjian, Jr., A.H., Armstrong, E.J. & Armstrong, A.W. Principles of Pharmacology:
 The pathophysiologic basis of drug therapy, 2nd edition, Lippincott. Williams and Wilkins, 2007,
ISBN 0781783550, £35.00
(An integrated pharmacology text based on systems. Some information on therapeutic uses of drugs)

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 Guyton, A.C., Human Physiology and Mechanisms of Disease, 6th Edition, W.B. Saunders, 1997,
ISBN 0721632998
 Schmidt, R.F. & Thews, G., Human Physiology, 2nd Edition, Springer-Verlag, 1989, ISBN
3540194320
 Waller, D. & Renwick, A., Principles of Medical Pharmacology, Balliere Tindall, 1994, ISBN
0702016136
 Waller, D.G. Medical Pharmacology and Therapeutics, 2nd Edition. Elsevier Sanders, 2005, ISBN
0702027545
You will also find relevant information in many of the Biochemistry textbooks recommended last
semester in the Biological molecules and Metabolism modules.

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FORMATIVE ASSESSMENT
A formative assessment will be held in Session 9. The session will consist of a paper sat under
examination conditions but followed by a debriefing session with the module leader. Students will be
able to bring study materials and textbooks for consultation to maximise the usefulness of this
session. The formats of sub-questions in the formative assessment and the time available to answer
will be similar to those to be used in the End of Semester Assessments (ESAs).

ON-LINE FORMATIVE ASSESSMENT

To permit you assess your learning within this module at any time, a series of self-marking formative
assessments are available on the module web pages in the Moodel, Semester 3,Membranes &
Receptors.

REVISION SESSION
A voluntary attendance revision session will be held in session 12. This will be hosted by the module
leader who will respond to questions from those gathered. Given the size of the group that may
attend, some students have reported in feedback from previous years that posing questions in the
session can be intimidating. For this reason, students may wish to submit questions for this session in
advance, either via the module Discussion Board or by e-mail to the module leader.

SUMMATIVE ASSESSMENT OF MODULE CONTENT

Module content will be assessed within the End of Semester Assessments (ESAs). In addition,
material from this module will be assessed as part of the End of Phase 1 Assessment at the end of
semester 6.

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 MEMBRANES AND RECEPTORS MODULE
SESSION 1 - LIPIDS, PROTEINS AND MEMBRANE STRUCTURE

AIMS
The aim of this session is to introduce the structure and dynamics of biological
membranes.
LEARNING OUTCOMES
By this session you should be able to:
 list the main kinds of lipids and general properties of fatty acids. Describe the
properties of amphipathic molecules and explain the process of formation of lipid
bilayers
 distinguish peripheral from integral membrane proteins and explain the forces
associating them with the membrane
 describe in general terms the mechanism of membrane insertion of integral proteins
and the features of these proteins which explains their topology in the membrane.
Discuss membrane asymmetry
 discuss the influence of unsaturated fatty acids and cholesterol on membrane fluidity.
Describe the main features of the fluid mosaic model of membrane structure and
explain the restrictions on protein movement in the membrane, including potential
interactions with cytoskeletal elements

PRIVATE STUDY
 Group work commenced in the study session should be completed.

 Complete the private study sheet on „Body fluids: regulation of composition and
volume‟ in preparation for the study session in Session 2.

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MEMBRANES AND RECEPTORS - SESSION 1
LIPIDS, PROTEINS AND MEMBRANE STRUCTURE
LECTURE 1.1 - THE MEMBRANE BILAYER

AIM
To introduce the basic structure of biological membranes. At the end of the lecture you
should be familiar with the lipid bilayer model for membrane structure.

General functions of biological membranes (cells and organelles)

1. Continuous highly selective permeability barrier.
2. Allows control of the enclosed chemical environment
3. Communication - control the flow of information between cells and their
environment
4. Recognition - signalling molecules, adhesion proteins, immune surveillance
5. Signal generation in response to stimuli - electrical, chemical

Different membranes have specialised functions

e.g. Plasma membrane - all of the above functions.
e.g. Mitochondrial membrane - energy conservation by oxidative phosphorylation

Membrane Composition - Varies with source but generally membranes contain

approximately: 40% lipid, 60% protein and 1-10% carbohydrate (dry weight). N.B. the
membrane bilayer is a hydrated structure and hence 20% of total membrane weight is
water.
Membrane lipids - amphipathic molecules - i.e. they contain both hydrophilic and
hydrophobic moieties. Distribution varies depending on cell type

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Phospholipid - predominant lipids - e.g. phosphatidylcholine

Head groups
- a range of polar head groups are employed - choline, amines, amino acids, sugars
Fatty acid chains
- enormous variety, C16 and C18 most prevalent
- unsaturated fatty acid side chains (double bonds) in the cis conformation introduce a
kink in the chain which reduces phospholipid packing. 3

Plasmalogens

Sphingomyelin - the only phospholipid not based on glycerol. In the membrane the
conformation of sphingomyelin resembles other phospholipids.

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Glycolipids - sugar containing lipids
- Cerebrosides - head group sugar monomers
- Gangliosides - head group oligosaccharides (sugar multimers)
Similararity of membrane lipid structures

Figure above taken from http://en.wikipedia.org/wiki/File:Membrane_lipids.png, with permission

Cholesterol - plasma membrane lipid, 45% of the total membrane lipid.

Distribution of different lipids is tissue specific and related to function

Lipid Bilayer
Amphipathic molecules form one of two structures in water, micelles and bilayers.
Bilayers are the favoured structure for phospholipids and glycolipids in aqueous

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media. Bilayer formation is spontaneous in water driven by the van der Walls
attractive forces between the hydrophobic tails. The co-operative structure is stabilised
by non-covalent forces; electrostatic and hydrogen bonding between hydrophilic
moieties and interactions between hydrophilic groups and water.
Pure lipid bilayers have a very low permeability to ions and most polar molecules.

Dynamics in lipid bilayers

Membranes are fluid structures. Lipid molecules possess four permitted modes of
mobility in a lipid bilayer.
1. Intra-chain motion - kink formation in the fatty acyl chains
2. Fast axial rotation.
3. Fast lateral diffusion within the plane of the bilayer.
4. Flip-flop - movement of lipid molecules from one half of the bilayer to the other on
a one for one exchange basis.
Unsaturated double bonds in the fatty acid side chains disrupt the hexagonal packing
of phospholipids and so increase membrane fluidity. Cholesterol plays an important
role in stabilising the plasma membrane. (You will consider this again as part of the
work session)

Membrane proteins
Membrane proteins carry out the distinctive functions of membranes which include
enzymes, transporters, pumps, ion channels, receptors, and energy transducers. Protein
content can vary from approximately 18% in myelin (nerve cell „insulator‟) to 75% in
the mitochondria. Normally membranes contain approximately 60% dry weight of
protein.

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Mobility of proteins in bilayers
Three modes of motion permitted – conformational change, rotational and lateral - NO
FLIP-FLOP
Restraints on mobility
- lipid mediated effects - proteins tend to separate out into the fluid phase or
cholesterol poor regions.
- membrane protein associations
- association with extra-membranous proteins (peripheral proteins) e.g. cytoskeleton.

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MEMBRANES AND RECEPTORS - SESSION 1
LIPIDS, PROTEINS AND MEMBRANE STRUCTURE
LECTURE 1.2 - MEMBRANE PROTEINS, MEMBRANE ASYMMETRY AND
THE CYTOSKELETON

AIMS
To consider
- the distribution and role of proteins in membrane structure
- the importance of an asymmetric distribution of membrane proteins
- mechanisms for the correct insertion of membrane proteins into the lipid bilayer and
- the structure of the erythrocyte cytoskeleton.
Lipid mosaic theory of membrane structure (Singer - Nicholson Model)
Biological membranes are composed of a lipid bilayer with associated membrane
proteins which may be deeply embedded in the bilayer (integral) or associated with the
surface (peripheral).

Peripheral membrane proteins

- bound to the surface of membranes by electrostatic and hydrogen bond interactions.
These proteins can be removed by changes in pH or ionic strength.

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Integral membrane proteins
- interact extensively with the hydrophobic regions of the lipid bilayer. These proteins
can not be removed by manipulation of pH or ionic strength but require agents
(detergents, organic solvents) that compete for the non-polar interactions in the bilayer.

Asymmetrical orientation of membrane proteins

Asymmetrical orientation of proteins in biological membranes is important for
function e.g. a receptor for a hydrophilic extracellular messenger molecule, such as
insulin, must have its recognition site directed towards the extracellular space to be
able to function.

The erythrocyte membrane - a model plasma membrane

Erythrocyte ghosts can be prepared by osmotic haemolysis to release cytoplasmic
components. Analysis of ghost membranes by gel electrophoresis reveals over 10
major proteins. The major ones have been numbered 1, 2, 3, 4.1, 4.2, 5, 6 and 7 etc..
Most of these proteins are released when ghost membranes are treated with high ionic
strength medium or by changing the pH and are, thus, peripheral proteins. These
peripheral proteins must be located on the cytoplasmic face since they are susceptible
to proteolysis only when the cytoplasmic face of the membrane is accessible.
Protein bands 3 and 7 can only be dissociated from the red cell membrane by
detergents and are, thus, integral proteins. Both proteins contain covalently attached
carbohydrate units and are, thus, glycoproteins. The highly hydrophilic nature of the
extracellular carbohydrate groups acts to lock the orientation of the protein in the
membrane by preventing flip-flop rotation. A great variety of carbohydrate structures
is possible on different proteins. Specific carbohydrate groups on membrane proteins

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may be important for cellular recognition to allow tissues to form and in immune
recognition.

Cytoskeleton (Membrane skeleton)

The peripheral membrane proteins removed from erythrocyte membrane preparations
by low ionic strength washes compose a membrane skeleton on the cytoplasmic face
of the membrane. The erythrocyte cytoskeleton is a network of spectrin and actin
molecules. Spectrin is a long, floppy rod-like molecule. and subunits wind together to
form an antiparallel heterodimer and two heterodimers then form a head-to-head
association to form a heterotetramer of 22 . These rods are crosslinked into networks
by short actin protofilaments (~14 actin monomers), and band 4.1 and adducin
molecules which form interactions towards the ends of the spectrin rods. The spectrin-
actin network is attached to the membrane through adapter proteins. Ankyrin (band
4.9) and band 4.1 link spectrin and band 3 protein and glycophorin A, respectively.
Attachment of integral membrane proteins to the cytoskeleton restricts the lateral
mobility of the membrane protein.

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Haemolytic anaemias
The erythrocyte cytoskeleton is a very important structure in maintaining the
deformability necessary for erythrocytes to make their passage through capillary beds
without lysis. In the common dominant form of Hereditary Spherocytosis spectrin
levels may be depleted by 40 - 50%. The cells round up and become much less
resistant to lysis during passage through the capillaries and are cleared by the spleen.
The shortened in vivo survival of red blood cells and the inability of the bone marrow
to compensate for their reduced life span lead to haemolytic anaemia. Other forms of
hereditary spherocytosis also exist where mutated cytoskeletal elements with
dysfunctional binding sites for other components are expressed. Similarly, in
Hereditary Elliptocytosis, a common defect is a spectrin molecule that is unable to
form heterotetramers resulting in fragile elliptoid cells. Even simple treatment with
cytochalasin drugs, which cap the growing end of polymerizing actin filaments, can
alter the deformability of the erythrocyte.
Membrane protein synthesis directs protein orientation
Like cytosolic proteins, membrane proteins and those to be secreted or targeted to
lysosomes are synthesised against the messenger RNA template by ribosomes.
However, before synthesis progresses very far the translation of these proteins is halted
until the ribosome has been transferred to the rough ER (Figure). A characteristic
hydrophobic amino acid sequence of
18 - 30 amino acids flanked by basic residues at the N-terminus of the nascent
polypeptide, termed the signal or leader sequence, is recognised by a large
protein/RNA complex called the signal recognition particle (SRP). Binding of the
SRP to the growing polypeptide chain and the ribosome locks the ribosome complex
and prevents further protein synthesis while the ribosome is in the cytoplasm.
On the ER the SRP is recognised by a SRP receptor or docking protein. In making the
interaction with the docking protein, the SRP is released from the signal sequence of
the nascent polypeptide removing the inhibitory constraint on further translation. The

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signal sequence then interacts with a signal sequence receptor (SSR) within a protein
translocator complex (Sec61) in the ER membrane, which directs further synthesis
through the ER membrane. The ribosome becomes anchored to this pore complex,
through which the growing polypeptide chain is extruded. In the case of a secreted or
lysosomal protein, synthesis is completed and the nascent protein is translocated into
the lumen of the ER. For membrane proteins the passage of the protein through the
membrane must be arrested. The stop transfer signal for this is a region of highly
hydrophobic primary sequence in the growing polypeptide of between 18 and 22
amino acids long followed directly by charged amino acids which, in -helical form, is
long enough to span the hydrophobic core of the bilayer. This sequence forms the
transmembranous region of the protein. A lateral gating mechanism releases the
membrane protein from the protein translocator into the lipid bilayer. The ribosome
then presumably detaches from the ER and protein biosynthesis continues in the
cytoplasm. The result is a transmembrane protein with it‟s N-terminal directed in to
the lumen and it‟s C-terminal to the cytoplasm. For both secretory proteins and
membrane incorporated proteins, the signal sequence is cleaved from the new protein
by signal peptidases even before protein synthesis is completed.

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The mechanism explained above and in the Molecules, Genes and Disease module is
sufficient to explain how proteins with an N-terminal signal sequence may be
orientated with their N-terminus directed towards the ER lumen but does not explain
how those with a lumen-directed C-terminal may be orientated. Moreover, many
membrane proteins lack a cleavable N-terminal signal sequence but rather contain
internal start-transfer („signal‟) sequences, raising the question of how their orientation
is defined? The following description is not required for examination purposes but is
included for the inquisitive student. While start-transfer sequences may bind to the
translocator complex in either orientation, in principle, the positioning of positively
charged residues at either the N- or C-terminal end of the start-transfer sequence
defines their orientation, which in turn specifies the orientation of the mature protein.
Where positive residues are located at the N-terminal end, the C-terminal section
passes into the lumen, whereas if positive residues are located at the C-terminal end,
the N-terminal section of the protein passes into the lumen. Binding of the positive

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residues within signal and start-transfer sequences on the cytoplasmic side of the
protein translocator complex provides an explanation that fits all scenarios.

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Post-translational processing
The nascent chain is further processed as it passes from the ER and through the cis to
trans Golgi. The new protein continues along the secretory pathway until the secretory
vesicle fuses with the plasma membrane. At this point secreted proteins are released
from the cell and membrane proteins are delivered such that the regions of the protein
that were located in the cytoplasm during synthesis remain with this orientation.

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MEMBRANES AND RECEPTORS MODULE - STUDY SESSION 1

AIMS
This work sheet should be completed during the work session in Session 1 and the
associated private study period. The aim of this work is to develop your understanding
of the asymmetric structure and dynamic nature of membrane bilayers.

QUESTION 1
Four modes of mobility are permitted for membrane phospholipids. What are they?
1. ........................................................
2..........................................................
3. ........................................................
4. ........................................................

QUESTION 2
Experimental investigation of the rate of transverse diffusion (flip-flop) of
phospholipids in a bilayer membrane was carried out using a paramagnetic analogue of
phosphatidylcholine, called spin-labelled phosphatidylcholine as shown below.

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The nitroxide (NO) group in spin-labelled phosphatidylcholine gives a distinctive
paramagnetic resonance spectrum whose signal is recognised easily in an appropriate
spectrometer. This signal disappears when nitroxides are converted into amines by
reducing agents such as ascorbate.
Lipid vesicles containing phosphatidylcholine (95%) and the spin-labelled analogue
(5%) were prepared by sonication and purified by gel-filtration chromatography. The
amount of signal from the paramagnetic resonance spectrum decreased to 50% of its
initial value within a few minutes of the addition of ascorbate. What does this result
indicate?
When a second aliquot of ascorbate was added there was no detectable change in the
signal within a few minutes but there was a slow exponential decay with a half-time of
6.5 hours. How would you interpret these changes in the amplitude of the
paramagnetic signal?

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QUESTION 3
It may take up to one day for a phospholipid to move from one lamaella of a lipid
bilayer to the other, while the same phospholipid may move an equivalent distance in
the plane of the bilayer in 2.5 s. How do you account for this difference in mobility ?

QUESTION 4
What is the role of cholesterol in the plasma membrane? How does cholesterol
decrease membrane fluidity at high temperatures and increase fluidity at low
temperatures? 12

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QUESTION 5
The platelet-derived growth factor receptor (PDGF-R) is a plasma membrane protein
that binds the growth factor, platelet-derived growth factor. This protein is identified in
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as a polypeptide with an
apparent molecular size of 180 kDa. Membrane preparations of cells were prepared as
outlined in the table and were subjected to the following labelling protocols followed
by SDS-PAGE to separate the cell proteins:
a) Lactoperoxidase/125Iodine - a non-penetrating protein labelling reagent that labels
proteins on accessible tyrosine residues with radioactive 125iodine.
b) Galactose oxidase - a non-penetrating carbohydrate labelling reagent. Transfers
radioactive 3H onto galactose residues.
Labelling of a 180 kDa polypeptide was assessed.
In addition, the following qualitative assays were performed in each membrane
preparation:
c) specific binding of 125I-labelled platelet-derived growth factor peptide (125I-
PDGF)
d) release of a peptide containing the N-terminal amino acid sequence of the receptor
on treatment with trypsin.
e) binding of a rhodamine-labelled antibody (fluorescent) which recognises a short
amino acid sequence in the C-terminal of the PDGF-R protein.

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What can be concluded concerning the orientation of the PDGF-R protein in the
plasma membrane? Draw a model of the proposed membrane topology of the PDGF-
R. 13

29
What further information would be required to confirm the topology of this protein in
the plasma membrane and the site of PDGF binding ?

Why is the assymetrical orientation of the PDGF-R important?

The C-terminal domain of the PDGF-R shares sequence similarity with protein
tyrosine kinase enzymes that catalyse the transfer of the terminal phosphate in ATP to
tyrosine residues in proteins. Also, it is known that receptor polypeptides need to

30
dimerize to achieve signal transduction. Propose how an extracellular PDGF signal
may be transduced into a cell and suggest why receptor dimerization is necessary.

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QUESTION 6
Apart from small changes of protein conformation only two modes of mobility are
permitted for integral membrane proteins. What are they ?
1. ..............................................................
2. ..............................................................
Why is the movement of protein in lipid bilayers more restricted that that of the lipid
constituents ?

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QUESTION 7
In a plasma membrane, a phospholipid diffuses 200 m sec-1 laterally on average.
Similarly, the average distances moved in one second by two transmembrane proteins,
rhodopsin and fibronectin receptor, are 130 m and 2.0 m, respectively. What might
account for the different mobilities of these two protein molecules? Why do you think
the mobility of these two proteins is different?

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 MEMBRANES AND RECEPTORS MODULE

SESSION 2 - MEMBRANE PERMEABILITY/ CELL VOLUME AND pH

REGULATION

AIMS

This session will introduce the major transport mechanisms that contribute to the
generation of ion gradients across cell membranes and that employ ion gradients to
provide the energy for the transmembrane transport of other ions or small polar
substances. Mechanisms of action of these processes and their role in cellular
physiology will be considered.

LEARNING OUTCOMES

By this session you should be able to:

 discuss the properties of solutes which affect their movement through membranes.
Distinguish passive diffusion, facilitated and active transport. Describe the general
features of channel proteins

 compare the proposed transport mechanisms of the plasma membrane Na+/K+-

ATPase, the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases, and the
Na+/Ca2+-exchanger in regulating ion concentrations inside cells

 describe the pumping of sugars, amino acids and ions by mammalian cells, using
symports and antiports driven by the Na+ gradient. Understand the regulation of
cytoplasm pH via the Na+/H+-exchanger and the Cl-/HCO3--exchanger

PRIVATE STUDY

 Complete study sheet on „Body fluids: regulation of composition and volume‟.

34
 Complete the private study sheet on „the transport of small polar molecules across
biological membranes‟.

 to research the basis of the resting membrane potential in preparation for Lecture 3.1
in Session 3

35
MEMBRANES AND RECEPTORS - SESSION 2

MEMBRANE PERMEABILITY

LECTURE 2.1 - ROLE OF MEMBRANES AS PERMEABILITY BARRIERS

AIMS

To consider the role of membranes as permeability barriers to small hydrophilic

molecules and to explore the protein-mediated mechanisms that allow the uptake or
extrusion of specific water-soluble molecules and ions.

Passive transport: diffusion

Non-polar molecules are able to enter and, therefore, diffuse across the hydrophobic
domain of lipid bilayers. The rate of passive transport increases linearly with
increasing concentration gradient.

Movement of water across membranes by osmosis

Permeability coefficients for most ions and hydrophilic molecules in lipid bilayers are
very low (< 10-10 cm s-1). Surprisingly, membranes are relatively permeable to water
(permeability coefficient = 5 x 10-3 cm s-1) and water will diffuse passively across
lipid bilayers up the concentration gradient of a solute, the osmotic gradient. In some
cells, e.g. kidney proximal tubule, the movement of water may be facilitated by
specific water channels, aquaporins.

Membranes as permeability barriers

The large free energy change that would be required for a small hydrophilic molecule
or ion to traverse the hydrophobic core of the lipid bilayer make the transverse
movement of hydrophilic molecules across an intact biological membrane a rare event.
Thus, membranes act as permeability barriers to all charged and hydrophilic
molecules.

36
The movement of ions and hydrophilic molecules across a membrane is mediated, and
regulated, by specific membrane transport systems. Transport processes have
important roles such as:

the maintenance of intracellular pH

the maintenance of ionic composition

regulation of cell volume

the concentration of metabolic fuels and building blocks

the extrusion of waste products of metabolism and toxic substances

the generation of ionic gradients necessary for the electrical excitability of nerve and
muscle

Facilitated diffusion

The presence of specific proteins in the bilayer can increase the permeability for a
polar substance enormously. For example, the permeability of Cl- through a
phosphatidylserine bilayer is very low. In the erythrocyte membrane this is increased
to ~107 fold. The protein responsible for the transport of Cl- is the Band 3 protein.
This protein does not just form a Cl- selective pore, but carries out a specific exchange
of Cl- for HCO3- which is essential to the function of the erythrocyte.

Models for facilitated transport include protein pores (channels), carrier molecules
(ping-pong) and protein flip-flop (unlikely thermodynamically). Facilitated transport is
a saturable process as each carrier can interact with only one or a few ions or
molecules at any moment and a finite number of transporters are present in the
membrane. Thus, as the concentration gradient increases a maximum rate of transport
will be measured when all the transporters are busy. Similar to enzymes, the
equilibrium point for the transported species is not altered by facilitated transport.

37
 Some pores are gated e.g.

 ligand-gated ion channels - open or close in response to ligand binding to a receptor

site (Sessions 5 and 6)

 voltage-gated ion channels - open and close in response to the potential difference
across the membrane (Sessions 3, 4 and 5)

 gap junction (connexin) - closed when cellular calcium concentration rises above 10
M or the cell becomes acid.

Distinction between passive and active transport

Whether the transport of an ion or molecule can occur spontaneously (passive

transport) or requires energy (active transport) is determined by the free energy change
of the transported species. The free energy change is determined by the concentration
gradient for the transported species and by the electrical potential across the membrane
bilayer when the transported species is charged.

38
Active transport

To overcome unfavourable chemical or electrical gradients the movement of the

transported ion or molecule must be coupled to a thermodynamically favourable
reaction. The free energy to drive active transport can come either directly or indirectly
from the hydrolysis of ATP, electron transport or light. Some cells may spend up to
30-50% of their ATP on active transport.

e.g. Na+-K+-ATPase (Na+ pump) pumps 3 Na+ ions outwards, 2 K+ ions inwards,
against the respective concentration gradients, at the expense of one ATP molecule
hydrolysed. N.B. if the pump runs in reverse it can act as an ATP generator.

In mitochondria, a gradient of H+ ions in employed to drive ATP synthesis via an

ATP-dependent proton transporter.

39
Sometimes the transport of one substance is linked to the concentration gradient for
another via a co-transporter. This is known as secondary active transport, as the
primary energy source, e.g. hydrolysis of ATP, is used indirectly. Membrane
transporters may be driven by gradients of ATP, phosphoenolpyruvate, protons and
sodium ions, light and high-potential electrons. Often a sodium gradient across a
membrane is employed.

Cotransport systems

Na+- glucose co-transport system of the small intestine and kidney (symport). Entry of
sodium provides the energy for the entry of glucose.

Na+- Ca2+-exchange - Inward flow of sodium down its concentration gradient drives
outward flow of Ca2+ up its concentration gradient (antiport).

Na+- H+- exchange - Inward flow of sodium down its concentration gradient leads to
cell alkalization by removing H+ (antiport).

Transporter terminology

When one solute molecule species is transported from one side of the membrane to the
other, the transporter is called a uniport. Other transporters are referred to as co-
transporters, when the transfer of one solute molecule depends on the simultaneous
or sequential transfer of a second solute in the same direction (symport) or in the
opposite direction (antiport).

40
41
MEMBRANES AND RECEPTORS - SESSION 2

MEMBRANE PERMEABILITY

LECTURE 2.2 - ATP-DEPENEDNT ION PUMPS AND ION EXCHANGERS

AIMS

 To outline the major physiological roles of

 Sodium-potassium ATPase (Na+-K+-ATPase, Na pump)

 Plasma membrane Ca2+ ATPase (PMCA)

 Sarco(endo)plasmic reticulum ATPase (SERCA)

 Sodium calcium exchange (NCX)

 Sodium hydrogen exchange (NHE)

 Anion exchange (AE)

 To consider how ion transporters work together in cell physiology

 To consider how ion transport contributes to

 Control of resting intracellular Ca2+ concentration

 Cellular pH regulation

 Cell volume regulation

 Renal bicarbonate reabsorption

 Renal Na+ handling

42
Na+-K+-ATPase (Na pump)

Forms Na+ and K+ gradients

Drives many secondary active transport processes

o Ion homeostasis, [Ca2+]i, pHi, cell volume, resting membrane potential, nutrient
uptake

Control of intracellular Ca2+ concentration

There is an ~20,000 fold difference in Ca2+ concentration across the plasma

membrane (~2 mM/50- 100 nM (extracellular/intracellular). High [Ca2+]i is toxic to
cells and therefore needs to be controlled.

43
Sodium calcium exchanger (NCX)
The NCX exchanges 3 Na+ for 1 Ca2+ and is, therefore, electrogenic with current
flowing in the direction of the Na+ gradient. In depolarised cells, the normal mode of
operation of NCX is inhibited and its mode of operation reverses, i.e. to bring Ca2+
into the cell. In this way NCX makes a contribution to Ca2+ influx during the cardiac
action potential (see session 4 and CVS module) and can contribute to Ca2+ toxicity
during periods of ischaemia.

44
Ion transporters in cellular pH regulation

When cellular buffering capacity is exceeded, cellular pH is controlled by the activity

of a variety of plasma membrane transporters. Acidification can be opposed by
expelling H+ ions or the inward movement of bicarbonate ions. Alkalinisation is
opposed by expelling bicarbonate via the anion exchanger.

45
Cell volume regulation

Electroneutral transport of ions allows the osmotic strength of the cytoplasm to be

varied without effect on the membrane potential (see session 3). Cells extrude ions in
response to cell swelling and influx ions in response to cell shrinking. Water follows.
Different cell types use particular combinations of transporters to achieve the
regulation they need.

46
The concerted action of transporters

By working together, ion channels can achieve physiological endpoints that would not
be possible if they worked in isolation, e.g. bicarbonate reabsorption in the proximal
tubule of the kidney, Na+ reabsorption in kidney tubules.

Almost all of the Na+ that appears in the glomerular filtrate is reabsorbed from the
kidney nephron. The driving force for this reabsorbtion is the low intracellular Na+

47
concentration that is maintained by Na+-K+-ATPase activity in tubular cells. Several
transport mechanisms are involved in Na+ reabsorbtion at different locations in the
nephron.

Where fluid loss is required to treat oedema or hypertension, block of one or more of
the Na+ reabsorbtion mechanisms with diuretic drugs can be used to increase Na+
excretion to produce a hyperosmotic urine and, hence, the excretion of water.

48
49
MEMBRANES AND RECEPTORS - STUDY SESSION 2

BODY FLUIDS: REGULATION OF COMPOSITION AND VOLUME

AIMS - The aims of this study session and the associated private study are to develop
an appreciation of the need to regulate the composition and volume of the different
body compartments and to consider the membrane transport mechanisms involved.

EXERCISE - Use the questions as a guide to your reading in standard physiology and
biochemistry text books. You will find some relevant sections in several of the
textbooks held in the Library. Particularly useful texts are:

Koeppen, B.M. & Stanton, B.A. Berne & Levy: Principles of Physiology, 6th Edition,
Wolfe Publications, 2008, ISBN 9780323073622

Guyton, A.C., Human Physiology and Mechanisms of Disease, 6th Edition, W.B.
Saunders, 1997, ISBN 0721632998

Widmaier, E.P., Raff, H. & Strang, H. Vander‟s Human Physiology: the mechanisms
of body function, 11th Edition, McGraw-Hill, 2005, ISBN 9780071283663

Answers to some questions may not be found easily in available texts. Where this is
the case discuss possible answers with your colleagues and test these in consultation
with the tutors.

50
QUESTION 1 - Describe the fundamental differences in composition between
intracellular and extracellular fluids?

QUESTION 2 -

(a) What is the approximate total aqueous volume contained within an average 70 kg
medical student.

(b) What proportion of this fluid is intracellular and extracellular?

(c) In what compartments is the extracellular fluid distributed? What is the volume of
fluid in each compartment?

51
QUESTION 3 - What are the most important membrane transport mechanisms
involved in the control of intracellular Na+, K+ and Ca2+ concentrations?

QUESTION 4 - Describe the immediate consequences of a sudden increase/decrease

of the extracellular sodium concentration?

QUESTION 5 - In what ways do you think cells might use membrane transport
systems to maintain a constant cell volume? (This subject is poorly covered in standard
physiology textbooks. The answer to this question requires a consideration of the
principles involved only).

52
QUESTION 6 - What are the consequences of an increase in the permeability of
blood capillaries to plasma proteins?

QUESTION 7 - In what ways can membrane transport processes contribute to the

regulation of intracellular pH?

53
MEMBRANES AND RECEPTORS - PRIVATE STUDY A SESSION 2

THE TRANSPORT OF SMALL POLAR MOLECULES ACROSS

BIOLOGICAL MEMBRANES

AIMS

To understand how membrane transport processes can mediate the transport of small
polar molecules across biological membranes.

EXERCISE

Information may be found in the standard biochemistry and physiology textbooks.

QUESTION 1 - Draw an annotated diagram to describe how different glucose

transporters in intestinal epithelial cells operate together to transport glucose from the
gut into the blood against the uphill concentration gradient for glucose. Note: this
mechanism for glucose transport is also employed in kidney epithelial cells.

QUESTION 2 - How does the uptake of glucose from the blood into adipose, brain,
liver and skeletal muscle cells differ that in intestinal and kidney epithelial cells ?

54
QUESTION 3 - How does insulin stimulate the rate of uptake of glucose into adipose
tissue and skeletal muscle ?

QUESTION 4 - What prevents the efflux of glucose from cells in tissues such as
adipose and skeletal muscle when the circulating glucose concentration falls to resting
levels in the post-adsorptive period after a meal ?

55
QUESTION 5 - Apart from glucose, what other metabolites use the sodium gradient
for their uptake into cells against the concentration gradient?

56
MEMBRANES AND RECEPTORS MODULE

SESSION 3- THE RESTING CELL MEMBRANE

AIMS

The aims of this session are:

 to develop an understanding of the membrane potential in cells

 to outline how they are set up and how they may be changed by mechanisms
involved in cellular signalling.

LEARNING OUTCOMES

By this session you should be able to:

 describe the ionic basis of membrane potential and the differences in ionic
composition of intra- and extra-cellular fluids

 understand what is meant by membrane depolarization or hyperpolarization and

the roles of the major ion-specific channels in the plasma membrane

PRIVATE STUDY

Private study time should be used to:

 complete the work sheet associated with this session in preparation for the
tutorial in Session 5.

 to research the basis of the action potential and nerve impulse conduction in
preparation for Lectures 4.1 and 4.2 is Session 4.

 Start thinking about your presentations for Sessions 6 and 7. See Assignment
Presentation booklet.

57
MEMBRANES AND RECEPTORS - SESSION 3

THE RESTING CELL MEMBRANE

LECTURE 3.1 - THE RESTING MEMBRANE POTENTIAL

AIMS

This session should develop your understanding of the membrane potential of cells;
how they are set up and may be changed by mechanisms involved in cell signalling.

By the end of the session you should be able to:

 outline what a membrane potential is, how the resting potential of a cell may be
measured, and the range of values found

 understand the concept of selective permeability, and explain how the selective
permeability of cell membranes arises

 describe how the resting potential is set up given the distribution of ions across
cell membranes.

 understand the term equilibrium potential for an ion, and calculate its value form
the ionic concentrations on either side of the membrane.

 define depolarization and hyperpolarization, and explain the mechanisms that

may lead to each of these

 explain how changes in ion channel activity can lead to changes in membrane
potential, and outline some of the roles of the membrane potential in signalling
within and between cells.

 outline how ligand-gated channels can give rise to synaptic potentials.

58
THE RESTING MEMBRANE POTENTIAL

All cells have an electrical potential difference across their plasma membrane.
Changes in this membrane potential underlie the basis of signal transmission in the
nervous system and in many other cells.

Measuring the Membrane Potential

Membrane potentials can be measured using a very fine micropipette - a

microelectrode - that will penetrate the cell membrane.

Selective Permeability of the Cell Membrane

Membrane potentials are set up because the membrane is selectively permeable to

different ions. The permeability of the membrane to ions occurs by way of channel
proteins; membrane-spanning transport proteins that allow ions to permeate.

59
These ion channels are characterized by:

1. Selectivity: the channel lets through only one (or a few) ion species. Channels
selective for Na+, K+, Ca2+ , Cl- , and with non-selective cation permeability are
known.

2. Gating: the channel can be open or closed by a conformational change in the protein
molecule.

3. A high rate of ion flow that is always down the electrochemical gradient for the ion.

So, depending on which types of channel are open, the resting membrane can be
selectively permeable to certain ion species.

Setting up the Resting Potential

60
At rest the membrane has open K+ channels, so is selectively permeable to K+ . K+
will begin to diffuse out of the cell down its concentration gradient. Since anions
cannot follow, the cell will become negatively charged inside. This membrane
potential will oppose the outward movement of K+, and the system will come into
equilibrium.

The Equilibrium Potential for K+

How big will the membrane potential be for given K+ concentrations on either side?
Imagine a model system, in which a membrane perfectly selective for K+ ions
separates two solutions with different K+ concentrations (in each case balanced by an
anion A- that cannot pass through the membrane).

61
The system will rapidly come into equilibrium so that the electrical (dotted line) and
diffusional (solid line) forces balance one another and there is no net movement of K+.
The membrane potential at which this occurs is called the potassium equilibrium
potential or EK. It can be calculated from the Nernst equation:

where V is the membrane potential, R is the gas constant, T is the temperature in o K,

Z the valency of K+ (+1), F is Faraday's number, and [K+]o and [K+]i are the outside
and inside concentrations of K+. It is common to work out the constants and convert
the natural logarithm to log10, giving, at 37oC:

62
The Nernst equation may be written for other ions as well, e.g. Na+, Ca2+, Cl-

The living cell

For the concentrations above, EK works out at -95 mV. Open K+ channels dominate
the resting permeability of many cells, so the resting membrane potential (RP) is quite
close to EK. The membrane is not perfectly selective, however, mainly because other
types of channel are also open, and so the RP is rather less negative than EK. In
skeletal muscle, the resting membrane is highly permeable to Cl- as well as K+, and
resting potential lies close to both EK and ECl.

The dependence of the RP on K+ permeability means that changing EK will change

the RP. Increasing [K+]o makes EK more positive and so changes the membrane
potential in the same direction.

63
MEMBRANES AND RECEPTORS - WORK SHEET SESSION 3

EQUILIBRIUM POTENTIALS
Aims - The aim of this session is to illustrate lecture 3.1 on the resting membrane
potential. The exercises will help you to consider the contribution to the resting
membrane potential of the major ionic species that are distributed across the membrane
of cells. You will also begin to assess the consequences of a change in permeability to
different ionic species for the membrane potential in preparation for lecture 3.2
concerning changing membrane potentials and Session 4 on electrical excitability.

The equilibrium potential for an ion, Eion is given by the Nernst Equation

where R is the Gas constant, T the absolute temperature, F Faraday‟s number and Z the
valency (+1 for K+, -1 for Cl- etc, [ion]out and [ion]in are the extracellular and
intracellular concentrations of the ion.

Working out the constants at 37°C, and changing the logarithm to base 10

64
You may find it helpful to refer to text books or your tutors for some of these
questions. Some of these questions are designed to start you thinking about concepts to
be introduced in the next two lectures concerning the electrical excitation of
membranes employed in electrical signalling.

EXERCISE 1.

a) What is responsible for the unequal distribution of inorganic ions between the
intracellular and extracellular fluid?

b) Resting cell membranes are selectively permeable to K+. Given the concentration
gradient that exists across the plasma membrane, which direction would you predict
that K+ ions will move?

65
c) What effect will this have on membrane potential and why?

d) Using the Nernst equation, calculate the K+ equilibrium potential (EK) for cells
with intra- and extracellular fluid compositions as given in the Table above.

e) The actual membrane potential of a nerve cell, when measured with a

microelectrode, was found to be -75 mV. Why was the measured membrane potential
different to that calculated for EK?

f) What would happen to the membrane potential if there was an increase in the
permeability of the membrane to K+ ions ?

g) What contribution does the Na+-K+-ATPase make to the maintenance of the resting
membrane potential?

66
EXERCISE 2.

a) Replace Cl- for K+ in the Nernst equation and calculate the chloride equilibrium
potential (EBClB). Note: the charge on the ion being considered is negative. This
should be taken into consideration in your calculation.

b) What can you conclude when comparing ECl with EK ? What contribution does Cl-
permeability makes in nerve cells to fixing membrane potential (relative to that of
K+)?

c) Some neurotransmitters act to increase Cl- conductance in the postsynaptic cell.

What are the consequences of an increased Cl- conductance for the membrane
potential.

67
EXERCISE 3.

a) Calculate ENa

b) During the initial phase of the action potential (electrical impulse involved in
electrical excitation) in nerve and muscle plasma membranes the Na+ permeability
increases so much that it becomes very much higher than that of K+. What can you
predict in relation to the membrane potential ?

c) The membrane potential is restored rapidly to resting levels in nerve and muscle
cells after an action potential. How do you think how this is achieved?

d) What do you predict would be the effect on membrane excitability of increasing the
permeability to Cl- ions?

68
EXERCISE 4.

a) Calculate ECa. Note: the valency of Ca2+ is +2. This should be taken into
consideration in your calculation.

b) During the heartbeat, myocardial Ca2+ channels open and result in a substantial
increase in Ca2+ permeability. In which direction does the Ca2+ flow ?

EXERCISE 5.

a) In a hyperkalaemic patient, the plasma K+ concentration was found to be 7.5 mM.

Assuming a similar value for the interstitial fluid, what would the consequences be for
the resting cell membrane?

b) Predict what the clinical consequences of such a changes might be.

69
MEMBRANES AND RECEPTORS - SESSION 3

THE RESTING CELL MEMBRANE

LECTURE 3.2 - CHANGING MEMBRANE POTENTIALS

Changes in membrane potentials underlie many forms of signalling between and

within cells.

There are two basic terms used to describe changes from the resting level:

Depolarization: a decrease in the membrane potential, so that the inside of the cell
becomes less negative.

Hyperpolarization: an increase in the membrane potential, so that the inside of the

cell becomes more negative.

Changing Membrane Permeability to Ions

The ionic distribution between cytoplasm and extracellular fluid gives positive
equilibrium potentials for Na+ and Ca2+, while those for K+ and Cl- are negative. If
the membrane permeability for one type of ion is increased by opening channels for
that ion, the membrane potential will move towards the equilibrium potential for that
ion. Thus:

Opening Na+ or Ca2+ channels will depolarize cells.

Opening K+ or (usually) Cl- channels will hyperpolarize cells.

Dealing with Real Cell Membranes that are not perfectly selective

When channels for more than one ion species are open these ions will contribute to the
membrane potential. How important each ion is will depend on how easily it can get
through the cell membrane relative to other ions - dependent on the number of

70
available channels and how easily they let the ion through. An expression that often
approximates what will happen quite well is the GHK equation (for Goldman-
Hodgkin-Katz).

Vm is the membrane potential and PNa, PK, and PCl are the relative permeabilities to
these ions.

Controlling Channel Activity

The number of open channels of different types underlies the overall selectivity of the
cell membrane. Channel opening is in turn controlled by the gating mechanisms that
open or close the channels involved. Channels are gated in two main ways:

Ligand gating: the channel is opened (or closed) by binding of a chemical ligand,
which may be an extracellular transmitter or an intracellular messenger.

Voltage gating: the channel opens or closes in response to changes in the membrane
potential.

Synaptic potentials

In fast synaptic transmission, the receptor is also a ligand-gated ion channel.

71
In slow synaptic transmission, the receptor is not itself an ion channel, but signals to
the channel in one of two ways, both involving a GTP-binding protein:

1. Within the membrane:

72
73
MEMBRANES AND RECEPTORS MODULE

SESSION 4 - ELECTRICAL EXCITABILITY

AIMS

The aims of this session are:

 to develop an understanding of the basis of electrical signalling in excitable

cells,

 to understand the permeability changes of the plasma membrane associated with

the action potential and

 to consider the role of nerve myelination

LEARNING OUTCOMES

By this session you should be able to:

 describe the properties of voltage-gated ion channels, the general features of

electrical excitability of membranes, and permeability changes associated with the
action potential

 discuss factors affecting conduction velocity in nerves

PRIVATE STUDY

Private study time should be used to

 develop your understanding of saltatory conduction in myelinated nerve fibres

and the effects of demyelination. A private study sheet is provided.

You should also:

 complete the work sheet associated with this session in preparation for the
tutorial in Session 5.

74
  start thinking about your presentations for Sessions 6 and 7. See Assignment
Presentation booklet

 continue thinking about your presentations for Session 6 and 7. See Assignment
Presentation booklet.

MEMBRANES AND RECEPTORS - SESSION 4

ELECTRICAL EXCITABILITY

LECTURE 4.1 - THE ACTION POTENTIAL AND ITS PROPERTIES

AIMS

 To understand the properties of the action potential and its ionic basis

 To be able to describe the associated changes in membrane ionic permeability

 To be able to describe the basis of the all-or-nothing law and refractoriness in

terms of these changes in permeability

 To be able to describe some molecular properties of ion channels

 To understand the action of local anaesthetics

Properties of the nervous impulse - the all-or-nothing law of excitable cells.

75
76
77
This is done using a technique called voltage-clamp. The membrane potential is
controlled and the currents flowing through the membrane is measured. This gives a
much clearer measurement of the effect of voltage on the number of Na+ and K+
channels open at different membrane potentials. Both of these channel types are
voltage-gated which means that depolarisation will cause them to open. During
maintained depolarisation Na+ channels close by a mechanism called inactivation.

Once a certain membrane potential is reached a positive feedback occurs as Na+

channels begin to open. This is the threshold for action potential generation.

78
79
The molecular nature of voltage-gated channels has been determined. Na+ and Ca2+
channels are similar, their main pore forming subunit is one peptide consisting of 4

80
homologous repeats. Each repeat consists of 6 transmembrane spanning domains with
one of these domains being able to sense the voltage field across the membrane.

81
82
Local anaesthetics, such as procaine, act by binding to and blocking Na+ channels,
thereby stopping action potential generation.

Local anaesthetics block conduction in nerve fibres in the following order:

 small myelinated axons

 non-myelinated axons

 large myelinated axons

They are weak bases and cross the membrane in their unionised form. They block Na+
channels easier when the channel is open and also have a higher affinity to the
inactivated state of the Na+ channel.

83
MEMBRANES AND RECEPTORS MODULE - WORK SHEET SESSION 4

AIMS: - This work session should develop your understanding of the way in which
action potentials occur in excitable cells, of some of their fundamental properties, and
of the effects of certain drugs on the action potential.

By the end of this session you should be able to:

 illustrate the change in membrane potential that occurs during an action

potential

 describe how the ionic movements underlying the action potential occur

 outline the properties of voltage-activated ion channels

 explain the basis of refactoriness in nerve and muscle

 explain how local anaesthetics act

EXERCISE

1. The basis of the action potential

a) Sketch the relationship between membrane potential and time during an action
potential, labelling the scales appropriately.

84
b) The action potential is often described as all-or-nothing. What does this mean? Can
you think of any consequences for the coding of information as it is transmitted by
nerve fibres?

c) Which ion channels are involved in generating an action potential?

85
d) Briefly describe the molecular nature of the channels you list.

e) What do you understand by the terms activation and inactivation, applied to a

voltage-activated ion channel?

f) Indicate on your diagram on page 44 the way in which the open probability of the
channels involved changes during the action potential.

g) Describe how ions move as a consequence of channel opening during the action
potential.

86
2. How much ion moves to generate an action potential?

For a nerve fibre 1μm in diameter, a calculation of the Na+ entry needed to generate
one action potential (based on a membrane capacitance of 1 μF/cm2 and a voltage
change of 100 mV) shows that this Na+ entry will increase the intracellular [Na+] by
about 40 μM (4 x 10-5 M). The resting intracellular [Na+] is about 10-12 mM.

If the Na+/K+ pump were to be blocked (for example by using ouabain), what would
be the consequences for nerve conduction of:

a) A single action potential in this nerve fibre?

b) A train of 1,000 action potentials?

3. Accommodation and refractoriness

a) Explain the term accommodation as applied to nerve.

87
b) What do you understand by the terms absolute refractory period and relative
refractory period of a nerve fibre.

c) Explain how the properties of ion channels lead to the absolute and relative
refractory periods you defined above.

4. Drugs affecting action potentials

a) How might a drug act to block the production of action potentials?

b) Indicate how local anaesthetics act to block action potentials of peripheral nerves.
Name one such drug.

c) Tetrodotoxin (from the Japanese puffer fish) blocks voltage-gated Na+ channels and
occasionally causes poisoning in Japan. 4-aminopyridine blocks voltage-gated K+
channels. Both compounds are often used in experimental studies of nerve. What
effect would you expect each to have on the action potential?

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MEMBRANES AND RECEPTORS - SESSION 4

ELECTRICAL EXCITABILITY

LECTURE 4.2 - CONDUCTION OF THE NERVE IMPULSE

Learning outcomes – you should be able to:

• Describe the results of extracellular recording and how this can be used to measure
conduction velocity

• Explain how axons are raised to threshold

• Explain the local circuit theory of propagation

• Explain how conduction velocity is linked to fibre diameter

• Explain the implications of myelination for conduction

• Describe certain consequences of demyelination

Electrical stimulation occurs under a cathode (negatively charged); excitability will

be reduced under an anode (positively charged). This can be used to stimulate an axon
or group of axons to threshold, thus initiating an action potential.

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Conduction velocity is calculated by measuring the distance between the stimulating
electrode and the recording electrode and the time gap between the stimulus and the
action potential being registered by the recording electrode:

conduction velocity = distance / time

How is the action potential conducted along an axon?

 A change in membrane potential in one part can spread to adjacent areas of the
axon

 This occurs because of local current spread shown diagrammatically below

 Conduction velocity is determined by how far along the axon these local
currents can spread

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  When local current spread causes depolarisation of part of the axon to threshold
then an action potential is initiated in that location

The further the local current spreads down the axon the faster the conduction velocity
of the axon will be. Properties of the axon that lead to a high conduction velocity
include:

 A high membrane resistance

 A low membrane capacitance

 A large axon diameter (this leads to a low cytoplasmic resistance)

Capacitance, C, is the ability to store charge. This is a property of the lipid bilayer. A
high capacitance takes more current to charge (or a longer time for a given current)
and can cause a decrease in spread of the local current, especially with brief current

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pulses. The membrane resistance depends on the number of ion channels open. The
lower the resistance the more ion channels are open and the more loss of the local
current occurs across the membrane, thus limiting the spread of the local current effect.
A diagram indicating these points is shown below:

These local currents cause the action potential to propagate down the axon. Note that
the action potential will not begin to go backward because an area of axon that has just
fired an action potential is refractory, i.e. it cannot fire another action potential until it
has recovered from being refractory, see below:

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The myelin sheath

Conduction velocity is increased considerably by myelination of axons. Large

diameter axons such as motoneurones are myelinated, smaller ones such as C-fibres
(sensory neurones) are not. The effect of myelin is to reduce the capacitance and
increase the resistance of the axon. Myelin is formed by special cells:

Schwann cells - these myelinate peripheral axons

Oligodendrocytes - these myelinate axons in the CNS

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Demyelination

There are certain diseases where areas of some axons can lose their myelin sheath. The
most well known condition is multiple sclerosis. This is a disease of the immune
system where myelin is destroyed in certain areas of the CNS. This can have dramatic
effects on the ability of previously myelinated axons to conduct action potentials
properly. This can lead to decreased conduction velocity, complete block or cases
where only some action potentials are transmitted.

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MEMBRANES AND RECEPTORS - SESSION 4

PRIVATE STUDY

AIMS: Using this study sheet you should develop your understanding of saltatory
conduction in myelinated nerve fibres and of the consequences of demyelination, at
least at a cellular level. Further understanding of the consequences for motor, sensory
and other functions of the nervous system will be dealt with in the neurobiology
module.

By the end of this session you should:

 understand how conduction occurs in unmyelinated and myelinated nerve;

 understand how the ionic movements examined earlier in this Session occur in
myelinated nerve fibres;

 understand how and when myelination occurs;

 know at least one condition in which demyelination occurs;

 understand the consequences of demyelination.

1. Conduction in unmyelinated nerve

Draw a diagram to show the way in which an action potential can propagate along an
unmyelinated nerve fibre.

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What carries the current that allows propagation of the action potential?

2. The nature of saltatory conduction

Describe what is meant by the term saltatory conduction.

How is the myelin sheath formed and what is the composition of myelin?

What properties of myelin permit saltatory conduction to occur?

Describe where the ionic movements that generate action potentials occur in
myelinated nerve fibres.

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Draw the relationship between conduction velocity and fibre diameter for myelinated
and unmyelinated nerve fibres, labelling the scales appropriately. Indicate on your
diagram the range of fibre diameters found for the two fibre types.

What is the explanation for the fact that at small diameters, unmyelinated fibres
conduct faster than myelinated fibres?

How long does it take the nervous impulse to travel from one node of Ranvier to the
next?

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Are myelinated or unmyelinated nerve fibres easier to stimulate using stimulating
electrodes, for example applying current to human peripheral nerves through the skin?

3. Structure and cell biology of myelinated nerve fibres

Draw a diagram of a myelinated nerve fibre, and indicate the following:

The internodal distance

The relative thickness of the myelin sheath

What cells form the myelin sheath of nerve fibres in the:

a) Central nervous system?

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b) Peripheral nervous system?

When during development does myelination occur?

Some myelinated nerve fibres are able to regenerate from the central end if cut. Does
this occur in the peripheral or central nervous systems? And what is the rate of such
regeneration?

What is known of the distribution of ion channels in myelinated nerve?

4. Effects of demyelination

What conditions may lead to demyelination of nerve fibres?

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What is thought to cause this pathological demyelination?

What are the consequences for conduction of the nervous impulse of demyelination
that is:

a) Partial?

b) Complete?

Will demyelination make nerve fibres easier or more difficult to stimulate with
currents applied with stimulating electrodes?

What is the maximum internodal delay that can occur during propagation. How is this
delay related to the duration of the action potential?

What might be the effect of treating a demyelinated nerve fibre with an agent that
blocks voltage-gated potassium channels?

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MEMBRANES AND RECEPTORS MODULE

SESSION 5 - EFFECTS OF ELECTRICAL SIGNALS - LIGAND GATED

CHANNELS

AIMS

 The aims of this session are:

 to develop an understanding of how electrical signals at the plasma membrane

are converted into other cellular events,

 to consider the steps involved in synaptic transmission,

 to consolidate information given on membrane potential and electrical

excitability (tutorial 1)

 to follow up on your private study, to integrate information on the control of

intracellular calcium ion concentration (tutorial 2)

LEARNING OUTCOMES

By this session you should be able to:

 explain the concept of ligand-gated channels, and describe the steps of

neurotransmission at the mammalian neuromuscular junction
 Discuss the importance of the control of intracellular Ca2+ concentration

PRIVATE STUDY
 Complete the private study sheet on „Receptors‟ in preparation for Lecture 6.1,
Session 6.
 Continue with preparations for presentations in Sessions 6 and 7

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  Complete the private study revision sheet on „Control of intracellular calcium
concentration‟.

MEMBRANES AND RECEPTORS - SESSION 5

ELECTRICAL EXCITABILITY
LECTURE 5.1 - THE CELLULAR RESPONSE TO ACTION POTENTIALS

AIMS
 To understand how action potentials open Ca2+ channels in cell membranes
 To be able to describe some aspects of the diversity of Ca2+ channels
 To be able to describe events underlying fast synaptic transmission
 To be able to describe some properties of ligand gated ion channels, with
nicotinic acetylcholine receptors as an example
 To understand the action of two types of blockers of nicotinic receptors

At the nerve terminal there are Na+ channels, K+ channels and Ca2+ channel

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104
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Ca2+ channels are located close to vesicle release sites. The increase in [Ca2+]I
following an action potential reaching the motor nerve terminal activates a group of
proteins associated with the vesicle to promote exocytosis of ACh.

The ACh will bind to the nicotinic ACh receptor on the post-junctional membrane to
produce an end-plate potential; this depolarisation in turn raises the muscle above
threshold so that an action potential is produced in the muscle membrane.

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The nicotinic acetylcholine receptor is an example of a ligand-gated ion channel.
Pharmacological agents that competitively block nicotinic acetylcholine receptors do
so by binding at the molecular recognition site for ACh.

Myesthenia gravis is an autoimmune disease targeting nACh receptors.

 Patients suffer profound weakness

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 Weakness increases with exercise

 Caused by antibodies directed against nAChR on postsynaptic membrane of

skeletal muscle

 Antibodies lead to loss of functional nAChR by complement mediated lysis and

receptor degradation

 Endplate potentials are reduced in amplitude leading to muscle weakness and

fatigue

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MEMBRANE AND RECEPTORS MODULE – SESSION 5

LECTURE 5.2 – CONTROL OF INTRACELLULAR CALCIUM

CONCENTRATION

AIMS AND OBJECTIVES

The aim of this lecture is to provide you with an understanding of the „tool-box‟ that
cells have at their disposal to handle the Ca2+ ion. Under resting or basal conditions,
the intracellular (cytoplasmic) free [Ca2+] ([Ca2+]i) is maintained at a very low level
compared to the surrounding extracellular fluid. However, changes in [Ca2+]i are used
to regulate an extremely wide variety of cellular events. This lecture will explore the
mechanisms by which the basal [Ca2+]i is achieved and how [Ca2+]i can be elevated
to alter cell function. For changes in [Ca2+]i to be used as a signalling event, the
[Ca2+]i must also be rapidly restored to basal levels. Furthermore, elevations of
[Ca2+]i that are too great or occur for too long are detrimental to the health of the cell,
emphasising the need to tightly control [Ca2+]i. This lecture will also explore the
mechanisms by which [Ca2+]i is restored following a Ca2+ signalling event.
Although some elements of the Ca2+ signalling tool-box are ubiquitous, not all cells
will express all the „tools‟ that will be discussed. In addition, the relative importance of
each may vary between different cell types and even within a specific cell type under
different conditions. Within the lecture some examples will be used to illustrate the
flexibility of the tool-box.
At the end of this lecture you should be aware of the major mechanisms by which cells
are able to regulate their [Ca2+]i and be aware of examples of how changes in [Ca2+]i
can be used as an intracellular signalling mechanism to regulate cellular physiology.

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Cellular Ca2+ handling
There are many Ca2+-sensitive processes in cells. For example, alterations in [Ca2+]i
are responsible for or regulate: fertilization, proliferation, secretion, neurotransmission,
metabolism, contraction, learning and memory, apoptosis and necrosis. As Ca2+
cannot be metabolised the cell has to regulate [Ca2+]i by mechanisms based largely on
moving Ca2+ into and out of the cytoplasm.

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Setting-up and maintaining the gradient

This relies on:

1. Relative impermeability of the plasma membrane

2. The ability to expel Ca2+ across the plasma membrane using:

a) Ca2+-ATPase

b) Na+-Ca2+ exchanger

3. Ca2+ buffers

4. Intracellular Ca2+ stores:

a) rapidly releasable

b) non-rapidly releasable

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How is the [Ca2+]i elevated and returned to basal levels?

In most cells, basal [Ca2+]i is around 100nM. When alterations in [Ca2+]i are used to
regulate aspects of cellular activity the global [Ca2+]i can reach concentrations around
1μM. Some Ca2+-dependent processes appear to require an even higher [Ca2+] and it
is believed these can be achieved due to microdomains. These are areas where the
[Ca2+] is in excess of that measured globally, for example, immediately around an
open, Ca2+-selective ion channel.

Changes in [Ca2+]i:

1. Ca2+ influx across the plasma membrane (ie. altered membrane permeability):

a) Voltage-operated Ca2+ channels (VOCC) (voltage-gated Ca2+ channels – VGCC)

b) Receptor-operated ion channels (ionotropic receptors)

2. Ca2+ release from „rapidly-releasable‟ stores:

a) G-protein-coupled receptors (GPCRs)

b) Ca2+-induced Ca2+ release (CICR)

3. Non-rapidly releasable stores (mitochondria)

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You should now understand the mechanisms by which cells are able to regulate their
[Ca2+]i and have an appreciation of how changes in [Ca2+]i can be used to alter
cellular function. Remember that the relative importance of these mechanisms will

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differ depending on the cell type and the circumstances in which the cell may find
itself.

MEMBRANES AND RECEPTORS – SESSION 5 - PRIVATE STUDY 1

RECEPTORS

AIMS

The aims of this private study and study session are to introduce the concept of cellular
receptors and, in overview, to find out about the various roles of receptors in cell
physiology and to consider the different molecular mechanisms for transducing an
extracellular message into an intracellular response.

EXERCISE

Use the questions as a guide to your reading in standard biochemistry, physiology and
pharmacology textbooks.

TIP: Simply looking up 'receptor' is likely to be unproductive. It is suggested that

indexes are scanned for 'x' receptor where x is any hormone or neurotransmitter
binding to the receptor eg. adrenaline, acetylcholine, insulin,

Lecture 6.1 in Session 6 will review this area and consolidate your private work.

QUESTION 1 - How are extracellular chemical signals recognised at target tissues?

QUESTION 2 - Define the term "ligand"

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QUESTION 3 - Devise a definition of a receptor

QUESTION 4 - What are the similarities and differences between ligand binding sites
on receptors and active sites and regulatory sites in enzymes?

QUESTION 5 - List as many cellular processes as you can that involve cellular
receptors.

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QUESTION 6 - In cellular signalling, what different mechanisms do receptors employ
to transduce an extracellular chemical signal into an intracellular event?

MEMBRANES AND RECEPTORS - SESSION 5, PRIVATE STUDY 2

CONTROL OF INTRACELLULAR CALCIUM CONCENTRATION –

INTEGRATION & REVISION

AIMS:

To find out about the control of intracellular calcium ion concentration, [Ca2+]i.

To integrate relevant information presented in earlier sessions.

OBJECTIVES:

Relevant objectives in the module include:

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compare the proposed transport mechanisms of the plasma membrane Na+/K+-
ATPase, the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases, and the
Na+/Ca2+-exchanger in regulating ion concentrations inside cells

describe the various receptor-effector mechanisms; receptors linked to ion channels, to

G-proteins or to protein kinases. Understand the concepts of second messengers and
cascades. Discuss the importance of cyclic nucleotides and the control of intracellular
Ca2+ concentration

explain the concept of ligand-gated channels, and describe the steps of

neurotransmission at the mammalian neuromuscular junction

REFERENCES:

Standard biochemistry, physiology and pharmacology textbooks will contain most of

the information you need for this exercise. You may also find it useful to refer to the
following:

Barritt, G.J. 1992 Communication within animal cells. Oxford Science Publications.
Chapters 6, 7 and 13.

EXERCISE:

Answer the following questions in preparation for tutorial 2 in session 7.

QUESTIONS:

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 1. What are the approximate Ca2+ concentrations of extracellular fluid and the
cytoplasm in resting cells? What is the concentration difference between
intracellular and extracellular fluid?

2. How is this concentration difference maintained?

3. By what general mechanisms can [Ca2+]i be elevated ? Give an example of each

mechanism.

4. List some cellular events that are dependent on raised [Ca2+]i.

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5. What is the function of calmodulin in transmitting a Ca2+ signal to cellular
components ?

6. Annotate the diagram below to show how the mechanism for raising cytoplasmic
Ca2+ concentration in a cell in response to acetylcholine might be different depending
on whether a nicotinic or a muscarinic M1 receptor are present. Which of these
responses would you expect to be faster?

7. How is the release of sarcoplasmic reticulum stores of Ca2+ coupled to membrane

depolarization in skeletal muscle?

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8. With reference to the contribution of Na+-Ca2+-exchange to the control of [Ca2+]i,
what could be the consequences for [Ca2+]i of membrane depolarization or a raised
intracellular Na+ concentration?

9. Describe the changes in [Ca2+]i in cardiac ventricular cells during the cardiac cycle
? How do voltage-sensitive Ca2+ channels, calcium-induced calcium release (CICR)
from the sarcoplasmic reticulum, Na+-Ca2+-exchange, and plasma membrane and
sarcoplasmic reticulum Ca2+-ATPases contribute to these changes?

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MEMBRANES AND RECEPTORS MODULE

SESSION 6 - RECEPTORS AND MEMBRANE TURNOVER

AIMS

The aims of this session are:

 to consider the structure and function of receptor proteins

 to introduce the principles of receptor-mediated endocytosis

 to deliver presentations for Assignment I. The aim of these presentation sessions

is to begin to introduce topics relating to the autonomic nervous system, that is
covered later in the module, and give you an opportunity to improve
presentation skills

LEARNING OUTCOMES

By this session you should be able to:

 describe and compare biological communication processes involving hormones,

local mediators and neurotransmitters

 give an overview of the role of membrane proteins as receptors for responding

to external stimuli, and for the uptake of external molecules via endocytosis

 contrast in general terms the mechanisms of polypeptide hormones and the

steroid and thyroid hormones

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PRIVATE STUDY

This time should be used to:

 research mechanisms employed by receptors to transduce chemical messages

across a membrane in preparation for Lectures 7.1 and 7.2 in Session 7, and

 complete preparation of presentation assignments II for Session 7.

MEMBRANES AND RECEPTORS - SESSION 6

RECEPTORS AND MEMBRANE TURNOVER

LECTURE 6.1 - RECEPTORS IN CELL SIGNALLING. RECEPTOR

STRUCTURE - COMMON STRUCTURAL MOTIFS.

AIMS

The principles of communication between cells via chemical messengers in the

endocrine and nervous systems will be considered. The role of receptors in transducing
the information carried by an extracellular hydrophilic signalling molecule across a
hydrophobic cellular membrane bilayer will be introduced. The concept of receptor
super-families, based on common structural motifs, and the structure of the four major
classes of receptors involved in cellular signalling via hormones, local mediators and
neurotransmitters will be introduced.

CHEMICAL SIGNALLING

Chemical signals may be classified according to their functions into hormones

(signalling between cells in different tissues via the circulation), neurotransmitters
(signalling at specialised cell junctions in the nervous system, synapses) and local
chemical mediators (signalling between adjacent cells in the same tissue). A single

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molecule may fall into more than one of these categories depending on where it is
synthesized and released and its site of action.

LIGAND

A ligand is any small molecule that binds specifically to a receptor site. Ligand
binding may produce an activation of a receptor. In this case the ligand is termed an
agonist. Alternatively, a ligand may combine with a receptor site without causing
activation. This type of ligand is termed an antagonist because it would oppose the
action of an agonist. Agonists which stimulate a receptor but are unable to elicit the
maximum cell response possible are termed partial agonists.

RECEPTOR

A receptor is a molecule that recognises specifically a ligand or family of ligands, and

which in response to ligand binding brings about regulation of a cellular process. In the
unbound state a receptor is functionally silent. Thus, catecholamine (e.g. adrenaline)
binding to a -adrenergic receptor (-adrenoceptor) brings about the activation of the
enzyme, adenylyl cyclase, and a cascade of signalling events in the cell. Equally,
binding to an LDL-receptor sets in train the internalization of cholesterol into the cell.

ACCEPTORS
N.B. Many molecules whose activities are modified by the binding of small chemicals,
including drugs, are not strictly receptors under this definition. If their basic function
can be carried out without the interaction of a ligand then they are not, by definition, a
receptor. For example, the enzyme dihydrofolate reductase is inhibited by the binding
of the drug, methotrexate, and is sometimes referred to as the methotrexate receptor.
This enzyme operates normally in the absence of methotrexate. Equally, the voltage-
gated Na+ channel opens in response to an electrical event, but can be modulated by
the binding of local anaesthetic agents and a variety of neurotoxic molecules, is often
referred to as the receptor for these agents. Dihydrofolate reductase and sodium
channels both operate in the absence of any signalling molecule. More accurately,

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these molecules should be referred to as "acceptor" molecules because their basic
function can occur without the interaction of a ligand.

SPECIFICITY OF RESPONSE

For a cell to respond to any chemical messenger it must produce specific receptor
proteins which recognise and produce a response to the signalling molecule. If the
signalling molecule is hydrophilic the signal recognition site of the receptor must be
present on the extracellular face of the cell surface. Interaction of the signalling
molecule with its specific receptor must then result in the activation of a cellular
process. If the signalling molecule is hydrophobic it will be able to gain access to the
cell through the lipid bilayer by diffusion but an intracellular receptor is still required
to transduce the signal into a cellular response. No specific receptor, no response in the
tissue! The presence or absence of a specific receptor in a cell governs the
responsiveness of a cell to any signalling molecule.

CLASSIFICATION OF RECEPTORS
Receptors are classified according to the specific physiological signalling molecule
(agonist) that they recognise. Sub-classification is often made on the basis of their
ability to be selectively activated by agonist molecules. Sub-classification is also often
made on the basis of the affinity (a measure of tightness of binding) of a series of
antagonists.

PROPERTIES OF RECEPTOR BINDING SITES

Analogies can be drawn between receptor binding sites and the active sites and
regulatory sites of enzymes.

Similarities

Binding at both receptor sites and enzyme sites is specific.

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The specificity of binding is governed by the shape of the binding cleft in the receptor
or enzyme site. It is the specificity of binding which confers specificity to the
regulation of processes in which receptors are involved or the specificity for substrate
of an enzyme. Binding to both receptors and enzymes is most often reversible.
Ligand binding to receptor and regulator binding to enzyme allosteric sites both induce
a conformational change and a change in the activity of the molecule (ligand and
substrate molecules may also 'induce a fit').
There is no chemical modification of ligand in receptor binding sites or enzyme
regulatory sites.

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Differences

The affinity of ligand binding at receptor sites is generally higher than the binding of
substrates and regulators to enzyme sites. The concentration of ligand that half fills all
available receptor sites (the dissociation constant, KD) is generally in the nanomolar
(10-9M) to micromolar (10-6M) concentration range. Often the concentration of
substrate that half fills available enzyme active sites (Michaelis constant, KM) is in the
micromolar (10-6M) to millimolar (10-3M) concentration range.

The ligand bound to a receptor site is not modified chemically whereas substrate
bound in an enzyme active site is modified in a chemical reaction catalysed by the
active site.

ROLE OF RECEPTORS IN CELLULAR PHYSIOLOGY

Examples include such processes as:

Signalling by hormones and local chemical mediators

Neurotransmission

Cellular delivery (low density lipoprotein, transferrin)

Control of gene expression (steroids, thyroid hormones),

Release of intracellular calcium stores (Inositol 1,4,5-trisphosphate receptors)

Immune responses

SIGNAL TRANSDUCTION

Some hydrophobic signalling molecules are able to cross the plasma membrane freely
and interact with intracellular receptors to bring about changes in cellular activity (e.g.
steroid and thyroid hormones). Most signalling molecules are hydrophilic and are

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unable to cross the plasma membrane. To exert their effect, hydrophilic signalling
molecules must interact with specific receptor proteins at the cell surface.

Common mechanisms to transduce an extracellular hydrophilic signal into an

intracellular event include:

1. Membrane-bound receptors with integral ion channels

2. Membrane-bound receptors with integral enzyme activity

3. Membrane-bound receptors which couple to effectors through transducing proteins

1. MEMBRANE-BOUND RECEPTORS WITH INTEGRAL ION CHANNELS -

LIGAND-GATED ION CHANNELS

Agonist binding to ligand-gated ion channels results in a change in conformation and

opening of a gated channel which permits the flow of ions down an electrochemical
gradient. This transduces the signal into an electrical event at the plasma membrane.

Subunit structure of the „classical‟ ligand gated ion channel family

Several receptors belong to the „classical‟ ligand-gated ion channel family that have
similar pentameric subunit structures, e.g. nicotinic acetylcholine receptors (nAChR),
gamma aminobutyric acid receptors (GABAAR), glycine receptors (GlyR). Subunits
have four transmembrane domains, one of which (M2) forms the lining to the channel
pore.

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2. MEMBRANE-BOUND RECEPTORS WITH INTEGRAL ENZYME
ACTIVITY.

Agonist binding to the extracellular domain of these receptors causes a conformational

change which activates an intrinsic enzyme activity contained within the protein
structure of the receptor, e.g. tyrosine kinase-linked receptors, guanylyl cyclase-linked
receptors. Examples include the atrial natriuretic peptide (ANP) receptor coupled to
guanylyl cyclase and growth factor receptors such as receptors for insulin, epidermal

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growth factor (EGF) and platelet derived growth factor (PDGF) linked to directly to
tyrosine kinase.

Tyrosine kinase-linked receptors

Binding of hormone to extracellular binding sites activates a protein kinase activity in

the cytoplasmic domain of the receptor protein, which autophosphorylates (catalyses
the transfer of a phosphate group from ATP onto its own structure) tyrosine residues
on the cytoplasmic domain of the receptor. Phosphorylated receptor tyrosine residues
are recognised either by transducing proteins, e.g. insulin receptor substrate-1 (IRS-1),
or directly by enzymes containing phosphotyrosine recognition sites, Src-homology-2
(SH2) domains. On association with receptor or transducing protein, effector enzymes
become activated allosterically, or possibly by tyrosine phosphorylation by the
receptor kinase, thus transducing the message into an intracellular chemical event.

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3. MEMBRANE-BOUND RECEPTORS WITH NO INTEGRAL ENZYME OR
CHANNEL ACTIVITY - SEVEN TRANSMEMBRANE DOMAIN
RECEPTORS (7TMDR)

Seven transmembrane domain receptors (7TMDR) couple to effector molecules via a

transducing molecule, a GTP-binding regulatory protein (G-protein). This family of
receptors is also known as the G-protein-coupled receptor (GPCR) family. Effectors
may be enzymes, e.g. adenylyl cyclase (ATP cyclic AMP), phosphatidylinositol 4,5-
bisphosphatase (phosphatidylinositol 4,5-bisphosphate inositol 1,4,5-trisphosphate and
diacylglycerol) or ion channels e.g. Ca2+ channels and K+ channels (see Lectures 2.1.
and 3.2)

A wide variety of extracellular signalling molecules utilize specific seven

transmembrane domain receptors and, thus, there is an extensive superfamily of
proteins with this common structure. Examples include: muscarinic acetylcholine
receptor (mAChR, stimulated by muscarine), adrenoceptors, dopamine receptors, 5-

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hydroxytryptamine (5-HT) receptors, opioid receptors, peptide receptors (e.g.
substance P, angiotensin), purine receptors (e.g. ATP), light receptors (rhodopsin),
smell and taste receptors and many others. Often a number of different types of G-
protein-coupled receptor exist for a particular agonist, each with its own pharmacology
e.g. M1-5 mAChRs.

Receptor binding results in a conformational change which activates GDP/GTP

exchange in GTP-binding regulatory proteins (G-proteins, see lecture 7.1) which
transduce the message on to an enzyme or channel in the membrane.

4. INTRACELLULAR RECEPTORS

Hydrophobic ligands, such as the steroid hormones, cortisol, oestrogen and

testosterone, and the thyroid hormones T3 and T4, penetrate the plasma membrane and
bind to monomeric receptors in the cytoplasm or nucleus. In the resting state these
receptors are stabilized by association with heat shock or chaperone proteins. The
activated receptor dissociates from the chaperone protein and translocates to the
nucleus where it binds to control regions in DNA defined by specific sequences,
thereby regulating gene expression. Compared to receptors that activate channels or
enzymes (reviewed above) the effects of intracellular receptor activation are relatively
slow in onset as transcription and translation are required.

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AMPLIFICATION

The concentration of many extracellular signalling molecules is very low (10-12-10-6

M). In each of the above mechanisms there is the possibility of molecular
amplification. For example, by stimulating the activity of an enzyme, the binding of a
chemical signal molecule to a single receptor can cause the modification of hundreds
or thousands of substrate molecules. A cascade of such catalytic events can produce
further amplification.

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CELLULAR ACTIVATION AND INHIBITION

Responses to receptor activation can lead to cellular activation or inhibition depending

on the receptor. For example, in cardiac pacemaker cells noradrenaline acting on
1-adrenoceptors produces an increased heart rate, while acetylcholine acting on M2
muscarinic receptors produces a slowing of heart rate.
Hepatocytes, insulin stimulates the synthesis of glycogen from glucose, while glucagon
stimulates glycogen breakdown.

MEMBRANES AND RECEPTORS - SESSION 6

RECEPTORS AND MEMBRANE TURNOVER

LECTURE 6.2 - PRINCIPLES OF RECEPTOR MEDIATED ENDOCYTOSIS

AIMS

The aim of this lecture to consider how relatively large, hydrophilic molecules can
enter cells by associating with a cell surface receptor. This process is called receptor-
mediated endocytosis. By the end of the lecture you should understand, in principle,
how this process can contribute to the uptake of metabolites, the passage of large
molecules across cells, the control of receptor number at the cell surface and the entry
of membrane-enveloped viruses.

PHAGOCYTOSIS

In mammals, phagocytosis is found only in specialized cells, i.e. macrophages and

neutrophils. In response to binding of a particle to receptors in the plasma membrane,
the cell extends pseudopods which permit further receptor interactions and membrane

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evagination and particle internalization via a „membrane-zippering‟ mechanism.
Internalized phagosomes fuse with lysosomes to form phagolysosomes in which the
particulate material is degraded.

This process permits the clearance of damaged cellular materials and invading
organisms for destruction.

PINOCYTOSIS - The invagination of the plasma membrane to form a lipid vesicle.

This permits the uptake of impermeable extracellular solutes and retrieval of plasma
membrane. Pinocytosis can be sub-divided into two forms, fluid-phase and receptor
mediated endocytosis.

RECEPTOR-MEDIATED ENDOCYTOSIS (RME) - Specific binding of

molecules to cell surface receptors permits the selective uptake of substances into the
cell.

UPTAKE OF CHOLESTEROL - AN EXAMPLE OF RME

Low density lipoproteins (LDL) originate in the liver and consist of a core of
cholesterol molecules esterified to fatty acid, surrounded by a lipid monolayer layer
containing phospholipids, cholesterol and a single protein species, apoprotein B.

Animal cells that require cholesterol synthesize cell surface receptors (LDL-receptor)
that recognise specifically apoprotein B. Within 10 minutes of binding, the LDL
particle is internalised and delivered to the lysosomes where the cholesterol is released
from the cholesterol esters.

LDL-receptors are localized in clusters over coated-pits (2% of cell surface). Coated
pits invaginate and pinch off from the plasma membrane to form coated vesicles.
Coated vesicles are quickly uncoated. The uncoated vesicles then fuse with larger
smooth vesicles called endosomes. The pH of the endosome is maintained between
approximately 5.5-6.0 by an

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ATP-dependent proton pump. At this pH the LDL-receptor has low affinity for the
LDL particle and the two dissociate. The endosome is also known as the Compartment
for the Uncoupling of Receptor and Ligand (CURL). The transmembranous receptors
are sequestered to a domain within the endosome membrane which buds off as a
vesicle and recycles the LDL-receptor to the plasma membrane. Theendosomes
containing the LDL fuse with lysosomes such that the cholesterol can be hydrolysed
from the esters and released into the cell. Thus, the LDLs and their receptors are sorted
from each other in the endosome.

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COAT STRUCTURE
The association of the coat proteins is energy-independent and, therefore, coated-pit
formation is spontaneous. The minimum structure that can be formed is a three legged
structure called the triskelion, containing clathrin (180 kDa) and two light chains (~
35 kDa) in the ratio 3:2:1. It is proposed that the triskelions associate to form a basket-
like structure consisting of hexagons and pentagons. The smallest enclosed structure
would consist 8 hexagons and 12 pentagons. Since assembly is spontaneous, uncoating
must be driven. This is carried out by an ATP-dependent uncoating protein which
binds and stabilizes the freed coat proteins. The clathrin coat is attached to the plasma
membrane by a number of integral membrane adapter proteins which form
associations both with the clathrin and receptors, locating the receptors over the coated
pit.

MUTATIONS AFFECTING THE LDL-RECEPTOR

Naturally occurring mutations of the LDL-receptor have been identified in some
patients with hypercholesterolaemia. Three types of mutations, when found in a
homozygous individual lead to three phenotypes:

1. Receptor deficiency. Mutations that prevent expression of LDL receptor.

2. Non-functional receptor. No binding of LDL. Normal coated pits and

internalization.

3. Receptor binding normal. No internalization due to a deletion in the C-terminal of

the receptor that makes the interaction with the coated pits. LDL-receptors are found
distributed over the whole cell surface in these patients.

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UPTAKE OF FERRIC (Fe3+) IONS BY TRANSFERRIN
Two Fe3+ ions bind to apotransferrin forming transferrin in the circulation.
Transferrin, but not apotransferrin, binds to the transferrin receptor at neutral pH and is
internalized as above. On reaching the acidic endosome, the Fe3+ ions are released
from the transferrin but at this pH the apotransferrin remains associated with the
transferrin receptor. The complex is sorted in the CURL for recycling back to the
plasma membrane, where at pH 7.4, the apotransferrin dissociates from the transferrin
receptor again.

UPTAKE OF OCCUPIED INSULIN RECEPTORS

Most receptors internalized by RME are located over the coated pits, but some, such as
the insulin receptor, only congregate over the coated pits when agonist is bound.
Insulin binding probably induces a conformational change in the insulin receptor that
allows it to be recognised by the coated pit. In the endosome insulin remains bound to
the receptor and the complex is targeted to the lysosomes for degradation. This
mechanism allows for the reduction in the number of insulin receptors on the
membrane surface (down-regulation) which desensitizes the cell to a continued
presence of high circulating insulin concentrations.

TRANSCYTOSIS
Some ligands that remain bound to their receptors may be transported across the cell,
e.g. maternal immunoglobulins to the foetus via the placenta, transfer of
immunoglobulin A (IgA) from the circulation to bile in the liver. During transport of
IgA the receptor is cleaved, resulting in the release of immunoglobulin with a bound
‟secretory component‟ derived from the receptor.

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FOUR MODES OF RECEPTOR-MEDIATED ENDOCYTOSIS

Receptors for different ligands enter the cell via the same coated pits and the pathway
from coated pits to the endosome is common for all proteins that undergo endocytosis.
Different modes of this process can be defined on the basis of the destination of
internalized receptor and ligand (see table below). Receptors targeted to different
cellular destinations by short amino acid motifs, are sorted within the CRL to discrete
regions of membrane, which buds off which bud off into transport vesicles.

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VIRUSES AND TOXINS

Membrane-enveloped viruses and some toxins exploit endocytic pathways to enter

cells after adventitious binding to receptors in the plasma membrane. Once in the
endosome, where the acid pH is favourable, the viral membrane is able to fuse with the
endosomal membrane, thereby, releasing the viral RNA into the cell where it can be
translated and replicated to form new viral particles.

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MEMBRANES AND RECEPTORS MODULE

SESSION 7 - SIGNAL TRANSDUCTION IN BIOLOGICAL MEMBRANES

AIMS

The aims of this session are:

 to develop an understanding of how signalling molecules are recognised by cells

and how an appropriate response is co-ordinated in specific cells

 to consider, in particular, signal transduction pathways involving G-protein

coupled receptors

 to deliver presentations for Assignment II

LEARNING OUTCOMES

By this session you should be able to:

 describe the various receptor-effector mechanisms; receptors linked to ion

channels, to G-proteins or to protein kinases. Understand the concepts of second
messengers and cascades. Discuss the importance of cyclic nucleotides and the
control of intracellular Ca2+ concentration

PRIVATE STUDY

Private study time should be used to:

 prepare the plots and consider answers for the data handling exercise for the
study session in Session 8.

146
 revise the module content so far. Why not try the formative assessments
available on the module web pages?

147
M.B., Ch.B. - MEMBRANES AND RECEPTORS - SESSION 7

SIGNAL TRANSDUCTION IN BIOLOGICAL MEMBRANES

LECTURE 7.1 – RECEPTOR-EFFECTOR SIGNALLING VIA G PROTEINS

AIMS

To understand how signalling molecules (e.g. hormones, neurotransmitters, growth

factors and environmental stimuli such as light and odours) are recognized by cells and
how an appropriate response is coordinated. The lecture will concentrate on an
important general type of a signal transduction pathway which consists of 3
fundamental components: a receptor, a guanine nucleotide binding protein (or G
protein) and an effector molecule, which may be an enzyme (e.g. adenylyl cyclase) or
an ion channel.

What is Signal Transduction?

Almost every cell in the body is capable of responding to external cues which instruct
the cell to alter its activity in some way (e.g. to contract/relax, increase/decrease
secretion, differentiate/divide, etc.). Although some signalling molecules can enter the
cell to cause such changes (e.g. glucocorticoids, thyroid hormone), the vast majority
cannot freely cross membrane barriers and therefore must exert their actions at the
external surface of the cell by binding to receptors located within the plasma
membrane.

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A number of general mechanisms are now recognized whereby the binding of a
signalling molecule („ligand‟) to its specific cell-surface receptor can bring about an
intracellular response. One family of receptors (G protein-coupled receptors) alter
the activities of effectors, which may be second messenger-generating enzymes (e.g.
adenylyl cyclase) or ion channels, via activation of one or more types of guanine
nucleotide binding proteins (G proteins). G protein-coupled receptors constitute an
important receptor superfamily responsible for an enormous diversity of cellular
functions, including muscle contraction, stimulus-secretion coupling, catabolic and
anabolic metabolic processes and light, smell and taste perception.

How do G proteins work?

The family of G proteins which transduce signals generated by agonists binding to and
activating receptors all have a common general structure. G proteins are
„heterotrimeric‟, that is they are made up of three distinct subunits termed (alpha),

149
(beta) and (gamma). The - and -subunits bind tightly to each other and function as a
single unit. The mechanism by which G proteins are regulated by receptor activation,
and how the activated G protein can alter effector activity is illustrated below:

The G protein -subunit has a guanine nucleotide binding site which binds GTP and
slowly hydrolyses it to GDP (i.e. the -subunit possesses GTPase activity). Under basal
conditions the G protein is present at the inner face of the plasma membrane
predominantly in its heterotrimeric form with GDP bound to the -subunit. Activated
receptor (i.e. the agonist-receptor complex) has a high affinity for this form of the G
protein and a protein-protein interaction occurs which leads to GDP being released by
the Gα-subunit and GTP binding in its place (i.e. the receptor acts as a guanine
nucleotide exchange factor (GEF)). The binding of GTP to the G-subunit decreases the
affinity of α-GTP for the receptor and for the G-subunit. Thus, both -GTP and subunits
are released and each can interact with effectors. The effector interaction is terminated
by the intrinsic GTPase activity of the -subunit which hydrolyses GTP to GDP. When

150
this occurs the affinity of the G-subunit for a G-subunit increases and the G
heterotrimer is reformed and awaits reactivation by an agonist-activated receptor to re-
initiate the cycle.

The G protein can be thought of as an on/off switch and a timer - the on switch is
receptor-facilitated GDP/GTP exchange and the timer/off switch is governed by the
length of time taken for GTP hydrolysis on the G-subunit. There is increasing evidence
that the timer function may not be a fixed property of the G, but may also be regulated
by other cellular proteins (e.g. RGS proteins – see diagram above).

What are the Cellular Targets for Activated G proteins?

Much of what we now understand about G protein-mediated signalling systems was

first elucidated through attempts to understand how hormones such as adrenaline bring
about the formation of the second messenger adenosine cyclic 3‟,5‟-monophosphate
(cyclic AMP). The intermediary role of the G protein (in this case Gs, where „s‟
indicated that one effect of the s-GTP is to stimulate adenylyl cyclase) between the
receptor (e.g. a -adrenoceptor) and effector (e.g. adenylyl cyclase) is illustrated below:

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Thus, in addition to the originally elucidated activation of adenylyl cyclase by Gs to
generate cyclic AMP, an adenylyl cyclase inhibitory G protein family (Gi) has also
been established. Like Gs, Gi proteins have additional effects independent of adenylyl

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cyclase inhibition, including effects on ion channels and signalling pathways involved
in growth and differentiation.

G protein families which exert their actions on effectors other than adenylyl cyclase
have also been discovered. Thus, a G protein family, called Gq/11, preferentially
interact with the membrane bound enzyme phospholipase C causing hydrolysis of a
minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2),
to generate 2 second messengers - inositol 1,4,5-trisphosphate (InsP3) and
diacylglycerol (DAG). In addition, the light-sensing protein rhodopsin, present in
mammalian retinal rod cells, activates a G protein (called transducin or Gt) which in
turn activates a phosphodiesterase enzyme which hydrolyses the second messenger
cyclic GMP to 5'-GMP. A similar signal transduction cascade is thought to be involved
in the colour-sensing retinal cone cells (involving cone pigment rhodopsins and Gt2).

Experimental Manipulation of the G protein Cycle

One important experimental means of investigating the type of G protein a receptor

may interact with is to establish what effect two bacterial toxins, pertussis toxin and
cholera toxin, have on receptor-effector coupling. Pertussis toxin contains an enzyme
(ADP-ribosyl transferase) activity which specifically modifies (and inactivates) Gi-
type proteins, “uncoupling” receptor-effector linkage, while cholera toxin contains a
similar ADP-ribosyl transferase enzyme activity which specifically modifies Gs-type
proteins, in this case leading to irreversible activation.

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M.B., Ch.B. - MEMBRANES AND RECEPTORS MODULE - SESSION 7

SIGNAL TRANSDUCTION IN BIOLOGICAL MEMBRANES

LECTURE 7.2 - EFFECTOR MECHANISMS IN INTRACELLULAR

SIGNALLING

AIMS

To understand how the activation or inhibition of effector molecules (e.g. an enzyme

or an ion channel), following activation by signalling molecules, leads to specific
cellular responses. Particular attention will be paid to effectors which are enzymes
which generate second messenger molecules (e.g. cyclic AMP, inositol 1,4,5-
trisphosphate (IP3)). Important and clinically relevant examples will be used to
illustrate how such signal transduction pathways bring about physiological changes in

154
key cell function and how such pathways can be pharmacologically manipulated to
therapeutic advantage.

Cellular Targets for Activated Receptors

Receptor activation can lead to the generation of a signal directly (e.g. in ligand-gated
ion channels where the receptor/effector functions reside within a single oligomeric
protein assembly; in steroid/thyroid hormone receptors where the activated receptor
migrates to directly interact with DNA to promote/inhibit transcription in the nucleus).
However, receptor and effector functions are often fulfilled by distinct proteins, with
the activated receptor directly or indirectly interacting with a separate effector
molecule. Thus, tyrosine kinase-linked receptors upon autophosphorylation can
interact with enzymes (e.g. phospholipase C) at the inner face of the plasma
membrane. Similarly, G protein-coupled receptors link to effectors via a G protein
which defines the effector target.

Second messenger-generating Effectors for G protein-coupled Receptors

Adenylyl Cyclase is an integral plasma membrane protein which can be either

activated (via Gs) or inhibited (via Gi) by activation of different receptors (e.g.
(nor)adrenaline acts at -adrenoceptors to activate, or at 2-adrenoceptors to inhibit
adenylyl cyclase; dopamine can act via D1- (stimulatory) or D2- (inhibitory)
receptors). The enzyme hydrolyses cellular ATP to generate cyclic AMP. Cyclic AMP
interacts with a specific protein kinase (cyclic AMP-dependent protein kinase, or
PKA) which can in turn phosphorylate a variety of other proteins within the cell to
affect (either positively or negatively) their activities.

155
In this way receptors which activate adenylyl cyclase and increase cellular cyclic AMP
levels can cause increased glycogenolysis and gluconeogenesis in the liver, increased
lipolysis in adipose tissue, relaxation of a variety of types of smooth muscle and
positive inotropic and chronotropic effects in the heart.

Phospholipase C is the effector enzyme in another near-ubiquitous cell signalling

pathway involving hydrolysis of a minor plasma membrane phospholipid,
phosphatidylinositol 4,5-bisphosphate (PIP2), to generate 2 second messengers -
inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 exerts its effects
by interacting with specific intracellular receptors on the endoplasmic reticulum (ER)
to allow Ca2+ to leave the lumen of the ER and enter the cytoplasm. One consequence
of this is the activation of Ca2+-sensitive protein kinases. DAG also interacts with a
family of protein kinases (protein kinase Cs), which are activated by this second
messenger.

A large number of G protein-coupled receptors can activate this pathway (e.g.

acetylcholine via M1 and M3 muscarinic cholinoceptors; histamine via H1-receptors;

156
noradrenaline via 1-adrenoceptors; serotonin via 5-HT2-receptors) and phospholipase
C activation is mediated by a distinct family of G protein collectively termed Gq. This
signalling pathway is responsible for an array of important responses including
vascular, GI tract and airways smooth muscle contraction, mast cell degranulation and
platelet aggregation.

Cyclic GMP phosphodiesterase activity: Unlike the above this is a specialized

mechanism found in the photoreceptive cells of the retina (rods and cones). In this case
the breakdown (rather than the synthesis) of a second messenger, cyclic GMP, is
regulated by activation of cyclic GMP phosphodiesterase by Gt (transducin) following
excitation of rhodopsin by a photon of light. In the dark, levels of cyclic GMP are
sufficient to open a second messenger-operated ion channel which allows Ca2+ and
Na+ to enter the cytoplasm. On exposure to light, activation of cyclic GMP
phosphodiesterase causes a decrease in cyclic GMP leading to channel closure and
membrane hyperpolarization, thus altering the signal output to the CNS.

Protein Phosphorylation as a Common Mechanism in Signal Transduction

Increases in cyclic AMP, cyclic GMP, diacylglycerol and Ca2+ exert all, or at least a
part, of their actions via interactions with specific serine/threonine protein kinases (so
called, because these kinases phosphorylate specific serine and/or threonine amino
acid residues within the target proteins):

Protein kinase Second messenger

cAMP-dependent protein kinase cyclic AMP

(PKA)

cGMP-dependent protein kinase cyclic GMP

(PKG)

157
 protein kinase C (PKC) diacylglycerol

Ca2+/calmodulin-dependent Ca2+
protein kinase (CaM-Kinase)

Each protein kinase causes phosphorylation of a distinct family of target proteins (e.g.
enzymes, ion channels, transporters, structural proteins, etc.), whose activities are
increased, decreased or unaltered by this covalent modification. Some examples of
how different protein kinases are involved in mediating second messenger effects are
given below.

Signal Amplification is a Critical Requirement of a Signal Transduction Pathway

For an extracellular stimulus (which may amount to only a few molecules of a

hormone interacting with appropriate cell surface receptors) to generate an intracellular
response, amplification of the signal is essential. Therefore an important role of the
receptor-G protein-effector signalling system is to allow such amplification to occur.
Amplification of the initial signal can be achieved at a number of levels:

1. Activated receptor can cause (sequential) GTP/GDP exchange on more than one G
protein

2. An activated G-GTP/free Gβγ can activate multiple effector molecules

3. Effector molecules act catalytically. Thus, activation of adenylyl cyclase by s-GTP

results in conversion of 100-1000s of molecules of ATP to cyclic AMP. Similarly, the
opening of an ion channel by -GTP allows 100-1000s of ions to move across the
plasma membrane.

158
Further amplification is often achieved through the mechanisms by which the second
messengers activate their cellular targets as these often involve an enzyme (e.g. cyclic
AMP-dependent protein kinase, protein kinase C) or a sequence of enzymes (i.e. an
enzyme cascade). It should be noted that both activation and de-activation of signalling
pathways is rapid, general occurring over a time-scale of a few seconds. Deactivation
is facilitated by a number of aspects of signalling pathways:

1. Once a receptor has productively interacted with a G protein the binding of the
agonist molecule is weakened and agonist-receptor dissociation is more likely to occur

2. Whilst activated, the receptor is susceptible to a variety of protein kinases which

phosphorylate the receptor and prevent it activating further G proteins (this is an
important part of the receptor desensitization phenomenon observed for most, but not
all, G protein-coupled receptors)

159
3. The active lifetime of a-GTP may be limited by cellular factors which stimulate the
intrinsic GTPase activity of the G subunit

4. Enzymic activities in the cell are such that the basal state is favoured. Thus, cells
contain high activities of enzymes which metabolize second messengers (e.g. cyclic
AMP is metabolized to the non-biologically active 5'-AMP by phosphodiesterases;
inositol 1,4,5-trisphosphate (InsP3) is metabolized to an inactive inositol bisphosphate
by a 5-phosphatase activity) rapidly returning their levels towards basal

5. Similarly enzymic cascades activated downstream of second messenger/protein

kinase activation are opposed by activities which act to reverse the second
messenger/protein kinase effect (i.e. target protein phosphorylation is reversed by
active cellular protein phosphatase activities).

Signalling Pathways in Action

1. Regulation of Chronotropy in the heart: The intrinsic rate at which the sinoatrial
(SA) node fires an action potential can be affected by acetylcholine released by the
parasympathetic nerves. The predominant acetylcholine receptor population in the SA
node is M2-muscarinic cholinoceptors; activation of these receptors increases the open
probability of K+-channels which have been shown to be directly regulated by both i-
GTP (and perhaps the βγ-subunits simultaneously released). Although M2-muscarinic
cholinoceptor activation will also inhibit adenylyl cyclase activity it is not known
whether this has any functional consequences. The increased plasma membrane K+-
permeability causes a hyperpolarization which slows (and if strong enough prevents)
the intrinsic firing rate resulting in a negative chronotropic effect.

2. Regulation of Inotropy in the heart: Sympathetic innervation of the cardiac

ventricles (and/or circulating adrenaline) can influence the force of contraction
(inotropy). Activation of -adrenoceptors (predominantly 1-adrenoceptors) increases

160
both cyclic AMP formation and the open probability of voltage-operated Ca2+-
channels (VOCCs). The increase in Ca2+ influx is brought about by two
complementary mechanisms. 1-Adrenoceptors activate adenylyl cyclase via s-GTP and
the increase in cyclic AMP activates cyclic AMP-dependent protein kinase which can
phosphorylate and activate the VOCC. In addition s-GTP can interact directly with
VOCCs - thus the direct and indirect actions at the level of VOCCs reinforce each
other and cause an increase in the magnitude of Ca2+-entry resulting in a positive
inotropic effect.

3. Arteriolar vasoconstriction: Sympathetic release of noradrenaline acts on 1-

adrenoceptors to stimulate phospholipase C and phosphoinositide turnover via a Gq
protein. The immediate effect is the generation of InsP3 which releases ER Ca2+ and
initiates a contractile response. The role of DAG is less clear, but it is thought that
activation of protein kinase C by DAG and the phosphorylation of key target proteins
is important for sustaining the vasoconstrictor response to noradrenaline.

4. Modulation of neurotransmitter release: G protein-coupled receptors located pre-

synaptically can influence the release of neurotransmitters at the synapse. For example,
pre-synaptic μ-opioid receptors can be stimulated, either by endogenous opioids or by
analgesics such as morphine, to couple to Gαi proteins. The Gβγ subunits liberated
from the Gαi-βγ heterotrimer interact with voltage-operated Ca2+ channels (VOCCs)
to reduce the entry of Ca2+ through these channels. This decrease in Ca2+ influx
inhibits the release of neurotransmitter from the pre-synaptic terminal, since
neurotransmitter release is a Ca2+-dependent process.

Further Reading

Pharmacology (6th Edition), Chapters 2-3. Rang HP, Dale MM, Ritter JM and
Flower R, Churchill Livingstone.

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MEMBRANES AND RECEPTORS MODULE

SESSION 8 - DRUGS AND RECEPTORS/PHARMACOKINETICS

AIMS

The aims of this session are:

 to develop an understanding of drug-receptor interactions

 to develop an understanding of how regulation of receptors impact on drug

action

 to consider the pharmacokinetics of drug administration

LEARNING OUTCOMES

By the end of this session you should be able to:

 Describe the difference between drug affinity, efficacy and potency

 Describe what is meant by the terms agonist, partial agonist and antagonist

 Distinguish competitive and non-competitive antagonism

 Understand the adaptive changes which can occur in receptor populations when
exposed to agonists and antagonists

 Appreciate whole body considerations of drugs reaching their sites of

therapeutic action, including principles of drug bioavailability and inactivation

162
PRIVATE STUDY
Private study time should be used to:

 complete the data handling exercise

 research the cellular mechanisms underlying receptor regulation and

desensitisation and their consequences on signal transduction and drug action. A
work sheet with questions together with some additional reading suggestions are
provided to guide you. Note, some of the key concepts in signal transduction
were covered in sessions 6 and 7, and the clinical implications of receptor
regulation will be described in Session 11, Lecture 11.1.
 revise the module content so far. Why not try the formative assessments
available on the module web pages?

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MEMBRANES AND RECEPTORS - SESSION 8

PHARMACOKINETICS

LECTURE 8.1 - PHARMACOKINETICS

AIMS:
Understand principles of drug formulation and administration, including use of
different sites of administration.
Be able to discuss oral bio-availability and factors affecting this.
Be able to describe mechanisms of drug elimination.
Define different ways in which drugs may interact.
Understand the differences between zero and first order kinetics (from Work Session).

Pharmacokinetics

Pharmacokinetics - "what the body does to a drug" as compared to

Pharmacodynamics - "what the drug does to the body"

4 questions should be asked :-

1) Is the drug getting into the patient? - the Pharmaceutical process

2) Is the drug getting to the site of action? - the Pharmacokinetic process

3) Is the drug producing the desired pharmacological effect? - the Pharmacodynamic

process

4) Is the pharmacological effect translated into a therapeutic effect? - the Therapeutic

process

164
I. The Pharmaceutical process

Getting the drug into the patient :-

1) Formulation of the drug - solid (tablet) or liquid

if solid, solubility and acid stability in stomach must be considered

Patient compliance is important (once daily dosage easier to remember).

2) Site of administration - e.g. local (eye, skin, inhalation, etc)

Systemic - (i) Enteral Sublingual, oral , rectal

(ii) Parenteral subcutaneous

165
  intramuscular

 intravenous

 inhalation

 transdermal

The term oral bioavailability is defined as the proportion of a dose given orally (or by
any other route other than IV) that reaches the systemic circulation in an unchanged
form.

Bioavailability can be expressed as (i) amount (depends on G.I. absorption and first
pass metabolism)

Gut absorption altered by food, disease.

(ii) rate of availability (depends on pharmaceutical factors and rate of gut absorption)

Amount - measured by area under curve of blood drug level vs. time plot

Rate - measured by peak height and rate of rise of drug level in blood

Therapeutic ratio - defined as maximum tolerated dose/ minimum effective dose

or LD50 / ED50

First pass metabolism - Blood from the gut reaches the liver by the portal system,
where the liver could metabolise the drug before it gets to the systemic circulation (e.g.
lignocaine,

opiates, propranolol, glyceryl trinitrate)

First pass metabolism is avoided by parenteral route

sublingual or rectal route

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II. Drug distribution

Volume of distribution the theoretical volume into which a drug has distributed
assuming that this occurred instantaneously (calculated as amount given / plasma
concentration at time '0' ).

Many drugs also bind to plasma proteins, and protein binding interactions could
occur.

It is the free level of drug that exerts an effect, not the total level.

These are important if (i) drug is highly bound to albumin (>90%)

(ii) drug has a small volume of distribution

(iii) drug has a low therapeutic index

Examples are warfarin and tolbutamide.

Object Drug (Class I drug) is used at a dose which is much lower than number of
albumin binding sites. Precipitant Drug (Class II drug) is used as a dose which is
greater than number of available

When a patient is taking one of the object drugs, adding on the precipitant drug will
temporarily lead to higher free levels of the object drug, and therefore, higher risk of
toxicity.

167
168
169
IV. Drug Elimination

1) Metabolism (e.g. by liver)

2) Excretion (e.g. by kidney)

A) Liver Metabolism -2 phases - (i) Phase I Oxidation, reduction, hydrolysis

(ii) Phase II Conjugation (glucuronide, acetyl, methyl, sulphate)

Phase I - carried out by mixed function oxidases in liver

Consists of NADPH cytochrome P450 reductase, cytochrome P450

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in liver microsomes, low substrate specificity, affinity for lipid soluble drugs,

Enzymes are inducible, and inhibitable (competitive, non-competitive)

Drug interactions with Warfarin are especially important.

Kidney Excretion of Drugs

Only the free unbound drug is filtered through glomerular tuft.
Drugs can be actively secreted by the tubule (e.g. penicillin)
Urine pH can determine how much of the drug is excreted. For weak acids (e.g.
aspirin), making the urine alkaline will make the drug ionised, so there will be less
tubular absorption because the charged drug stays in the tubule lumen.

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The converse is true for weak bases (e.g. amphetamine), where acid urine increases
excretion.

Acid urine will ionise a weak base, making the charged drug stay in the tubule lumen.

In renal disease,

1) If the drug or active metabolite is excreted as its main route of elimination, T½ is

prolonged. Therefore, lower the maintenance dose.

2) It takes 5 T½s to reach a new equilibrium every time you change the dose.

3) The loading dose is unchanged, unless volume of distribution changes (e.g. digoxin)

4) Protein binding of drugs is altered.

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MEMBRANE AND RECEPTORS MODULE – SESSION 8

DRUGS AND RECEPTORS

LECTURE 8.2 - DRUGS AND RECEPTORS

AIMS AND OBJECTIVES

The aim of this lecture is to provide you with an understanding of drug-receptor

interactions to eventually help you consider therapeutic decisions in clinical practice.
To this end the quantitative relationship between drug concentration and response are
presented and the concepts underlying agonist and antagonist drug action described.

At the end of this lecture you should know what is meant by the terms: affinity,
efficacy, potency, agonist, antagonist and partial agonist.

Medical pharmacology is the science of the interactions of chemicals (drugs) with

the human body. In many cases, drugs produce their effects by interacting with targets,
which are mostly (but not exclusively) proteins. Many of these protein targets are
receptors. These are specific proteins that normally respond to endogenous signalling
ligands such as neurotransmitters, hormones and local mediators. Many clinically
effective drugs interact with receptors, either mimicking (agonists) or blocking
(antagonists) endogenous ligands. We will consider drug-receptor interactions,
although the principles may be applicable to other targets.

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Affinity and efficacy

Some drugs bind to receptors AND cause a response – AGONISTS – these drugs have
both affinity and efficacy.

Some drugs bind to receptors but do not cause a response – ANTAGONISTS – these
have affinity ONLY. They block the effects of agonists ie. prevent receptor activation
by agonists.

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Note that efficacy is measured in relative terms (it has no absolute scale). Agonists
with different Emax values have different efficacy. However, agonists with the same
Emax values may not have identical efficacy – they could differ in affinity which
means that the relationship between receptor occupancy and functional response will

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be different for the two agonists – one may be more able to convert binding into
function.

Relationship between drug binding and functional response – spare receptors

Although this relationship resembles that predicted by the Law of Mass Action, the
relationship between binding (ie. Kd) and effect (ie. EC50) is complex.

Full and partial agonists

The efficacy of a drug describes its ability to activate a receptor and produce a
response; efficacy influences the relationship between the Kd and the EC50.

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Antagonists have no efficacy (ie. =0). Remember, antagonists do not produce a
response per se, they block the effect of an agonist. When comparing the ability of
different agonists to evoke responses in a tissue, it is sometimes observed that some
drugs cannot produce a maximal effect, even with full receptor occupancy. These types
of agonists are referred to as partial agonists. Note that the EC50 of a partial agonist
is equal to its Kd.

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Antagonists
Antagonists – block effects of agonists
1. Reversible competitive antagonism (commonest and most important in
therapeutics)
- depends on dynamic equilibrium between ligands and receptors. The inhibition is
surmountable (overcome) by addition of more agonist.

2. Irreversible competitive antagonism – occurs when the antagonist dissociates only

very slowly or not at all (eg. because of a covalent bond between ligand and receptor).
The inhibition is non-surmountable.

3. Non-competitive antagonism (generally post-receptor or allosteric) (not

considered here).

RECEPTOR CLASSES AND DRUG DEVELOPMENT

 Structure-activity relationships between drugs often identify different receptor

subclasses and allow specificity of therapeutic action. Studies of drug affinity
often provide an early clue to the existence of receptor subclasses.

 Multiple receptor subclasses have been identified for virtually every

endogenous ligand.

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  Different subclasses can often use different transduction mechanisms thus
allowing one endogenous ligand to initiate different functions.

 Subclasses are often distributed differently in tissues.

 Subclass-selective drug development is crucial to therapeutics.

SOME THINGS TO CONSIDER IN THE RELATIONSHIP BETWEEN DRUG

DOSE AND CLINICAL RESPONSE

Three factors to consider:

(1) Pharmacological potency and efficacy

(2) Variation in drug responsiveness due to:

Concentration and bioavailablility of drug at receptor (pharmacokinetics)

Concentration of endogenous receptor ligand

Alteration in receptor number or function

Changes in responses distant to receptor e.g. Transduction pathway

(3) Clinical selectivity, therapeutic versus toxic effects

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MEMBRANES AND RECEPTORS - WORK SESSION 8

QUANTITATION OF LIGAND-RECEPTOR INTERACTION

OBJECTIVE: To support material delivered in Lecture 8.2 (Receptors as

pharmacological and therapeutic targets). In particular to ensure understanding of the
following principles.

LEARNING OUTCOME: To understand the basic principles of ligand binding,

agonism and antagonism.

Exercise A – Basics – molarity and concentration.

a) What is the molarity of a solution of acetylcholine containing 0.073 mg/mL, where
the molecular weight of acetylcholine is 146.21?
b) How many mg/mL are required to prepare a 0.75mM solution of acetylcholine?

Exercise B - Comparison of ligand binding and receptor function – understanding

affinity, potency, the concept of „spare receptors‟ and the consequences of altered
receptor number on responses.
Information about the interaction of a ligand with a receptor can be obtained from
binding experiments in which increasing concentrations of a radioactively labelled
version of the ligand are incubated with a preparation containing the receptor (for
example, isolated membranes). When the binding reaction is at equilibrium the bound
and free radioligand can be separated and the amount of ligand that is bound to
receptors can be determined. As the concentration of radioligand is increased, more
receptors will be occupied until there are no „free‟ receptors. At any concentration of
radioligand, the occupancy of the receptor population can then be calculated with
reference to full occupancy. This can be represented graphically in the form of a
binding curve in which the occupancy is plotted on the y-axis (with full occupancy
set as either „1‟ or 100%) against the log10 of the radioligand concentration on
the x-axis (panel i).

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The ability of a ligand to cause a receptor-mediated functional response can also be
represented graphically in the form of a concentration-response curve (panel ii).
Usually the response is plotted on the y-axis and the log10 concentration (sometimes
the „dose‟, particularly for an in vivo experiment) is plotted on the x-axis.

a) Comment on the shapes of these curves.

b) What is the affinity (Kd) of drug X?

c) What is the potency (EC50) of drug X?
d) What level of receptor occupancy is necessary to cause 50% of the maximal
response?

e) Comment on the relationship between the Kd and EC50. How can you explain the
difference?

e) In chronic heart failure, the number of cardiac β1-adrenoceptors is reduced. Shown

below is a concentration-response curve showing the relationship between the
concentration of noradrenaline and the force of contraction (where contraction is
mediated through β1-adrenoceptors) in experiments that could have been conducted on
„normal‟ heart tissue. What are the possible consequences of reduced β1-adrenoceptor
number on this concentration-response relationship? Draw one or more curves to
illustrate your answer.

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Which of the drugs (A or B) has:

a) the highest affinity?

b) the greatest potency?

c) the highest intrinsic activity?

d) the greatest efficacy?

Effects of acetylcholine and three acetylcholine analogues on the contraction of

the isolated ileum

Experiment:
Guinea-pig ileum pieces were suspended in an organ bath in buffer at 37oC and
stimulated with either acetylcholine or an analogue of acetylcholine. The contractions
were measured and normalized to the maximum contraction produced by acetylcholine
(= 100%).

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 Contractile response Results: Concentration

(%, normalized to the maximum (M)

acetylcholine response)

Analogue 3 Analogue 2 Analogue 1 Acetylcholine

5 0 1 1 10-9

25 5 0 9 10-8

52 25 10 50 10-7

60 45 48 88 10-6

61 49 93 99 10-5

58 50 99 100 10-4

62 48 95 100 10-3

Calculations:

1. Construct concentration-response curves for acetylcholine and the three analogues

(on the same graph).

2. Determine the EC50 values for acetylcholine and each of the analogues.

Analogue 3 Analogue 2 Analogue 1 Acetylcholine

EC50 (Units?)

3. List acetylcholine and the analogues in order of potency, with the most potent first.

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4. List acetylcholine and the analogues in order of intrinsic activity, with the highest
activity first.

5. Given that acetylcholine is a full agonist, how would you describe each of the
analogues?

6. List acetylcholine and the analogues in order of efficacy, with the most efficacious
first.

7. What would be the intrinsic activity of an antagonist?

Exercise D – Understanding antagonism.

Effects of three experimental antagonists on acetylcholine-induced contraction of

the isolated ileum

The way in which an agonist concentration-response curve is modified by an

antagonist gives clues concerning the nature of the interaction. Some antagonists
produce a parallel shift in the agonist concentration-response curve, others may reduce
the maximum response.

This exercise is designed to illustrate the way in which the acetylcholine-stimulated

contraction of the ileum is inhibited by three antagonists.

Experiment:

Guinea-pig ileum pieces were suspended in an organ bath in buffer at 37oC and
stimulated with acetylcholine and the contractions measured. Concentration-response

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curves were constructed with all points normalized to the maximum response obtained
at 10-3 M acetylcholine (= 100%). Repeat curves were constructed in the presence of
three experimental antagonists, used at 10-8 M. The results are tabulated below.
Following extensive washing to remove antagonists, the agonist response curves were
repeated. Similar concentration response curves to the control were obtained for tissue
treated originally with antagonists 1 and 2. In contrast, the curve obtained for tissue
originally treated with antagonist 3 was unchanged.

3. Comment on the likely nature of the interaction of antagonists 1, 2 and 3 with the
receptor.

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4. Can you draw any conclusions about the relative affinities of antagonists 1 and 2?

Additional reading:

1. Human Pharmacology: Molecular-to-Clinical. (Part 1, Chapter 1). Ed: L.B. Wingard

& T.M. Brody, Mosby Year Book, 1998. ISBN 0815124562

2. Pharmacology 6th edition. (Chapter 1) Rang, H.P., Dale, M.M., Ritter, J.M. &
Flower. R. Churchill Livingstone, 2007. ISBN 0443069115.

MEMBRANE AND RECEPTORS MODULE – SESSION 8

DRUGS AND RECEPTORS

PRIVATE STUDY - REGULATION OF RECEPTORS

AIM

To recognise the importance of receptor regulation in signal transduction

To find out about the mechanisms and consequences of receptor desensitisation.

To integrate relevant information presented in earlier sessions.

OBJECTIVES

Describe what is meant by desensitisation, tolerance and supersensitivity.

Compare the mechanisms involved in homologous versus heterologous desensitisation.
Explain the basic processes involved in desensitisation of a G-protein coupled

receptors, using the Beta-adrenergic receptor as your example.

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REFERENCES

All standard pharmacology text books will contain most of the information you need
for this exercise. You may also find it useful to refer to the following:

Rang, Dale, Ritter & Flower, Pharmacology 6th Edition, Churchill Livingstone,
Chapter 1

Norman and Lodwick, Flesh and Bones of Medical Cell Biology, Chapter 27.

EXERCISE

QUESTION 1.

In graph (A) the response of a tissue over time to repeated applications of the same
concentration of a full agonist is shown. The tissue was allowed to recover for varying
amounts of time between each application, thus between the 2nd and 3rd application
the tissue was left for considerably longer than between the 1st and 2nd application.

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a. With reference to the first application of agonist in panel A, suggest reasons why the
peak response is not maintained but declines to a lower sustained plateau level during
application of the agonist.

b. What is the effect of frequent repeated drug application on the tissue response?

c. What is the effect of prolonged washing of the tissue following the second
application of drug?

d. What mechanisms may underlie receptor desensitisation?

c. The graphs in (B) and (C) illustrate what occurs to a response following
homologous desensitisation and heterologous receptor desensitisation, respectively.
What do these two terms mean? Explain how they relate to the size of the second
response in each graph.

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d. Give another mechanism by which a tissue may become desensitised in response to
drug application.

e. What is the effect of chronic drug application on the tissue response?

QUESTION 2.

Desensitisation of G-protein-coupled receptors (GPCRs) can result from

(i) modification of the receptor by phosphorylation

(ii) reversible receptor internalisation

(iii) down-regulation.

a. Using the beta-adrenergic receptor as your example, describe the signalling cascades
and proteins which underlie each of these mechanisms. Remember, a diagram can be
worth a 1000 words!

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b. Which of these mechanisms (i, ii or iii) is likely to have (1) the fastest onset, and (2)
slowest recovery?

c. Which mechanism (i, ii or iii) is most commonly associated with heterologous

desensitisation?

d. Which mechanism (i, ii or iii) is most likely to underlie drug tolerance?

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QUESTION 3.

What is the function of receptor recycling in receptor resensitisation?

QUESTION 4.

a. Prolonged treatment of hypertensive patients with -adrenoceptor antagonists can

lead to receptor supersensitivity. What does this mean?

b. Why can this cause a patient to suffer a heart attack should he (or she) suddenly stop
taking their medication?

QUESTION 5.

Case Report

A 55 year old male with a 40 year history of smoking 20+ cigarettes per day was given
a diagnosis of inoperable bronchogenic carcinoma with distant metastases. The patient
was referred to a pain management clinic by his oncologist as he was experiencing
pain that could not be controlled by simple paracetamol or non-steroidal anti-
inflammatory drugs. Morphine sulphate (MST) 60 mg daily in divided doses was
started in the pain clinic. From a pain management perspective (MST use and pain),
the course of his disease over the ensuing 4 months until his death is illustrated in the
Figure below. In the clinic, the effectiveness of his pain management was assessed
using a visual analogue scale (VAS). This is simply a 100 mm line with „no pain‟
labelled at 0 mm and „worst pain imaginable‟ labelled at 100 mm. Each day the patient
was asked to put a mark on this line to describe their pain at rest. On referral to the
pain management clinic VAS was 30 mm. The patient complained of nausea and
constipation during treatment that was most pronounced after about 10 weeks. Both
symptoms required treatment.

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a. How does MST use and pain score change during the progression of his disease?

b. Explain the change in MST use.

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c. Are there any consequences of the increased MST dose required to achieve adequate
pain relief?

d. Can you think of any ways that the dose of MST could be reduced to produce
acceptable pain relief?

e. What molecular mechanisms might underlie the changes seen in MST use by this
patient?

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MEMBRANES AND RECEPTORS MODULE

SESSION 9 – FORMATIVE ASSESSMENT

AIMS

The aim of this session is to give an opportunity for students to assess their progress in
the module so far.

PRIVATE STUDY
Private study time should be used to:

Complete the data handling exercise on pharmacokinetics in preparation for Session 10

Consolidate learning in any weak areas highlighted in the formative assessment. It is

better to do this now than leave it to just before the summative assessment

MEMBRANES AND RECEPTORS – STUDY SHEET

PRIVATE STUDY - SESSION 9 / STUDY SESSION – SESSION 10

DATA HANDLING EXERCISE ON PHARMACOKINETICS

In preparation for the study session in session 10, you should plot the graphs described
under points 1 and 2 below and attempt to provide answers to questions 3 – 5.
Reference to the lecture notes for lecture 17 should help you interpret the questions.
This preparation for the study session will enhance discussions with your tutors during
the study session.

In this exercise, you will investigate the kinetics that best describe the disappearance
of Drug A and Drug B from the circulation. Both drugs have been infused
intravenously into a human volunteer, and at time 0, the infusion was stopped.

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Questions:

1. Plot the time on the x-axis against drug level (for both A and B) on the y-axis.

2. Plot the time on the x-axis against Loge Drug level (for both A and B) on the y-axis.

3. Determine whether Drug A or Drug B is eliminated by linear (so-called „first-

order‟) kinetics, and which drug is eliminated by non-linear (so-called „zero-order‟)
kinetics.

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Linear kinetics - the plot of Loge Drug level against time is a straight line.

Non-linear kinetics - the plot of Drug level against time is a straight line.

4. For the drug with linear kinetics, estimate the half-life (T½) in hours.

For the drug with non-linear kinetics, estimate the rate of elimination of the drug
in
mg ml-1 h-1.

5. When the dose of Drug A or of Drug B is increased, try to predict what would
happen to plasma levels for each of these drugs. If you were prescribing Drugs A or B,
which of these would require greater care as you increased the dose ?

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GRAPH PAPER

MEMBRANES AND RECEPTORS MODULE

SESSION 10 – THE AUTONOMIC NERVOUS SYSTEM/

PHARMACOKINETICS

AIMS

The aims of this session are:

 to consider the anatomical structure of the ANS, the major neurotransmitters

involved and the sites of drug action

 to consider the pharmacokinetics of drug administration

 to illustrate pharmacokinetic principles using worked examples

LEARNING OUTCOMES

By this session you should be able to:

 understand the anatomical and pharmacological divisions of the autonomic

nervous system

 to compare and contrast the pathways of neurotransmitter synthesis, storage,

release and termination at adrenergic and cholinergic synapses; outline the
regulation of noradrenaline and acetylcholine release, and be able to list the
major drug classes used to affect autonomic nervous system function

 give example to illustrate the importance of ANS drugs in cardiovascular and

airways control in specific disease states

 appreciate whole body considerations of drugs reaching their sites of therapeutic

action, including principles of drug bioavailability and inactivation

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PRIVATE STUDY

Private study time should be used:

to work on the study sheet on asthma, hypertension and thyrotoxicosis in preparation

for tutorial 4 in Session 11.

Revise for End of Semester Assessment 2

MEMBRANES AND RECEPTORS MODULE - SESSION 10

THE AUTONOMIC NERVOUS SYSTEM

LECTURES 10.1 AND 10.2 - THE AUTONOMIC NERVOUS SYSTEM

AIMS

To understand the anatomical and pharmacological divisions of the autonomic nervous

system (ANS). To be able to compare and contrast neurotransmitter synthesis, storage,
release and termination of action at cholinergic and adrenergic synapses. To
understand the differing roles of pre- and post-synaptic receptors. To be able to list the
various mechanisms by which drugs can influence neurotransmission and the major
drug classes used to manipulate autonomic function.

What does the ANS do?

Apart from the motor innervation of skeletal muscle (the somatic efferent system), the
ANS conveys all of the central nervous system (CNS) efferent outputs to the body and
is responsible for regulating physiological functions largely outside the influence of
voluntary control.

Thus, the ANS controls smooth muscle (vascular and visceral); exocrine (and some
endocrine) secretions; rate and force of the heart, and may influence certain metabolic
pathways. Tissues are not necessarily innervated by both branches of the ANS,
however where this occurs, sympathetic and parasympathetic systems often have

198
opposing effects (e.g. in control of heart rate; GI smooth muscle motility, etc..), but
there are notable exceptions to this (e.g. ANS control of salivary secretion).

If generalizations are to be made, then it is reasonable to state that sympathetic activity

tends to predominate in stress (e.g. sympathetically mediated increases in the rate and
force of contraction in the heart during the “fight-or-flight” response), whereas
parasympathetic activity predominates in the basal state (e.g. suppression of cardiac
pacemaker firing by vagally-induced tone).

Anatomical Structure of the ANS

In outline, the anatomy of the ANS is relatively simple. In all cases the ANS conveys
information from the CNS to the neuro-effector junction (i.e. the point at which the
target cell/tissue is innervated) by two neurons arranged in series and termed pre-
ganglionic and post-ganglionic. The ANS consists of two anatomically defined
divisions, the sympathetic and parasympathetic systems.

 Sympathetic pre-ganglionic neurons have their cell-bodies in the lateral horn of

the grey matter of the spinal cord and emerge from the spinal cord in the
thoraco-lumbar region. They synapse with post-ganglionic sympathetic neurons
in the paravertebral chain of sympathetic ganglia, which lies bilaterally and
close to the spinal column, or for some pre-ganglionic sympathetic nerves
destined for the abdominal and pelvic viscera, in the abdominal
prevertebral/midline chain.
 Parasympathetic neurons emerge from the CNS either in cranial or sacral
(cranio-sacral) regions of the spinal column. The parasympathetic ganglia lie
close to the target organs and therefore the post-ganglionic fibres are short.

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Neurotransmitters in the ANS

 The principal transmitters involved in autonomic transmission are acetylcholine

(ACh) and noradrenaline (NA).

 All pre-ganglionic neurons are cholinergic (i.e. use ACh as the principal
transmitter). Sympathetic or parasympathetic pre-ganglionic release of ACh
activates nicotinic ACh receptors (nAChRs) present on post-ganglionic cells.

200
 Parasympathetic post-ganglionic neurons are also cholinergic. In this case
released ACh interacts with post-synaptic muscarinic ACh receptors (mAChRs)
in the target tissue.

 Most sympathetic post-ganglionic neurons are noradrenergic (i.e. use NA as the

principal transmitter). NA interacts with one or more types of adrenoceptor (i.e.
1, 2, 1, 2) present post-synaptically. However, in some cases (e.g. sympathetic
innervation of the sweat glands and piloerector muscles) the post-ganglionic
neurons are cholinergic, releasing ACh and exerting post-synaptic actions via
mAChRs.

 Other transmitters (commonly known as NANC - “non-adrenergic, non-

cholinergic” - transmitters) are also important in certain situations. NANC
transmitters are often co-released with either ACh or NA (Co-transmission).
Examples of NANC transmitters include ATP, 5-hydroxytryptamine (also
known as serotonin), nitric oxide (NO) and several neuropeptides including
neuropeptide Y; vasoactive intestinal peptide (VIP) and substance P.

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Drugs that interact with any of these steps can affect neurotransmission. Although we
will consider mainly drugs which act by blocking or mimicking neurotransmitter
action at the post-synaptic membrane (i.e. step 9 in diagram), other therapeutically
important sites of drug action can be pinpointed using this general diagram, and
examples of these will also be highlighted below.

Cholinergic Transmission

Acetylcholine is synthesized by the enzyme choline acetyltransferase from choline (an

essential dietary constituent) and the metabolic intermediate acetyl CoA in the
cytoplasm of cholinergic terminals. Although some of the ACh is degraded by

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cytoplasmic cholinesterase, the majority is transported into synaptic vesicles by an
indirect active transport mechanism similar to that described above for noradrenaline.
Cholinergic terminals possess numerous vesicles containing high concentrations (>100
mM) of ACh that can be released by Ca2+-mediated exocytosis.

Released ACh can interact with both pre- and post-synaptic cholinoceptors. However,
the opportunity to interact with receptors is limited by ACh in the synaptic cleft being
acted upon by cholinesterase, which rapidly degrades ACh to choline and acetate. The
activity of this enzyme is higher at fast (nicotinic) cholinergic synapses limiting the
synaptic cleft half-life of ACh to a few milliseconds. Most choline is recaptured by a
choline transporter present in the synaptic terminal.

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Agents that interfere with cholinergic transmission and are of therapeutic use generally
act by interaction with cholinoceptors. The only notable exception to this is the use of
cholinesterase inhibitors to decrease the rate of ACh degradation and so prolong the
lifetime of ACh within the synaptic cleft.

 Nicotinic cholinoceptor antagonists may have a preferential ganglion (e.g.

trimethaphan) or neuromuscular (e.g. tubocurarine, pancuronium) blocking
action. The former class of agent is rarely used clinically, whilst the latter are
used to cause muscle paralysis during anaesthesia.

 Muscarinic cholinoceptor agonists (see list below) vary in their

muscarinic/nicotinic selectivity and resistance to degradation by cholinesterase.
Note that no agent shows significant selectivity between muscarinic receptor
subtypes (i.e. M1, M2, M3 selectivity). The major clinical use is in the treatment
of glaucoma (raised intraocular pressure) where the agent (usually pilocarpine)
can be applied in the form of eye drops. Minor uses include suppression of atrial
tachycardia, increasing gastrointestinal activity after abdominal surgery, and
stimulation of bladder emptying.

 Muscarinic cholinoceptor antagonists (see list below) show little selectivity for
receptor subtypes, but vary in their peripheral versus central actions. Hyoscine
(methylscopolamine) is used as anaesthetic premedication as it decreases
bronchial and salivary secretions, prevents reflex bronchoconstriction, reduces
any bradycardia induced by the anaesthetic and also has a sedative effect. Local
application of a poorly absorbed muscarinic cholinoceptor antagonist (e.g.
ipratropium bromide) can be used to treat bronchoconstriction in asthmatics
where the constriction is caused by increased parasympathetic discharge.

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 Pupillary dilatation and paralysis of accommodation can be caused by
muscarinic cholinoceptor antagonists (e.g. homatropine, tropicamide)
facilitating opthalmoscopic examination and having a beneficial effect in
various (rare) eye conditions.

 Cholinesterase inhibitors (e.g. edrophonium, physostigimine, dyflos) differ in

their longevity of action and their peripheral versus central effects. They are
used to acutely reverse the effects of non-depolarizing neuromuscular blocking
agents used in anaesthesia, in the topical treatment of glaucoma and in the
treatment of myasthenia gravis. Recently, cholinesterase inhibitors (e.g. tacrine,
donepezil) have been introduced for the treatment of the early stages of
Alzheimer‟s disease.

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Adrenergic Transmission

Noradrenaline (and the other signalling molecules dopamine and adrenaline) is

synthesized from tyrosine within the nerve terminal (see diagram below). The rate-
limiting enzyme is tyrosine hydroxylase. The presence of phenylethanolamine N-
methyltransferase in the chromaffin cells of the adrenal medulla allows adrenaline to
be synthesized as the main product for release. The enzyme dopamine -hydroxylase is
located within synaptic vesicles and therefore newly synthesized dopamine is
transported into the vesicle prior to conversion to noradrenaline. The vesicular
transporter recognizes not only dopamine, but also noradrenaline allowing
noradrenaline to be recycled following release and re-uptake (see below). Under most
circumstances the cytoplasmic noradrenaline concentration is low, whilst the
intravesicular concentration is very high (0.5-1.0 M), this is possible because the
vesicular transporter exploits a H+-ATPase-generated cytoplasm:vesicle H+-gradient
to move catecholamines against their concentration gradient. Cytoplasmic
noradrenaline is susceptible to enzymic breakdown by monoamine oxidase (MAO).

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Noradrenaline release is triggered by depolarization of the nerve terminal membrane,
Ca2+-entry and fusion of vesicles with the pre-synaptic plasma membrane (Ca2+-
mediated exocytosis). Released noradrenaline can interact with both pre-and post-
synaptic adrenoceptors. However, the opportunity to interact with receptors is limited
by a high affinity reuptake system (called “Uptake 1”) which acts to rapidly remove
noradrenaline from the synaptic cleft, rapidly decreasing the localized concentration
increase following release and thus terminating its actions. Any noradrenaline escaping
from the synaptic cleft is removed from the extracellular space by another, widespread,
lower affinity re-uptake system (“Uptake 2”). Noradrenaline recaptured by the nerve
terminal has two fates: it can be re-vesiculated and therefore undergo further release/
reuptake cycles or it can be metabolized (initially by MAO).

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Drugs Acting on Adrenergic Nerve Terminals
-Methyl-tyrosine competitively inhibits tyrosine hydroxylase and, therefore, blocks de
novo synthesis of noradrenaline. Only clinical use is to inhibit noradrenaline synthesis
in pheochromocytoma (noradrenaline-secreting tumour).

-Methyl-DOPA is taken up by adrenergic neurones and is converted to α-methyl-

noradrenaline by the sequential action of DOPA decarboxylase and dopamine -
hydroxylase. Unlike the true neurotransmitter, -methyl-noradrenaline is poorly
metabolized and therefore accumulates in the synaptic vesicles of noradrenergic
terminals. It is released by Ca2+-mediated exocytosis, but differs from noradrenaline
in that it preferentially activates pre-synaptic 2-adrenoceptors reducing transmitter
release. The ability of α-methyl-DOPA to form the “false transmitter” -methyl-
noradrenaline has been exploited in the treatment of hypertension.

CarbiDOPA inhibits DOPA decarboxylase in the periphery, but not in the CNS
(because it does not cross the blood-brain-barrier). It is used in combination with L-
DOPA in the treatment of Parkinson‟s disease (a dopamine deficiency in the basal
ganglia).

Adrenergic blocking drugs (e.g. guanethidine, bretylium) are selectively

concentrated in terminals by Uptake 1. They act via a variety of mechanisms,
including a local anaesthetic action reducing impulse conduction and Ca2+-mediated
exocytosis, a partial blocking action on reuptake of neurotransmitter, and depletion of
noradrenaline from synaptic vesicles. They are now rarely used therapeutically,
because of severe side-effects (postural hypotension).

Indirectly-acting sympathomimetic agents (IASAs) (e.g. tyramine, amphetamine,

ephedrine) are structurally related to noradrenaline. Because they are only weak
agonists at adrenoceptors they are thought to exert their actions by other/additional
mechanisms. IASAs are recognised and transported into the adrenergic terminal by
Uptake 1 and are taken up into synaptic vesicles where they cause noradrenaline to

208
leak from the vesicle. The displaced noradrenaline can leak into the synaptic cleft by a
mechanism unrelated to Ca2+-mediated exocytosis. The extent to which noradrenaline
leaks into the synaptic cleft can be greatly enhanced by inhibition of the noradrenaline-
degrading enzyme MAO.

Uptake 1 inhibitors comprise an important class of therapeutic agents - the tricyclic

antidepressants (e.g. amitriptyline) - however, these agents exert their therapeutic
actions centrally and their possible peripheral actions (enhancement of sympathetic
actions to cause, e.g. tachycardia and cardiac dysrhythmias) are unwanted side-effects
avoided by choice of drug and dose.

Adrenoceptor agonist pharmacology has produced highly receptor subtype-selective

agents, therefore a knowledge of the pre- and post-synaptic adrenoceptor subtype

209
disposition may allow the rational use of adrenoceptor agonists to achieve specific
therapeutic ends. Important uses of adrenoceptor agonists are given below:

Selective 1-agonists (e.g. dobutamine) can cause positive inotropic and chronotropic
effects which may be useful in treating circulatory shock - however, all 1-agonists are
prone to causing cardiac dysrhythmias

Selective 2-agonists (e.g. salbutamol, terbutaline) are highly effective in reversing

bronchconstriction in asthmatics

Selective 1-agonists (e.g. phenylephrine, oxymetazoline) are used as nasal

decongestants. 1-agonists (though more usually adrenaline) may be given in
conjunction with a local anaesthetic injection to cause local vasoconstriction and so
retard the dissipation of the anaesthetic.

Selective 2-agonists (e.g. clonidine) can be used as anti-hypertensive agents. This

action is brought about partly through stimulation of inhibitory pre-synaptic receptors
which decrease noradrenaline release and partly through a centrally-mediated action

Adrenoceptor antagonists are also widely used therapeutically. Most useful drugs are
or -adrenoceptor-selective and increasingly drugs which distinguish and -subtypes are
being used to reduce the unwanted side-effect profiles associated with therapy.
Important uses of adrenoceptor antagonists are given below:

-adrenoceptor antagonists (e.g. phentolamine, and the irreversible blocker

phenoxybenzamine) are used to cause peripheral vasodilatation (i.e. oppose

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sympathetically-mediated vasoconstriction) in the treatment of peripheral vascular
disease. They are not used to treat hypertension because they cause postural
hypotension and reflex tachycardia.

Selective 1-adrenoceptor antagonists (e.g. prazosin) are used in the treatment of

hypertension, although postural hypotension and impotence are still common
unwanted side-effects.

-adrenoceptor antagonists (e.g. propranolol) or 1-adrenoceptor antagonists (e.g.

atenolol) are used to treat hypertension, cardiac dysrhythmias, angina and myocardial
infarction. Possible unwanted side-effects include bronchoconstriction (particularly
using non-selective β-adrenoceptor antagonists in patients susceptible to
parasympathetically-mediated brochospasm), bradycardia, cold extremities, insomnia
and depression. Some trials report that the use of partial agonists (e.g. alprenolol,
oxprenolol) cause fewer side-effects as they provide a low tonic stimulation of -
adrenoceptors whilst still blocking receptor stimulation by noradrenaline.

Figures 1-6 are taken from Rang & Dale ‘Pharmacology’ second edition (1991) or Rang, Dale & Ritter
‘Pharmacology’ fourth edition (1999), published by Churchill Livingstone.

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MEMBRANES AND RECEPTORS MODULE

PRIVATE WORK – SESSION 10

STUDY SESSION – SESSION 11

DRUGS AND THE AUTONOMIC NERVOUS SYSTEM

AIMS
The aims of this session are to integrate information on the autonomic nervous system
and the common disease states of asthma, hypertension and thyrotoxicosis in which
treatments targeted to the autonomic system can be used.

OBJECTIVES

to be able to list the major drug classes used to affect autonomic nervous system
function

give examples to illustrate the importance of autonomic nervous system (ANS) drugs
in cardiovascular and respiratory disease states

to consider some clinical examples of the application of pharmacological principles

introduced in this module

ASTHMA

Asthma is not only a common but increasingly prevalent disease. It is recognised to be

an inflammatory disease with an often complex aetiology. Although therapies can be
aimed towards preventing activation of the underlying inflammatory mechanisms,
treatments that prevent or reverse the life-threatening bronchoconstriction occurring
during an asthmatic attack contribute the greater proportion of the therapies given.

a. What are the principle clinical/pathological abnormalities in asthma?

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b. The major autonomic innervation of the human airways is parasympathetic – what
are the consequences of increased parasympathetic drive?

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c. Although there is very little sympathetic innervation of the human airways there is a
large population of (non-innervated) adrenoceptors in the airway – what subtype of
adrenoceptor are these, and where in the bronchial tree are they predominantly
situated?

d. What are the consequences of stimulating airways adrenoceptors? When might this
occur in normal physiology?

e. What are the main categories of drugs that are used to treat asthma?

f. Which of these drugs mimic the functions of the autonomic nervous system and how
do they act at a cellular level?

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g. A variety of adrenoceptor agonists have been used to treat asthma – briefly discuss
the advantages of using highly selective agents which display either short or long
durations of action.

h. Compared to muscarinic cholinoceptor antagonists, adrenoceptor agonists have a

greater therapeutic benefit to asthmatic patients. What advantage does adrenoceptor
agonist therapy confer over the use of muscarinic cholinoceptor antagonists?

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215
HYPERTENSION

a. What physiological reflexes are involved in the normal control of blood pressure?

b. Briefly explain what hypertension is. What are the clinical criteria for diagnosis?

c. What target sites for drug action can you define to control abnormally elevated
blood pressure?

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Mild hypertension is often asymptomatic, but has very serious consequences as it
predisposes affected individuals to several diseases, including heart attack, stroke and
heart failure. Prognosis is poor in untreated individuals.

d. Briefly explain what are the main classes of drug currently used in the treatment of
hypertension.

e. What are the major population of adrenoceptors that mediate vasoconstriction of the
vasculature?

f. Does antagonism of these receptors explain the principal anti-hypertensive action of

-adrenoceptor antagonists?

g. What are the main unwanted side effects of using α-adrenoceptor antagonists to treat
hypertension?

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Non-selective α-adrenoceptor antagonists are no longer used in hypertension due to
their side effects. Selective α1-antagonists are sometimes used, and have less effect on
cardiac function and gastrointestinal motility.

h. β-adrenoceptor antagonists appear to exert their anti-hypertensive action through a

number of possible mechanisms – briefly outline and rationalize at least 3 of these.

i. One ability of β-adrenoceptor antagonists is to inhibit the secretion of renin from the
juxtaglomerular cells in the kidney. Explain why such an action may have an anti-
hypertensive effect?

j. β-adrenoceptor antagonists also have unwanted side effects – what are these?

Non-selective α-adrenoceptor antagonists are no longer used in hypertension due to

their side effects. Development of new, longer acting, more α1-adrenoceptor selective
antagonists has led to a greater use of these compounds of late. These have fewer side
effects, particularly on cardiac function and gastrointestinal motility, and are
sometimes used, particularly as they may have beneficial effects on plasma lipid levels
(more HDL, less LDL).

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k. Why should so much consideration be given to side-effect profiles when treating
hypertension?

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THYROTOXICOSIS

a. What are the main symptoms and clinical signs found in patients with
thyrotoxicosis?

b. Compare and contrast the symptoms of thyrotoxicosis with those of simple anxiety.

c. Which of these symptoms are mediated by the autonomic nervous system? And
why?

d. What drug targeted to the autonomic nervous system can be used for the treatment
for thyrotoxicosis? Why is this drug effective?

e. What other treatments are used for thyrotoxicosis?

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MEMBRANES AND RECEPTORS MODULE

SESSION 11 – DRUGS AND THE AUTONOMIC NERVOUS SYSTEM (ANS)

AIMS

The aims of this session are:

 to consider clinical examples of the application of pharmacological principles

introduced in this module

 to consolidate understanding of the pharmacokinetics of drug administration

LEARNING OUTCOMES

By this session you should be able to:

 give example to illustrate the importance of ANS drugs in cardiovascular and

airways control in specific disease states

 appreciate whole body considerations of drugs reaching their sites of therapeutic

action, including principles of drug bioavailability and inactivation

PRIVATE STUDY

 Private study time should be used to revise for End of Semester Assessment 2

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MEMBRANES AND RECEPTORS MODULE

SESSION 12 – REVISION

AIMS

To revise module content

LEARNING OUTCOMES

As given in the Introduction in the module handbook and in the Phase 1 Course
booklet

STRUCTURE OF SESSION

Students are free to use time allocated to the Membranes and Receptors module this
morning in any way they wish. Attendance in the Medical School is not required
during this session and students may chose to undertake revision at home or at another
venue.

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The revision tutorial will be student-led. The module leader will be available to
answer any questions that students may bring. The session will last for as long as
there are still questions to be answered. Students may leave the session at any time,
once all of their own questions have been answered.

MEMBRANES AND RECEPTORS MODULE

PRESENTATION ASSIGNMENTS

AIMS

The presentation assignments are designed to cover important material in this course,
much of which will complement work in other sessions, particularly those centred on
the Autonomic Nervous System later in the module. The format of the assignments
will develop your skills in researching and distilling new information and give you
valuable experience in preparing and delivering specific scientific information at an
appropriate level through oral presentation.

ORGANIZATION

(1) Students will work in their normal study groups.

(2) Each study group will be given two topics to research, with the aim of producing
two oral presentations

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(3) Groups will decide who will be responsible for delivering each of the assignment
presentations in the form of a 7-8 min talk. Different students should make the
presentations in Session 6 and Session 7.

(4) Each talk will be given to groups of approximately 40 students and a tutor.
Presenters should note that their presentations are a learning opportunity for their peers
and, hence, that it is important to emphasise the core concepts in the subject material,
rather than to go into too much detail to „impress‟ the tutor.

Teamwork: It is very important that study groups work together in researching and
preparing the material for each assignment presentation. In addition, it is important
that each oral presentation is practised in one or more “dummy-runs” within the
group. This allows the presenter(s) to practise his/her presentation, the group a chance
to suggest improvements, and crucially, to ensure that the length of the presentation
fits the 7-8 min time-slot allowed. Presentations will be stopped by the tutor if they
overrun their allotted time. It should be noted that it is the responsibility of the whole
group, not just the presenter(s), to field questions about the presentation asked by
either other students or the tutor.

The Assignments: To help you structure your presentation a work book is provided
outlining issues which should be addressed in each of the talks. This format should
also help you to take relevant notes during the presentations.

Visual Aids: Presentations will be scheduled in tutorial rooms equipped with a

computer and data projector to permit PowerPoint presentations to be made. You
should aim to make your presentation with a minimum number of slides. A good rule-
of-thumb is 7 slides for a 10 minute presentation; therefore 5 slides should suffice.

Peer support for student learning:

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Handouts: In previous years, some students have wished to provide handout materials
to facilitate note taking for their audience. Please note that there are no Faculty funds
allocated for this purpose and so any costs incurred in this way cannot be reimbursed.

WebBoard: Alternatively, some students have posted their notes or PowerPoint

presentations on the module WebBoard. This is encouraged. To assist those wishing to
locate such materials within the WebBoard, it would be helpful if all resources for the
same presentation could be posted together under the same topic, e.g. „Presentation
ACh1‟, „Presentation ACh2‟, etc..

Assessment of assignment presentation content: New subject material is presented by

your peers during assignment sessions which illustrates core material and adds further
detail. A commonly asked question is „Will I need to know the subject material
covered by the assignment sessions for the second SAQ asessment in the module?‟
While it would be possible to achieve a satisfactory grade in the second assessment by
revising only the core material presented in the module, the assignment sessions are
designed to provide additional important details and integration within the module.
Thus, good satisfactory or excellent answers to an SAQ question may well include
information from these sessions.

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SESSIONS 6 AND 7 – PRESENTATIONS

ACETYLCHOLINE

Acetylcholine is a very important neurotransmitter in both the central and peripheral

nervous systems. Elsewhere in this and other modules, you will consider the role of
acetylcholine at the neuromuscular junction - the neuroeffector junction of the somatic
nervous system - in some detail. Therefore, for the purposes of these assignments we
will concentrate on acetylcholine as a transmitter in the autonomic nervous system
(ANS). Clearly much of what we learn with respect to acetylcholine in the ANS is also
applicable to this molecule acting as a transmitter in other systems.

PRESENTATION ACh1

Synthesis & Storage:

(1) Acetylcholine acts as the major neurotransmitter at four distinct general classes of
synapse in the autonomic nervous system. One such class of synapse is at the post-
ganglionic fibre-target tissue (neuroeffector) junction of the parasympathetic branch of
the ANS; what are the other three classes? Draw a simple diagram to illustrate where
acetylcholine acts as the major neurotransmitter in the ANS.

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(2) Acetylcholine is synthesised from choline (an essential component obtained in the
diet) and what other metabolic intermediate? What is the enzyme involved and where
does synthesis occur?

(3) How is acetylcholine "packaged" for release?

Regulation of Release:

(4) Parasympathetic discharge is primarily regulated by CNS initiation of the firing of

pre-ganglionic fibres which form synapses with the post-ganglionic neurones. Use the
outline given below to sketch the events which occur to cause release of acetylcholine
by pre-ganglionic cholinergic fibres synapses.

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(5) Give an example of an agent which interferes with the release of acetylcholine.

How is Neurotransmitter Action Terminated?

(6) For a neurotransmitter such as acetylcholine to act efficiently (i.e. for post-synaptic
effects to be exerted on a sub-second time-scale), the parasympathetic synapse must
possess mechanisms to rapidly terminate the action of acetylcholine. How is this
achieved?

(7) What happens to free choline and acetate present in synapses?

PRESENTATION ACh2

Cellular Targets:

(1) Briefly list some of the important sites (i.e. organs/tissues) of parasympathetic
innervation.

(2) What is the effect of increased parasympathetic discharge in the heart?

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(3) Acetylcholine is employed as the neurotransmitter at all parasympathetic nerve
synapses. Are there any other synapses in the autonomic nervous system that use
acetylcholine as neurotransmitter?

Cholinoceptors:

(4) Multiple forms of receptors for acetylcholine, termed "cholinoceptors", occur.

What naturally-occurring substances, which each either mimic or prevent some of the
actions of acetylcholine, were first used to show that two distinct classes of
acetylcholine receptor exist?

(5) Briefly, explain how the receptor subtype found at the ganglionic junction causes a
post-ganglionic response when activated by acetylcholine.

(6) What is the class of acetylcholine receptor found at the parasympathetic

neuroeffector junction?

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(7) This class of acetylcholine receptor be further divided into at least three subtypes
Each mediates its cellular action by changing the activity of enzymes which synthesise
second messengers, and/or by changing the opening of particular ion channels in the
plasma membrane. Indicate in the table below the predominant G-protein and effectors
that are involved in transducing the signal from different receptor subtypes List these
acetylcholine receptor subtypes and which enzymes/ion channels each sub-type
preferentially links to:

(8) Briefly describe the series of events that couple activation of M2 muscarinic
receptors to the activation of the effector(s).

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PRESENTATION ACh3

Physiologically important actions of acetylcholine:

(1) Release of acetylcholine by parasympathetic post-ganglionic fibres plays an

important role in the regulation of a number of tissues. For the tissues given below list
the major physiological actions of acetylcholine.

Cholinoceptor Pharmacology:

(2) Agents which mimic some or all of the actions (i.e. cause the same cellular
responses) of acetylcholine are termed cholinoceptor agonists. What might be the
advantage of synthesising cholinoceptor agonists which only interact with a particular
receptor subtype?

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(3) Are such agents available?

(4) If so, are they used clinically

Agents which can prevent the actions of acetylcholine are termed cholinoceptor
antagonists. If the action of such agents is competitive, then the antagonist interacts
with the cholinoceptor, occupying or blocking the agonist binding site, but having no
stimulatory effect on cholinoceptor activity per se.

(5) Muscarinic cholinoceptor antagonists are used therapeutically in a number of

clinical conditions. One clinical use of muscarinic cholinoceptor antagonists is to treat
gastrointestinal disorders. Also, these agents are frequently given as a premedication
for general anaesthesia. Why are muscarinic cholinoceptor antagonists used under
these circumstances?

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(6) What unwanted side effects limit the usefulness of these agents?

PRESENATION ACh4

How does understanding the autonomic innervation of the eye help to explain the
strategies used to treat glaucoma?

(1) Draw a simple diagram to illustrate the anatomy of the human eye relevant to sites
of sympathetic and parasympathetic innervation.

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(2) An abnormally raised intraocular pressure is termed glaucoma. Untreated this can
lead to irreversible damage of the eye and blindness. What are the most likely causes
of this condition?

(3) What are the consequences of increasing parasympathetic tone in the eye? Might
this be beneficial in decreasing intraocular pressure?

(4) Which receptor ligands are used clinically to treat glaucoma?

(5) If the desired effect is to increase the stimulation of muscarinic receptors, what
alternative non-receptor strategy can be adopted. Which agents are used clinically?

(6) What are the effects of increasing sympathetic tone in the eye? Is this most likely
to lead to an increased or decreased intraocular pressure?

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(7) What agents, active at adrenoceptors, are used clinically in the treatment of
glaucoma?

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SESSIONS 6 AND 7 – PRESENTATIONS

NORADRENALINE
Noradrenaline is an important neurotransmitter in the central and peripheral nervous
systems. For the purposes of these assignments we will concentrate on noradrenaline
as a transmitter in the autonomic nervous system (ANS). Clearly, much of what we
learn with respect to noradrenaline in the ANS is also applicable to noradrenaline
acting as a central transmitter.

PRESENTATION NA1

Synthesis & Storage:

(1) Consider the following: "Noradrenaline is the major neurotransmitter at the

neuroeffector junction of sympathetic post-ganglionic fibres" - Using a simple
schematic diagram of the autonomic nervous system, explain this statement.

(2) Are there any post-ganglionic synapses in the sympathetic nervous system at which
noradrenaline is not the transmitter?

(3) The "biogenic amines" (dopamine, noradrenaline and adrenaline) are all

236
synthesised from the amino acid tyrosine: outline the synthetic pathway. What are the
enzymes involved? Where does this pathway occur?

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(4) What determines whether a nerve terminal is "dopaminergic" (releases dopamine)
or "(nor)adrenergic" (releases noradrenaline)?

(5) How is noradrenaline “packaged” for release?

Regulation of Release:

(6) Sympathetic discharge is primarily regulated by central nervous system (CNS)

initiation of the firing of pre-ganglionic fibres which form synapses with the
noradrenergic post-ganglionic neurones. Briefly outline the sequence of events which
occur following release of acetylcholine by the pre-ganglionic fibre which lead to
noradrenaline release. Use the sketch of a noradrenergic synaptic varicosity below to
briefly outline the key features of post-ganglionic sympathetic innervation of target
issues.

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PRESENTATION NA2

Cellular Targets:

(1) Briefly list some of the important sites (i.e. organs/tissues) of sympathetic
innervation.

(2) In the heart, increased noradrenaline release causes both an increase in heart-rate
(positive chronotropy) and an increase in the force of each contraction (positive
inotropy). Explain the anatomical basis for these two distinct responses to sympathetic
innervation.

Adrenoceptors:

(3) Multiple forms of receptors for noradrenaline (and adrenaline), termed

"adrenoceptors", occur. All adrenoceptors belong to the family of receptor proteins

239
whose primary amino acid sequence crosses the plasmalemma seven times and which
exert their cellular effects by activation of sub-populations of G proteins (guanine
nucleotide binding proteins) within the cell. Complete the Table overleaf to show
which effector molecules (e.g. adenylyl cyclase, phospholipase C, etc.) are regulated
by the different subtypes (i.e. 1-, 2-, 1- and 2) of adrenoceptor. Are distinct types of G
protein involved in linking different receptor subtypes to each effector?

How is Neurotransmitter Action Terminated?

(4) For a neurotransmitter such as noradrenaline to act efficiently (i.e. for post-synaptic
effects to be exerted on a sub-second time-scale), the sympathetic synapse must
possess mechanisms to rapidly "clear" noradenaline from the synaptic cleft. How is
noradrenaline removed from the synaptic cleft? Outline some of the properties of the
mechanism which allows it to rapidly decrease extracellular noradrenaline levels.

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(5) What is the fate of noradrenaline removed from the synaptic cleft? Can
noradrenaline be re-packaged for re-use?

(6) Name the TWO major enzymes responsible for inactivation of noradrenaline?

(7) Which products of metabolism can be measured in the blood or urine as an indirect
index of sympathetic activity?

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PRESENTATION NA3

Physiologically important actions of noradrenaline:

(1) Complete the following Table illustrating the major physiological effects of
adrenoceptor activiation. The predominant actions of adrenoceptor stimulation in liver
and fat are given as examples.

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Adrenoceptor Pharmacology

(3) Agents which mimic some or all of the actions (i.e. cause the same cellular
responses) of noradrenaline/adrenaline are termed adrenoceptor agonists. What might
be the advantage of synthesising an adrenoceptor agonist which only interacts with a
particular receptor subtype?

(4) Outline the type of adrenoceptor agonist used to:

(5) Why are different agents required for these different therapeutic interventions?

(6) Agents which can prevent the actions of catecholamines - adrenoceptor

antagonists - do so by interacting with adrenoceptors, blocking agonist binding, but

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having no stimulatory effect on adrenoceptor activity per se. Adrenoceptor antagonists
are widely used therapeutically in a number of clinical conditions (and will be covered
in other parts of the Module). Give an example of the clinical use of an - and -
adrenoceptor antagonist.

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PRESENTATION NA4

-Methyltyrosine, -methyl DOPA and guanethidine have been used clinically to

inhibit sympathetic neurotransmission. Explain the mechanism by which each of
these agents affects noradrenaline release, and why their clinical usage has
declined.

(1) Which enzyme is specifically inhibited by -methyltyrosine? Why is this important

with respect to noradrenaline synthesis?

(2) What particular type of cancer is treated by administration of -methyl tyrosine?

(3) Instead of acting as an inhibitor, -methyl DOPA acts as a “false substrate” for
which biosynthetic enzyme? What product of -methyl DOPA metabolism accumulates
in noradrenergic terminals? If released what is the major action of this “false
transmitter”?

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(5) Guanethidine is a noradrenergic neurone blocking drug – what does this mean?
Briefly outline the hypotheses put forward to explain how guanethidine inhibits
noradrenaline release.

(6) Guanethidine is no longer used clinically – what unwanted side effects caused
withdrawal of this agent?

(7) Generally, the preferred tools for inhibiting noradrenergic transmission act at
adrenoceptors – why do you think adrenoceptor agonists and antagonists have become
the drugs of choice?

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(4) -methyl DOPA has been adopted as one clinical strategy for the treatment of
hypertension; explain the theory behind this therapeutic approach.

247