DOI: 10.1515/folmed-2017-0006
ORIGINAL ARTICLE, MEDICINE
Effects of the Novel High-affinity 5-HT(1B/1D)-receptor Ligand
Frovatriptan on the Rat Carotid Artery
Kremena E. Saracheva1,2, Natalia A. Prissadova3, Valentin I. Turiiski3, Valeri I. Slavchev3,
Atanas D. Krastev3, Damianka P. Getova4
1
Department of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Medical University of Plovdiv, Plovdiv, Bulgaria
of Pharmacology and Medical Toxicology, Faculty of Pharmacy, Medical University of Plovdiv, Plovdiv, Bulgaria
3 Department of Medical Physics and Biophysics, Faculty of Pharmacy, Medical University of Plovdiv, Plovdiv, Bulgaria
4 Laboratory of Neuropharmacology, Technological Center for Emergency Medicine (TCEMED), Plovdiv, Bulgaria
2 Department
Correspondence:
Kremena E. Saracheva, Department of Pharmacology and Clinical
Pharmacology, Faculty of Medicine,
Department of Pharmacology
and Medical Toxicology, Faculty
of Pharmacy, Medical University
of Plovdiv, 15A Vassil Aprilov Blvd.,
4002 Plovdiv, Bulgaria
E-mail: kremena_saracheva@
yahoo.com
Tel: +359 899 778 329
Received: 21 Aug 2016
Accepted: 18 Sep 2016
Published Online: 29 Nov 2017
Published: 27 March 2017
Key words: serotonin, migraine,
triptans, frovatriptan, carotid artery
Citation: Saracheva KE, Prissadova NA, Turiiski VI, Slavchev
VI, Krastev AD, Getova DP. Effects of the novel high-affinity
5-HT(1B/1D)-receptor ligand
frovatriptan on the rat carotid
artery.
Background: In blood vessels 5-HT stimulates sympathetic nerves, the endothelium and vascular smooth muscle cells. Triptans are specific anti-migraine drugs
and they activate the serotoninergic 5HT1b/d receptors causing vasoconstriction
of the cerebral vessels.
Aim: To evaluate the effect of frovatriptan on isolated rat carotid artery.
Methods: Contractile activity of the preparations was registered isometrically.
Krebs solution (pH = 7.4) was used for washing smooth muscle (SM) preparations
aerated with 95% O2 and 5% CO2 at 37°C. The 60-minute adaptation of tone level
of preparations was taken as a starting tone and the changes such as contraction
or relaxation were calculated using it.
Results: Frovatriptan (1×10-6 mol/l - 1×10-5 mol/l) induced a contraction, but at
higher concentrations it caused relaxation of the carotid artery. The L-norepinephrine contractile reaction was enhanced in the presence of frovatriptan. In the
presence of 5-HT2 receptor antagonist, methysergide, frovatriptan increased the
relaxation. In the presence of the specific α-1 receptor antagonist, prazosin, the
frovatriptan-induced relaxation decreased.
Conclusion: The observed contractile effect of frovatriptan is probably associated
with the main effect of the drug – activation of the serotoninergic 5HT1B /1D receptors causing vasoconstriction of the cerebral vessels and their anti-migraine
effect. At higher concentrations, frovatriptan, most likely via some non-specific
mechanism, could activate the following intracellular chain reaction: stimulation
of α1D could activate eNOS which may increase in the concentration of NO which
results in the final effect of relaxation.
Folia Medica 2017;59(1):31-36.
doi: 10.1515/folmed-2017-0006
BACKGROUND
Serotonin (5-hydroxytryptamine, 5-HT) exerts its
multiplicity of physiological effects through an
unsurpassed diversity of receptors. Some of these
effects are mediated through actions of 5-HT in
the central nervous system, whereas 5-HT also
has multiple and diverse effects through direct interaction with 5-HT receptors in different parts of
the cardiovascular system.1 In blood vessels 5-HT
stimulates sympathetic nerves, the endothelium and
vascular smooth muscle cells.2
Triptans are specific anti-migraine drugs gener-
31
ally recommended to patients whose conditions are
refractory to analgesics. The therapeutic effect of
triptans appears to be induced by activating the
serotoninergic 5-HT1B/1D receptors in the trigeminovascular system causing vasoconstriction of the
cerebral vessels and neuronal inhibition. Despite
their relative selectivity, the vasoconstrictive activity
of triptans also affects peripheral arteries, although
to a lesser extent.3
Triptans differ markedly in terms of their pharmacokinetic and pharmacodynamic properties. Some
triptans, such as sumatriptan, are considered to be
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K. Saracheva et al
AIM
To evaluate the effect of frovatriptan on isolated
rat carotid artery.
MATERIALS AND METHODS
Male Wistar rats with initial body weight in the
range of 220 to 280 g were provided by the Animal
House of Medical University Plovdiv, Bulgaria. The
rats were housed in standard laboratory conditions
(23-25°C, 50-55% humidity and 12/12h light/dark
cycle) and fed with standard commercial food and
given water ad libitum. At the beginning of the
experiments the animals were euthanized by overdose anesthesia (ketamine and xylazine). Smooth
muscle preparations of a. carotis communis 0.8-1.0
mm wide and 15 mm long were cut. All experiments were carried out according to the guidelines
of using laboratory animals in EU- guidelines/EEC
Directive of 1986.
Contractile activity of the preparations was
registered isometrically using Tenzo detectors
(Swema, Sweden). The initial mechanical stress of
the preparations reached by the stretch tensosystem
is a value corresponding to the tensile force of 10
mN. Krebs solution (pH = 7.4) used for washing
the SM preparations was continuously aerated with
a gas mixture of 95% O2 and 5% CO2 at 37°C. A
60-minute adaptation of tone level of preparations
was taken as a starting tone and changes such
as contraction or relaxation were compared to it.
During the adaptation the Krebs solution changed
several times. The drug-caused reactivity of the
SM preparations was measured and registered by
32
gain stage Microtechna (the Czech Republic) and
recorded using paper recorder (Linseis, Germany).
CHEMICALS AND SOLUTIONS
The following chemicals were used: acetylcholine
(Dispersa Baeschlin, Germany); DMSO (Sigma),
frovatriptan succinate monohydrate (Sigma), methysergide maleate salt (Sigma), L-norepinephrine
hydrochloride (Sigma), prazosin hydrochloride
(Sigma).
The Krebs solution had the following composition (mM): NaCl 120; KCl 5.9; CaCl2 2.5; MgCl2
1.2; NaH2PO4 1.2; NaHCO3 15.4 and glucose 11.5
(Merck).
STATISTICS
The data obtained were expressed as mean ± standard
error of the mean (SEM). The number of tissue
preparations used in each experiment is indicated
by n. Statistical differences were tested using the
Student’s t-test, and P<0.05 was considered significant. All statistical analyses were performed using
a specialized software SPSS, version 17.0 (SPSS
Inc. Chicago, IL).
RESULTS
EFFECTS OF FROVATRIPTAN ON THE CONTRACTILE ACTIVITY
OF SM PREPARATION OF CAROTID ARTERY.
At the concentration of 1×10-6 mol/l and 1×10-5
mol/l, frovatriptan induced contraction in the SM
preparations of carotid artery (Fig. 1).
0.4
Smooth muscles tonus, mN
fast-acting while also possessing greater risk of
adverse events. Frovatriptan is one of the newest
triptans and its distinct pharmacokinetic and pharmacodynamics profile reveals a clinical potential
due to the long duration of action (half-life is 26h)
and low incidence of side effects and drug interactions. The elimination half-life of frovatriptan is
five times greater than that of other triptans, while
the time to maximum concentration is similar to
that of other triptans when given orally.4 It has
high affinity to 5-HT1B/1D receptors and moderate
affinity to 5-HT7 receptors. It does not inhibit or
induce cytochrome P450, associate to a low rate
binding to plasma proteins, which in turn leads to
a lower risk of drug interactions.5
0.3
0.2
0.1
0
-0.1
1
2
3
4
5
-0.2
-0.3
-0.4
*
Concentration of frovatriptan, mol/l
Figure 1. Changes in the contractile activity of isolated
SM preparations of carotid artery after application
of frovatriptan at concentration of 1×10-6 mol/l (1),
5×10-6 mol/l (2), 1×10-5mol/l (3), and 5×10-5 mol/l (4)
and 1×10-4 mol/l (5), (n=9).
At concentrations of 5×10-5 mol/l and higher
frovatriptan provoked significant relaxation (Fig. 2).
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Effects of Frovatriptan on the Rat Carotid Artery
pared to the control reaction exerted by frovatriptan
alone (Fig. 4).
Smooth muscles relaxation, mN
0
Figure 2. Frovatriptan isometric record of smooth
muscle activation changes from isolated preparations
of carotid artery (n=6).
L-NOREPINEPHRINE ON THE
CONTRACTILE ACTIVITY OF SM PREPARATIONS OF CAROTID
ARTERY IN PRESENCE OF FROVATRIPTAN.
In the presence of frovatriptan at a concentration of
5×10-5 mol/l we observed a significant increase of
the contractile reaction of L-norepinephrine (1×10-5
mol/l) compared to the control reaction (Fig. 3).
0.4
0.3
0.2
0.1
0
2
Figure 3. Changes in the tone of isolated SM preparations induced by L-norepinephrine (1×10-5 mol/l (1))
and L-norepinephrine (2) in the presence of frovatriptan
(5×10-5 mol/l) (n=7). Statistically significant effects are
indicated by * (P<0.05).
EFFECTS
OF
5×10-5
MOL/L OF FROVATRIPTAN ON THE
SM PREPARATIONS OF CAROTID
ARTERY IN PRESENCE OF METHYSERGIDE.
Frovatriptan (5×10-5 mol/l) in the presence of the
5-HT2 receptor antagonist methysergide (1×10-5
mol/l) significantly increased the relaxation comCONTRACTILE ACTIVITY OF
Folia Medica I 2017 I Vol. 59 I No. 1
-0.4
-0.5
-0.6
*
-0.7
Concentration of frovatriptan, mol/l
OF
5×10-5
MOL/L FROVATRIPTAN ON THE CON-
TRACTILE ACTIVITY OF
SM
PREPARATIONS OF CAROTID
ARTERY IN PRESENCE OF PRAZOSIN.
In the presence of the specific alpha-1 receptor
antagonist, prazosin (1×10-6 mol/l), the relaxation
induced by frovatriptan (5×10-5 mol/l) significantly
decreased compared to that in the controls by
frovatriptan alone (Fig. 5).
0
Smooth muscles relaxation, mN
Smooth muscles tonus, mN
0.5
1
-0.3
Figure 4. Changes in the tone of SM preparations of
frovatriptan (5×10-5 mol/l) (1) and of frovatriptan combined with methysergide (1×10-5 mol/l)(2) (n=7). Statistically significant effects are indicated by * (P<0.05).
*
0.6
2
-0.2
EFFECTS
EFFECTS OF 1×10-5 MOL/L
0.7
1
-0.1
-0.05
-0.1
1
2
*
-0.15
-0.2
-0.25
-0.3
-0.35
-0.4
-0.45
-0.5
Figure 5. Changes in the tone of isolated SM preparation, induced by (1) frovatriptan (5×10-5 mol/l) and (2)
frovatriptan in the presence of prazosin (1×10-6 mol/l)
(n=7). Statistically significant effects are indicated by
* (P<0.05).
DISCUSSION
Studies using isolated blood vessels provide a
robust quantitative pharmacological comparison of
drugs and may identify subtle differences between
triptans. These differences are of scientific interest
since, in theory, all triptans belong to the same
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K. Saracheva et al
class (i.e. 5-HT1B/1D receptor agonists) and therefore
would be expected to have similar pharmacological
actions. It is important to remember that any differences detected using in vitro studies should not be
extrapolated and used as predictors or comparators
of drug effects when administered to man.
5-HT was recognized as a substance isolated
from blood serum (sero-) that could modify the
tone of smooth muscles (-tonin).6-8
Frovatriptan, which belongs to the group of
triptans, may act by constricting cranial vessels
through 5-HT1B receptors, by inhibiting peripheral
trigeminal nerve afferents that innervate the vessels
and pain-producing dura mater through 5-HT1D
receptors, or by inhibiting central trigeminal neuronal traffic through 5-HT1D receptors, or by a
combination of these mechanisms.9
Our results shows that at concentrations of
1×10-6 mol/l and 1×10-5mol/l frovatriptan induces
contractions in SM preparations of carotid artery.
This effect is probably related to the drug’s main
action – activation of serotoninergic 5-HT1B/1D
receptors causing vasoconstriction of the cerebral
vessels and their anti-migraine effect. It is well
known that a-adrenoceptors play an important role
in the regulation of vascular tone and blood pressure and that stimulation of the receptors mediate
constriction of the isolated carotid artery.10
There are 3 α-1 adrenergic receptor subtypes:
α 1A, α 1B, and α 1D, all of which signal through
the Gq/11 family of G-proteins and different subtypes show different patterns of activation. They
activate mitogenic responses and regulate growth
and proliferation of many cells. Catecholamines
like norepinephrine (noradrenaline) and epinephrine
(adrenaline) influence signaling pathways through the
α1-adrenergic receptor in the central and peripheral
nervous systems. α1A- and α1B-ARs localized to
the plasma membrane while α1D-ARs accumulate
intracellularly.11
There is evidence in the literature that the
activation of α1-adrenoceptors induces relaxation
in rabbit bronchial artery 12 and rat pulmonary
arteries, in a process involving the endothelial
production and release of nitric oxide (NO). In
the rat carotid artery, phenylephrine produce an
endothelium-dependent relaxation mediated by α1D
adrenoreceptors and involving the endothelial NO
synthase (eNOS).13
Frovatriptan possesses higher 5-HT1B agonist affinity and less affinity to α1D adrenoreceptors. Our
experiment showed that in its lower concentration
34
frovatriptan occupied by priority 5-HT1B receptors,
and only then the NO activation pathway of α1D
receptors could be possible. After all this a smooth
muscle contraction is observed in the 1×10-6 mol/l
and 1×10-5 mol/l drug concentrations.
Despite the drug’s lower α1-adrenoreceptor affinity in higher concentrations, it could be balanced
under the following intracellular pathway: activation
of α1D-ARs – NO synthesis – guanylate cyclase
activation – increasing of internal cGMP – activation
of protein kinase G (PKG). On the one hand PKG
could influence directly the contractile machinery
of smooth muscles14, on the other hand it could
block L-type Ca2+ channels (in terms of Ca2+ influx
reduction, necessary for muscle contraction)15 and
on third hand PKG could activate calcium pumps
whish also leads to reduction of intracellular calcium. The overall processes cumulation leads to
5-HT1B and α1D activation and subsequent smooth
muscle relaxation at a concentration of 5×10-5
mol/l of frovatriptan.
Such a hypothesis is supported by the results of
experiments conducted with methysergide (specific
inhibitor (5HT) receptor) and prazosin (specific
inhibitor of alpha-1 receptors). Our data shows
that the block 5-HT receptors by methysergide is
probably due to activation of the α1D receptors by
frovatriptan.
The blockade of α1 receptors by prazosin reduced
significantly the frovatriptan-induced relaxation
which confirms the probable involvement of α1D
receptors in the realization of its effects. As a result of experiments conducted in the presence of
frovatriptan (5×10-5 mol/l), the contractile response
to L-norepinephrine was significantly increased
compared to control reaction on the carotid artery.
Our results also suggest that frovatriptan predominantly at higher concentrations probably by
non-specific mechanism, which could activate the
following intracellular chain reaction: stimulation
on α1D → activation of eNOS → increase in the
concentration of NO16 and subsequent relaxation,
which is atypical for the triptans.
In conclusion, frovatriptan in concentrations of
1×10-6 mol/l, 1×10-5mol/l induces contractions in
the carotid artery which is due to the activation
of 5-HT1B / D receptors. At higher concentrations
(5×10-5 mol/l) frovatriptan induces relaxation atypical
for triptans. This effect could be a result from the
activation of α1D receptors. On the other hand, at a
concentration of 5×10-5 mol/l frovatriptan potentiates the effect of L-norepinephrine which in turn
Folia Medica I 2017 I Vol. 59 I No. 1
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Effects of Frovatriptan on the Rat Carotid Artery
can activate the following intracellular cascade:
stimulation of α1D receptors followed by activation
of eNOS and subsequently increased concentration
of NO with the final effect of relaxation.
Apparently frovatriptan initiates very complicated
and balanced pathways via 5-HT1B and α1D receptors,
which, depending on its concentration, could lead
to contraction or relaxation of the carotid artery.
ACKNOWLEDGEMENTS
This study is part of the Scientific Project SDP
14/2015 of MU-Plovdiv.
REFERENCES
1. Kaumann JA, Levy FO. 5-hydroxytryptamine receptors in the human cardiovascular system. Pharmacol
Ther 2006;111(3):674-706.
2. Geerts IS, Matthys KE, Herman AG, et al. Involvement of 5-HT1B receptors in collar-induced hypersensitivity to 5-hydroxytryptamine of the rabbit
carotid artery. Br J Pharmacol 1999;127(6):1327-36.
3. Roberto G, Piccinni C, D’Alessandro R, et al.
Triptans and serious adverse vascular events: Data
mining of the FDA Adverse Event Reporting System
database. Cephalalgia 2014;34(1):5-13.
4. Tullo V, Valguarnera F, Barbanti P, et al. Comparison
of frovatriptan plus dexketoprofen (25mg or 37.5
mg) with frovatriptan alone in the treatment of migraine attacks with or without aura: A randomized
study. Cephalalgia 2014;34(6):434-45.
5. Savi L, Mogavero S, Egan CG. Efficacy and pharmacokinetic activity of frovatriptan compared to
rizatriptan in patients with moderate-to-severe
migraine. Drug Des Devel Ther 2014;8:983-92.
6. Rapport MM, Green AA, Page IH. Serum vasoconstrictor, serotonin; isolation and characterization. J
Biol Chem 1948;176(3):1243-51.
7. Erspamer V, Asero B. Identification of enteramine,
the specific hormone of the enterochromaffin cell
Folia Medica I 2017 I Vol. 59 I No. 1
system, as 5-hydroxytryptamine. Nature 1952;169
(4306):800-1.
8. Page IH, McCubbin JW. Modification of vascular response to serotonin by drugs. Am J Physiol
1953;174(3):436-44.
9. Goadsby PJ. Serotonin 5-HT1B/1D receptor agonists in
migraine comparative pharmacology and its therapeutic implications. CNS Drugs 1998;10(4):271-86.
10. Willems EW, Heiligers JPC, De Vries P, et al. Alpha1adrenoceptor subtypes mediating vasoconstriction
in the carotid circulation of anaesthetized pigs: possible avenues for antimigraine drug development.
Cephalalgia 2001;21:110-9.
11. McCune DF, Edelmann SE, Olges JR, et al. Regulation of the cellular localization and signaling properties of the alpha(1B)- and alpha(1D)-adrenoceptors
by agonists and inverse agonists. Mol Pharmacol
2000;57(4):659-66.
12. Zschauer AO, Sielczak MW, Smith DA, et al. Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation. J Appl Physiol 1997;82(6):1918-25.
13. de Andrade CR, Fukada SY, Olivon VC, et al. Alpha1
D-adrenoceptor-induced relaxation on rat carotid
artery is impaired during the endothelial dysfunction
evoked in the early stages of hyperhomocysteinemia.
Eur J Pharmacol 2006;543(1-3):83-91.
14. Lee MR, Li L, Kitazawa T. Cyclic GMP causes
Ca2+ desensitization in vascular smooth muscle by
activating the myosin light chain phosphatase. J Biol
Chem 1997;272(8):5063-8.
15. Carvajal JA, Germain AM, Huidobro-Toro JP,
et al. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol
2000;184(3):409-20.
16. Lowry JL, Brovkovych V, Zhang Y, et al. Endothelial nitric-oxide synthase activation generates
an inducible nitric-oxide synthase-like output of
nitric oxide in inflamed endothelium. J Biol Chem
2013;288(6):4174-93.
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K. Saracheva et al
Эффект воздействия нового высокоэффективного 5-HT(1B/1D)рецептора лиганда фроватрипана на сонную артерию крысы
Кремена E. Сарачева1,2, Наталия A. Присадова3, Валентин И. Турийски3, Валери И.
Славчев3, Атанас Д. Крастев3, Дамянка П. Гетова4
1
Кафедра фармакологии и клинической фармакологии, Факультет медицины, Медицинский университет, Пловдив,
Болгария
2 Кафедра фармакологии и медицинской токсикологии, Факультет фармации, Медицинский университет, Пловдив,
Болгария
3 Кафедра медицинской физики и биофизики, Факультет фармации, Медицинский университет-Пловдив. Болгария
4 Лаборатория нейрофармакологии, Технологический центр неотложной медицины, Пловдив, Болгария
Адрес для корреспонденции:
Кремена E. Сарачева, Кафедра
фармакологии и клинической
фармакологии, Факультет
медицины, Медицинский
университет - Пловдив, Кафедра
фармакологии и медицинской
токсикологии, Факультет
фармации, Медицинский
университет - Пловдив, бул.
Васил Априлов 15A, 4002
Пловдив, Болгария
E-mail: kremena_saracheva@
yahoo.com
тел: +359 899 778 329
Введение: В кровеносных сосудах 5-HT стимулирует симпатические нервы,
эндотелий и клетки гладких мышц сосудов. Триптаны являются специфическими антимигренозными медикаментами, которые активируют серотонинэргические 5HT1B/1D рецепторы, вызывая вазоконстрикции сосудов головного мозга.
Дата получения: 21 августа
2016
Дата приемки: 18 сентября
2016
Дата онлайн публикации: 29
ноября 2016
Дата публикации: 27 марта
2017
Результаты: Фроватрипан (1×10-6 мол/л - 1×10-5 мол/л) индуцирует сокращение, но в более высоких концентрациях вызывает релаксацию сонной артерии. L-норэпинефриновая сократительная реакция была усилена наличием
фроватрипана. При наличии 5-HT2 рецептора антагониста, метисергида, фроватрипан усиливает проявление релаксации.При наличии специфического
α-1 рецептора антагониста, празосина, фроватрипан-индуцированная релаксация понижается.
Ключевые слова: серотонин,
мигрень, триптаны,
фроваптрипан, сонная артерия
Заключение: Установленный сократительный эффект фроватрипана вероятно связан с основным эффектом препарата – активацией серотонинэргических 5HT1B/1D рецепторов, вызывающих вазоконстрикции сосудов головного мозга и их антимигренозный эффект. В более высоких концентрациях
фроватрипан, вероятнее всего неизвестным неспецифическим механизмом
может активировать следующий интрацеллюлярный каскад реакций: стимуляция α1D может активировать eАОС (эндотелиальную синтазу оксида азота),
которая может повыситься в концентрации АО, что приводит к конечному
эффекту релаксации.
Образец цитирования: Saracheva KE, Prissadova NA, Turiiski
VI, Slavchev VI, Krastev AD, Getova
DP. Effects of the novel high-affinity 5-HT(1B/1D)-receptor ligand
frovatriptan on the rat carotid
artery.
Цель: Оценить эффект воздействия фроватрипана на изолированную сонную артерию крысы.
Методы: Сократительная активность препаратов зарегистрирована в изометрическом режиме. Раствор Кребса (pH = 7.4) использован для отмывания
препаратов гладких мышц (ГМ), аэрированных 95% O2 и 5% CO2 при 37°C.
60-минутная адаптация уровня цвета препаратов взята в качестве контрольного цвета и изменения в виде сокращения и релаксации измерены в соответствии с ним.
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doi: 10.1515/folmed-2017-0006
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