Research Interests: Kinetics, Aging, Neurochemistry, Calcium, cyclic AMP, and 8 moreAnimals, Spinal Cord, Sensory Neuron, Capsaicin, Rats, Chickens, Neurosciences, and cyclic GMP
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We investigated activation mechanisms of hepatic stellate cells (HSCs) that are known to play pivotal roles in the regeneration process after 70% partial hepatectomy (PHx). Parenchymal liver cells (PLCs) and non-parenchymal cells (NPLCs)... more
We investigated activation mechanisms of hepatic stellate cells (HSCs) that are known to play pivotal roles in the regeneration process after 70% partial hepatectomy (PHx). Parenchymal liver cells (PLCs) and non-parenchymal cells (NPLCs) were isolated and purified from the regenerating livers at 1, 3, 7, 14 days after PHx. Each liver cell fraction was stained by immunocytochemistry using an anti-desmin antibody as a marker for HSCs, anti-alpha-smooth muscle actin (alpha-SMA) as a marker for activated HSCs, and 5-bromo-2'-deoxyuridine (BrdU) for detection of proliferating cells. Tissue sections from regenerating livers were also analyzed by immunohistochemistry and compared with the results obtained for isolated cell fractions. One and 3 days after PHx, PLC-enriched fraction contained HSCs adhered to PLCs. The HSCs adhered to PLCs were double positive for BrdU and alpha-SMA, and formed clusters suggesting that these HSCs were activated. However, HSC-enriched fraction contained HS...
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Research Interests: Neurochemistry, Regulation, Cell Culture, Cell Differentiation, Glioma, and 10 morecyclic AMP, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Differentiation, Neuroblastoma, Hybrid Solar Cells, Two-Dimensional Gel Electrophoresis, G protein, Neurosciences, Gtp Binding Proteins, and Isomerism
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Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg... more
Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg of membrane protein and displayed a dissociation constant for this ligand of 30-40 nM. Prolonged exposure of these cells either to PGE1 or to iloprost, which is a stable analogue of prostacyclin, caused a 40-70% decrease in levels of the receptor. The remaining receptors were capable of interacting with the stimulatory G-protein (Gs) of the adenylate cyclase cascade, as saturation analysis of the binding of [3H]PGE1 indicated that they had a similar affinity for the 3H-labelled ligand, and because the specific binding of [3H]PGE1 to these receptors was still sensitive to the presence of poorly hydrolysed analogues of GTP. We have recently demonstrated that prolonged exposure of NG108-15 ells to PGE1 causes a cyclic AMP-independent loss of Gs alpha-s...
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Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane... more
Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but...
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Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like... more
Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased....
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Individual G-proteins are highly similar in primary sequence. It is thus pertinent to ask what degree of specificity of interaction each of these display with the various receptors and effector systems. Many of the identified G-proteins... more
Individual G-proteins are highly similar in primary sequence. It is thus pertinent to ask what degree of specificity of interaction each of these display with the various receptors and effector systems. Many of the identified G-proteins are co-expressed in a single tissue or cell. As the extreme C-terminus of the alpha-subunit of each G-protein appears to be a key domain for the interactions of receptors and G-proteins, we have generated a series of G-protein-selective anti-peptide antisera against this region and then have used these antisera to attempt to interfere with receptor-G-protein coupling. With this approach, we have demonstrated that delta-opioid receptor-mediated inhibition of adenylate cyclase in neuroblastoma x glioma (NG108-15) cell membranes is transduced specifically by Gi2 and in the same cell that alpha 2-adrenergic inhibition of Ca2+ currents is transduced by G0.
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Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this... more
Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS-PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gs alpha.
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Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta... more
Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively acti...
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Nine distinct alpha subunits of guanine nucleotide binding proteins (G-proteins) have now been identified by cDNA cloning. Each of these functions to allow transduction of information between hormone-activated receptors in the plasma... more
Nine distinct alpha subunits of guanine nucleotide binding proteins (G-proteins) have now been identified by cDNA cloning. Each of these functions to allow transduction of information between hormone-activated receptors in the plasma membrane and effector systems which are either ion channels or enzymes which regulate the intracellular concentration of second messengers. As the individual G-proteins are highly similar in primary sequence, it is pertinent to ask what degree of specificity of interaction each of these display with the various receptors and effector systems. Specificity of tissue location defines that the rod and cone transducins (TD1 and TD2, respectively) act as the coupling proteins between rhodopsin and cone opsins and their cyclic nucleotide phosphodiesterase effectors and that G(olf) is the G-protein which tranduces signals from odorant receptors to adenylate cyclase in olfactory sensory neurones. However, many of the other identified G-proteins are co-expressed ...
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Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, G...more
A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of... more
A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeli...
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The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify... more
The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved on receptor activation by ligand. In addition, these methods can be used to probe the specificity of the interaction and to yield information about the stoichiometries involved.
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1. Int J Biochem. 1990;22(7):701-7. Guanine nucleotide binding proteins in neuroblastoma x glioma hybrid, NG108-15, cells. Regulation of expression and function. Milligan G, McKenzie FR, McClue SJ, Mitchell FM, Mullaney I. ...
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ABSTRACT Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human ß2 adrenoceptor cDNA under the control of the ß actin promoter.... more
ABSTRACT Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human ß2 adrenoceptor cDNA under the control of the ß actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (ßN22) but not one expressing only low levels of the receptor (ßN17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg2+ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn2+. The elevated basal activity was partially reversed by addition of the ß-adrenoceptor antagonist propranolol. Agonist activation of ßN22 but not ßN17 cells led to a large selective down-regulation of cellular Gsa levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [3H] forskolin was achieved with substantially greater potency (some 30 fold) in ßN22 cell membranes than in ßN17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [3H] forskolin binding which was equipotent in each of ßN22, ßN17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of Gsa down-regulation in each cell type. Expression of an epitope tagged variant of Gsa in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type Gsa. Isolation of clones of NCB20 cells expressing high levels of the ß2 adrenoceptor also resulted in a specific agonist-induced down-regulation of Gsa.
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ABSTRACT It is now appreciated both that G-protein-linked receptors and signal transducing heterotrimeric G-proteins consist of large multi-member superfamilies and that regulation of a signal transduction cascade can be produced by a... more
ABSTRACT It is now appreciated both that G-protein-linked receptors and signal transducing heterotrimeric G-proteins consist of large multi-member superfamilies and that regulation of a signal transduction cascade can be produced by a variety of means following activation of a G-protein by a receptor. To begin to unravel the complexities of this regulation it is clearly important to be able to define the molecular identity of the G-protein or G-proteins activated by a receptor and to assess the quantitative importance of such interactions for the integration of signals produced by a receptor agonist. Substantial progress has been made towards these goals in recent years and the purpose of this short review will be to discuss the use and potential limitations of some of the currently most widely used approaches.
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ABSTRACT : Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2-adrenoceptor under the control of a β-actin promoter. Two clones were selected for detailed analysis:... more
ABSTRACT : Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2-adrenoceptor under the control of a β-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose-response curves to isoprenaline in clone D1 indicated that half-maximal down-regulation of Gsα was produced by ∼1 nM agonist. Measurement of Gs mRNA levels in both clones, however, using both reverse transcriptase-polymerase chain reaction and northern blotting revealed no significant change following treatment with isoprenaline.