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At least 775 records · Page 43Linked to original sources
PubMed1994

Reduced sensitivity to ethanol of Gs alpha and Gi/o alpha in the cerebral cortex of alcoholic patients.

In this study, we examined the quantity and quality of G proteins in membrane preparations from cortical regions (frontal, parietal, temporal and occipital cortex) in post-mortem brains obtained from subjects with chronic alcoholism and controls matched with respect to age and post-mortem delay (PMDT). Immunoblot analysis showed that the level of GsH alpha (52 kDa) was significantly decreased in temporal cortical membranes from alcoholics compared with controls, while no differences were observed in the amount of any G protein in frontal, parietal and occipital regions. Additionally, ethanol enhanced photoaffinity guanine nucleotide binding to both G alpha and Gi/o alpha in human cortical membranes. The percentage of increase in ethanol-stimulated photoaffinity GTP labeling of Gs alpha and Gi/o alpha was decreased in alcoholic patients in all cortical regions. These observations indicated that subsensitivities to ethanol-induced stimulation in G protein may contribute to impaired trans-signaling in cortical membranes of chronic alcoholics.

Alcoholism↗
PubMed1997

Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor.

Cannabinoids act at the CB1 receptor to inhibit adenylate cyclase activity via a pertussis toxin-sensitive G-protein. Within the striatum, CB1 receptors have been shown to be localized on the same neurons as Gi-coupled dopamine D2 receptors. In this study we have examined the interactions of CB1 and D2 receptors on adenylate cyclase. In striatal neurons in primary culture, both the CB1 receptor agonist [3-(1, 1-dimethylheptyl)-11-hydroxy-Delta8tetrahydrocannabinol] (HU210) and the D2 receptor agonist quinpirole inhibited forskolin-stimulated cAMP accumulation when applied separately. In contrast, HU210 and quinpirole in combination augmented cAMP accumulation. This augmentation was blocked by the CB1 receptor antagonist SR141716A or the D2 antagonist sulpride. Pertussis toxin treatment of striatal neurons prevented the inhibition of cAMP accumulation by D2 receptors but unmasked a cannabinoid receptor-mediated stimulatory effect on cAMP accumulation. The cannabinoid receptor-stimulated accumulation of cAMP was blocked in a concentration-dependent manner by SR141716A, suggesting that the response was regulated through the CB1 receptor. Similar augmentation of cAMP accumulation after pertussis toxin treatment was observed in Chinese hamster ovary (CHO) cells transfected with, and stably expressing, the CB1 receptor. This stimulation of cAMP was not Ca2+-sensitive and was unaffected by a range of protein kinase inhibitors. Treatment of the pertussis toxin-treated cells with cholera toxin before CB1 receptor activation amplified the stimulatory pathway, suggesting that this response was mediated through a Gs-type G-protein. Stimulation of cAMP accumulation was not observed after pertussis toxin treatment of CHO cells expressing the human CB2 receptor, suggesting that this novel signaling pathway is unique to the cannabinoid CB1 receptor.

Animals↗
PubMed2004

Changes in PKA activity and Gs alpha and Golf alpha levels after amphetamine- and cocaine-induced behavioral sensitization.

Neuroadaptations in the cAMP signal transduction system have been proposed to play a critical role in psychostimulant-induced behavioral sensitization. Studies relevant to this hypothesis have provided conflicting evidence, however, because repeated cocaine and amphetamine treatment has been alternately reported to either increase or decrease protein kinase A (PKA) activity in the nucleus accumbens and dorsal striatum. In an attempt to reconcile these disparate findings, PKA activity and the stimulatory G-protein subunits, G(salpha) and G(olfalpha), were measured after rats had received a sensitization-inducing regimen of amphetamine or cocaine. Results showed that seven consecutive daily injections of amphetamine or cocaine produced locomotor sensitization and caused a reduction in accumbal PKA activity. A challenge injection of amphetamine on the test day also reduced dorsal striatal PKA activity. G(salpha) levels were unaffected by psychostimulant exposure, while G(olfalpha) levels in the nucleus accumbens declined significantly after amphetamine or cocaine pretreatment. Because repeated amphetamine and cocaine treatment both induced behavioral sensitization and decreased accumbal PKA activity, it appears that psychostimulant-induced increases in PKA activity are not necessary for the ultimate expression of behavioral sensitization.

Amphetamine↗
PubMed2002

Identification and properties of a Gs protein in catfish liver membranes.

The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5'-O-(gamma-thiotriphosphate) (GTPgammaS) (maximal activation of about eightfold at 10(-5) M; half-maximal activation at 1.31 x 10(-7) M), and reduced by the competitive inhibitor of GTP, GDPbetaS (70% maximal inhibition at 10(-4) M; half-maximal inhibition at 1.98 x 10(-7) M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 microM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [(35)S]GTPgammaS binding assay. At 100 microM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20 degrees, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPgammaS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 microM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a G(s)alpha-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-G(s)alpha antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein alpha(s) subunits in the liver of an ectothermal vertebrate.

Adenylyl Cyclases↗
PubMed2003

Evidence for a significant role of a Gs-triggered mechanism unrelated to the activation of adenylyl cyclase in the cyclic AMP-independent relaxant response of guinea-pig tracheal smooth muscle.

Cyclic AMP is a key molecule in the regulation of airway smooth muscle tone. Increased cyclic AMP leads to relaxation of this smooth muscle and its inhibition results in the muscle contraction. A constitutive role for cyclic AMP in the contraction and relaxation of airway muscle is supported by the observations that direct activators of adenylyl cyclase, such as forskolin and membrane-permeable cyclic AMP analogues, relax this smooth muscle potently. This traditional view of the role for cyclic AMP is the basis for the idea that relaxation of airway smooth muscle mediated through adenylyl cyclase-linked, G(s)-coupled receptors, including the beta(2)-adrenoceptor, is achieved mainly by the elevation of cyclic AMP content [cyclic AMP-dependent mechanism(s)]. However, recent pharmacological and biochemical evidence raises a fundamental question concerning the role of cyclic AMP; can G(s)-coupled receptor-mediated relaxation of tracheal smooth muscle be attributed exclusively to cyclic AMP-dependent mechanism(s)? In the present study, we show that cholera toxin (CTX, 5 microg/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein G(s), relaxes guinea-pig tracheal smooth muscle. CTX also elevates tissue cyclic AMP content by about 30-fold and this is practically abolished by an adenylyl cyclase inhibitor, SQ 22,536 (100 microM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of G(s) is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. G(s)-triggered, cyclic AMP-unrelated cellular mechanism(s) seem(s) to play a substantial role in smooth muscle relaxation mediated through adenylyl cyclase-linked receptors. This mechanism may account in part for the cyclic AMP-independent relaxant response of tracheal smooth muscle.

Adenylyl Cyclases↗
PubMed2000

Differential gene transcriptional regulation of Gi isoforms and Gs protein expression in diabetic rat hearts.

Many cardiac diseases can be associated with alterations in the function and quantity of G proteins. We examined the gene expressions and protein levels of Gi-1alpha, Gi-2alpha, Gi-3alpha and G(s alpha) in ventricular myocardial preparations from rats 4-6 weeks after induction of diabetes with streptozotocin in comparison with those from age-matched control rats. Diabetic rat myocardium exhibited reductions in the protein levels of Gi-2alpha and Gi-3alpha by 22+/-2% and 57+/-2%, respectively. In diabetes, 22% and 53% reductions in myocardial mRNA levels of Gi-2alpha and Gi-3alpha were observed. Although a faint protein signal of Gi-1alpha was detectable, no apparent expression of mRNA for Gi-1alpha was found in either control or diabetic myocardium. The reduced protein and mRNA levels of Gi-2alpha and Gi-3alpha were prevented by insulin therapy. No change was found in the protein and mRNA levels of G(s alpha) in diabetic myocardium. In conclusion, diabetes leads to a differential regulation of protein expressions of G(i alpha) isoforms and G(s alpha) in ventricular myocardium. The reduced expression of Gi-2alpha and Gi-3alpha proteins can be explained, at least in part, by the decreases in the transcriptional levels.

Animals↗
PubMed2005

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein.

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.

Amino Acid Sequence↗
PubMed2004

Gene expression profile of the Gs-coupled prostacyclin receptor in human vascular smooth muscle cells.

Migration and proliferation of medial smooth muscle cells (SMC) in the arterial intima contributes to the development of atherosclerotic plaques and restenotic processes after coronary angioplasty. Prostacyclin (PGI2)-mediated stimulation of cyclic adenosine 3'5'-monophosphate (cAMP) signaling is believed to be important for maintaining SMC in a quiescent state. In order to identify new cellular targets of PGI2/cAMP action, we have used microarray screening to examine changes in the transcriptional profile in human vascular SMC in response to exposure to the stable PGI2 mimetic iloprost. We have identified 83 genes with significantly altered expression after iloprost (100 nM) exposure for 6 hr. Fifty-one genes were upregulated, among them stanniocalcin precursor (18.8+/-2.7), zinc finger transcription factor (7.8+/-2.0), hyaluronan synthase 2 (6.8+/-1.8), cyclooxygenase 2 (4.7+/-0.8), dual specific phosphatase (3.9+/-0.5) and vascular endothelial growth factor (2.3+/-0.4). Thirty-two genes were reduced, among them cystein-rich angiogenic protein (-14.9+/-1.3), monocyte chemotactic protein 1 (-7.4+/-1.1) and plasminogen activator inhibitor PAI-1 (-4.5+/-0.5). By means of semi-quantitative RT-PCR, time-courses of gene expression were established. The present study identified genes not hitherto recognized to be targets of PGI2 action, providing further insight into its cAMP-mediated effects on SMC growth, migration and matrix secretion.

Arteriosclerosis↗
PubMed2006

Mutations in the Gs alpha gene causing hormone resistance.

G-protein-coupled receptors (GPCRs) and G proteins mediate the effects of a number of hormones of relevance to endocrinology. Genes encoding these molecules may be targets of loss- or gain-of-function mutations, resulting in endocrine disorders. The only mutational change of G proteins so far unequivocally associated with endocrine disorders occurs in the Gsalpha gene (GNAS1, guanine nucleotide binding protein alpha stimulating activity polypeptide 1), which activates cyclic AMP (cAMP)-dependent pathways. Heterozygous loss-of-function mutations of GNAS1 in the active maternal allele cause resistance to hormones acting through Gsalpha-coupled GPCRs, whereas somatic gain-of-function mutations cause proliferation of endocrine cells recognizing cAMP as mitogen. This review will focus on inactivating mutations leading to hormone resistance syndromes, i.e., pseudohypoparathyroidism types Ia and Ib.

Chromogranins↗
PubMed2003

High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels.

Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca(2+) channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca(2+) which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G(s), G(i) and G(q)-linked receptors in the same cell population.

Animals↗
PubMed2004

Characterisation of Gs activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine.

Herein, we examined the direct coupling of human dopamine D1 receptors to G(s) proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists ( approximately 100%) in stimulating guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding to G(s) in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTP gamma S binding to G(s) with a pK(B) (9.29) close to its pK(i) (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G(s) activation with pK(B)'s of 6.7 and 7.3, respectively, values close to their pK(i) values at these sites. In conclusion, the use of an anti-G(s) protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.

Animals↗
PubMed1998

Loss of activation of Gs but not Gi following expression of an alpha2A-adrenoceptor-Gi1alpha fusion protein.

Both the porcine alpha2A-adrenoceptor and a fusion protein between this receptor and a pertussis toxin-resistant form of Gila were stably expressed in Rat-1 fibroblasts. The agonist UK14304 mediated a biphasic regulation of adenylyl cyclase activity via the isolated receptor with inhibition of the enzyme activity at low concentrations of the compound which was subsequently reversed at higher concentrations. By contrast, stimulation of the fusion protein with this agonist could only produce inhibition of enzyme activity. This inhibition was produced by activation of endogenous Gi rather than the fused alpha subunit of Gi1, as pertussis toxin treatment obliterated inhibitory regulation of adenylyl cyclase via the fusion construct. Pertussis toxin treatment potentiated stimulation of adenylyl cyclase via the isolated receptor but such treatment was unable to uncover capacity of the fusion protein to produce such an effect.

Adenylate Cyclase Toxin↗