Inhibitory GTP-binding Protein Research Articles

Effects of lithium ion on ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin, islet-activating protein

Studies were made on the effects of Li + on ADP ribosylation of inhibitory GTP-binding (G 1) protein by islet-activating protein (IAP), pertussis toxin. The ADP ribosylation of 40–41 kDa proteins of the membranes of rat cardiac ventricular cells by IAP was reduced by the addition of a nonhydrolyzable analog of guanine nucleotide. GTPγS, indicating that these proteins included G 1 protein. The addition of LiCl (0.5–10 mM) to the membrane fractions of the cells attenuated the ADP ribosylation of the G i protein of the cell membranes by IAP dose-dependently. The effects of LiCl were reversible. Of the monovalent ions tested. Li + inhibited the ADP ribosylation of the protein by IAP most strongly. The effects of LiCl (2 mM) were observed even in the presence of 150 mM KCl. Moreover, LiCl decreased the ADP ribosylation of purified G 1 protein by IAP. These results support that G 1 proteins are one of the targets for the therapeutic effects of lithium.

Biochemical Mechanism of Caffeine Tolerance

Most of the biological actions of caffeine are possibly mediated through its antagonistic effects to adenosine. Adenosine activates an inhibitory GTP-binding protein (Gi). One of the physiological actions of Gi is the inhibition of cAMP formation. Caffeine overcomes this action thus leading to elevation of cAMP. Firing of neurons and the release of neurotransmitters is also inhibited by adenosine. Caffeine overcomes this effect, thus producing increased CNS-activity. During long term administration of caffeine many functions of the organism develop tolerance including cardiovascular and central nervous systems. Present evidence suggests that caffeine tolerance following continuous severe coffee ingestion is the response of the body against caffeine through the upregulation of adenosine receptors.

Involvement of pertussis‐toxin‐sensitive G protein in muscarinic‐receptor‐mediated inhibition of K+‐activated 4‐nitrophenylphosphatase activity of cardiac sarcolemma

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.

Cyclic AMP‐Dependent Induction of Serotonin N‐Acetyltransferase Activity in Photoreceptor‐Enriched Chick Retinal Cell Cultures: Characterization and Inhibition by Dopamine

The activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme in the melatonin biosynthetic pathway, was examined in low-density monolayer cultures of chick embryo retinal cells prepared with three levels of photoreceptor enrichment. In cultures prepared from embryonic day 8 retinas (E8), photoreceptors represented approximately 30% of the total cell population, whereas in those prepared from embryonic day 6 retinas (E6), approximately 70% of the cells were photoreceptors. In E8 retinas treated with kainic acid to destroy neurons (E8K), the relative content of photoreceptors was increased to approximately 50%. NAT activity was detectable in the cultures under all conditions studied, and was markedly increased by drugs that increase intracellular cyclic AMP levels and cyclic AMP-dependent protein kinase activity: 8-bromocyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). Consistent with the hypothesis that NAT is localized in photoreceptors, the effects of the stimulatory treatments were significantly greater in E6 and E8K cultures than in E8 cultures. The stimulation of NAT activity in E6 cultures was inhibited by actinomycin D and cycloheximide, suggesting the involvement of RNA and protein synthesis. Dopamine inhibited the induction of NAT activity by forskolin and IBMX, but not that elicited by 8-bromocyclic AMP. The dopamine-mediated suppression of activity was significantly inhibited by pertussis toxin and by spiperone and sulpiride, both D2-dopamine receptor antagonists, but not by SCH 23390, a D1-dopamine receptor blocker, or antagonists of alpha-adrenergic, beta-adrenergic, or serotonergic receptors. Because the inhibitory effect of dopamine on E6 and E8K cultures was at least as great as that on E8 cultures, the results suggest that dopamine acts on D2-like receptors on photoreceptors. The receptors appear to be coupled to adenylate cyclase through an inhibitory GTP-binding protein and to mediate inhibition of cyclic AMP synthesis and consequent induction of NAT activity.

Phospttorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase inhibits dissociation off the trimer into three subunits of the protein

Phospttorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase inhibits dissociation off the trimer into three subunits of the protein

μ‐Opioid Receptors and Not K‐Opioid Receptors Are Coupled to the Adenylate Cyclase in the Cerebellum

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (greater than 75% mu-opioid receptors) and the guinea pig cerebellum (greater than 80% kappa-opioid receptors). In the mu-preparation, but not in the kappa-preparation, opioids inhibited the basal and the forskolin-stimulated adenylate cyclase activity in a dose-dependent manner and stereospecifically. The inhibition was in the 20-30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a mu-opioid binding site, with the effect being elicited by non-selective (etorphine and morphine) and mu-selective (Tyr-D-Ala-Gly-Me-Phe-Gly-ol) agonists, whereas delta- and kappa-selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.

Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.

We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits.

O-135 - Effects of lithium ion on inhibitory GTP-binding proteins

O-135 - Effects of lithium ion on inhibitory GTP-binding proteins

Activation of protein kinase C alters the interaction of α 2-adrenoceptors and the inhibitory GTP-binding protein (G i) in human platelets

The effect of 12-tetradecanoyl phorbol 13-acetate (TPA) on the hormonal modulation of adenylate cyclase was studied. The effect of epinephrine (α 2-adrenergic action) was markedly diminished in membranes from TPA-treated platelets as compared to the controls. Interestingly, the inhibitory effect of guanylyl imido diphosphate (Gpp(NH)p) was not altered. Neither the number of α 2-adrenoceptors nor their affinity for [ 3H]yohimbine were affected by the treatment with TPA. In control platelets, 77% of the receptors were in a high-affinity state for epinephrine and 22% in a low-affinity state; Gpp(NH)p shifted the receptor affinity towards the low-affinity conformation. In membranes from TPA-treated platelets, the receptors were in the low-affinity state and no further decrease in affinity was induced by Gpp(NH)p. Our data suggest that activation of protein kinase C in platelets blocks the hormonal inhibition of adenylate cyclase by interfering with the receptor-G i interaction.

Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1

The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.

Effects of lithium ex vivo on the GTP-mediated inhibition of calcium-stimulated adenylate cyclase activity in rat brain

The aim of this study was to investigate the effects of chronic lithium treatment on calcium (Ca 2+)-stimulated adenylate cyclase activity in rat striatum and hippocampus, and to elucidate the effect of lithium treatment on the neurotransmitter/GTP-mediated inhibition of Ca 2+-stimulated enzyme activity in the two brain areas. Lithium treatment, which gave a serum-lithium concentration of 0.9 ± 0.16 mmol/l, enhanced Ca 2+-stimulated enzyme activity in the hippocampus but reduced this activity in the striatum. Serotonin (5-HT) dose dependently reduced Ca 2+-stimulated adenylate cyclase activity in the hippocampus, and chronic lithium administration reduced the ability of 1 μM 5-HT to inhibit Ca 2+-stimulated enzyme activity. Furthermore, the 5-HT-induced GTP-mediated inhibition of Ca 2+-stimulated adenylate cyclase activity in the hippocampus was markedly decreased by lithium. Increasing concentrations of dopamine in the striatum did not, however, affect Ca 2+-stimulated adenylate cyclase activity and the inhibition of enzyme activity observed with increasing concentrations of GTP was not influenced by chronic lithium treatment. These results demonstrate that lithium ex vivo exerts dual and region-specific effects on Ca 2+-stimulated adenylate cyclase in the brain. Furthermore, long-term administration of lithium could reduce the inhibitory effect of 5-HT on adenylate cyclase in the hippocampus, by influencing the inhibitory GTP-binding protein. The effects of lithium on serotonergic and dopaminergic neurotransmission could be involved in the therapeutic actions of lithium in manic-depressive illness.

Protein kinase C stimulates adenylate cyclase activity in prolactin‐secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP‐binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.

Mechanisms of hormone-induced desensitization of adenylate cyclase.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

α2‐Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK‐N‐SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein Gi alpha-2 and a 38-kDa phosphorylated protein in the human platelet.

By using antibodies specific for alpha subunits of inhibitory GTP-binding proteins (Gi alpha polypeptides) to probe Western blots of whole platelet protein, we detected Gi alpha-2 as the predominant Gi alpha species present in platelets. The subcellular compartmentalization of distinct Gi alpha-2-immunoreactive polypeptides coupled to thrombin and alpha 2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by highspeed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either alpha-thrombin or epinephrine, we observed that a greater proportion of Gi alpha-2 was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of Gi alpha-2 in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive Gi alpha-2 from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of approximately 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general Gi alpha antiserum AS/7 but not with the Gi alpha-2 antiserum LE/3. These findings suggest a role for specific localization of Gi alpha proteins in epinephrine-induced platelet responses.

Site of Pertussis Toxin-Induced ADP-Ribosylation on Gi Is Critical for Receptor Modulation of GDP Interaction with Gi

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: Possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2- 3H] inositol were permeabilized by ATP 4−, exposed to guanine nucleotides and resealed by Mg 2+. Addition of guanosine 5′-0-(3-thio triphosphate) (GTPγS) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [ 3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5′-0-(2-thiodiphospate) (GDPβS) or by neomycin. Surprisingly, addition of GDPβS (100–600 uM) on its own resulted in a dose-related increase in [ 3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP>GTPγ S>ATP>CTP. The stimulatory effect of UTP, ATP and CTP, but not GTPγS or GDPβS, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTPγS decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.

Pertussis toxin effects on transmitter release from perivascular nerve terminals

Pertussis toxin (PT) was used to investigate the possible involvement of an inhibitory GTP-binding protein (G protein) in neuromuscular transmission at the sympathetic nerve terminal of guinea pig mesenteric artery. When the intracellular microelectrode technique was used, the amount of transmitter released was evaluated by recording the amplitude of excitatory junction potentials (EJPs) in the vascular smooth muscle (VSM) cells. EJPs were evoked by perivascular nerve stimulation with brief square pulses. The amplitude of EJPs was depressed by exogenously applied norepinephrine (NE) (greater than or equal to 10(-7) M) or histamine (greater than or equal to 10(-7) M). However, these effects of NE and histamine on EJP amplitude were almost completely blocked in PT-pretreated VSM cells (2 x 10(-7) g/ml PT, 24 h at 21 degrees C). These effects of PT pretreatment were time and temperature dependent. In contrast, there was no significant change in the resting membrane potential (-70.1 +/- 2.1 mV) or input resistance (11.9 +/- 0.4 M omega) in the VSM cells after PT pretreatment. These results suggest that the effects of NE and histamine on EJP amplitude may be mediated by PT-sensitive G proteins in the presynaptic nerve terminal.

Coupling of inhibitory GTP binding protein to somatostatin receptors on rat cerebrocortical membranes

We studied the interaction between somatostatin receptors and inhibitory GTP binding protein in rat cerebrocortical membranes. Guanine nucleotides reduced [125I-Tyr1] somatostatin binding to cerebrocortical membranes in a dose-dependent manner with rank order of potency being guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) greater than GTP greater than GMP. Maximum reduction of the binding to 32% of control was observed in the presence of 10(-5) M Gpp(NH)p. Scatchard analysis of the labeled somatostatin binding revealed that the decrease in the binding by Gpp(NH)p was due to the decrease in the binding affinity for somatostatin. Divalent cations, such as Mg++, Mn++, and Ca++, caused an increase in labeled somatostatin binding to membranes with the maximum binding observed at a concentration of 10, 10, 1 mM, respectively. However, Na+ decreased a labeled somatostatin binding in a dose-dependent manner, and half maximum inhibition of the binding was observed at 10 mM Na+. Moreover, Gpp(NH)p and Na+ lowered labeled somatostatin binding in an additive fashion. When cerebrocortical membranes were treated at 37 degrees C for 40 min with various concentrations of Islet-Activating-Protein (IAP), which had been preactivated with dithiothreitol, subsequent labeled somatostatin binding to the membranes was decreased in a dose-dependent manner. 30 micrograms/ml IAP treatment caused a decrease in the binding to 50% of control, which was characterized by the decreased binding affinity without a significant change in the binding capacity. Furthermore, exposure of IAP plus NAD to cerebrocortical membranes caused ADP-ribosylation of a membrane protein with Mr = 41,000 on autoradiogram. Such an IAP treatment of cerebrocortical membranes abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. These results suggest that somatostatin receptors in the brain couple to inhibitory GTP binding protein, which mediates adenylate cyclase inhibition by somatostatin.

P-017 - The influences of LiCl on the inhibitory GTP-binding protein

P-017 - The influences of LiCl on the inhibitory GTP-binding protein