Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.     

Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G-protein

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1-3 nM yielding half-maximal stimulation. Binding of 3H-bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP-gamma-S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G-protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.     

Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins

We studied the consequences of infection of L6E9 myoblasts with T. cruzi on the adenylate cyclase complex to test the hypothesis that infection alters the functional properties of the guanine nucleotide regulatory proteins, Ns and Ni. Stimulating activities of adenylate cyclase due to isoproterenol, isoproterenol plus Gpp(NH)p, or forskolin (activities mediated by Ns) are not altered by infection. However, inhibitory activities mediated by Ni [Gpp(NH)p, acetylcholine, and adenosine inhibition of forskolin-dependent adenylate cyclase activity] are compromised by infection. The reduction in adenosine's inhibition of forskolin-dependent adenylate cyclase activity is seen throughout the effective concentration range of adenosine. Pertussis toxin does not change basal or stimulated adenylate cyclase activity in infected cells compared with normal uninfected cells, nor does it alter the inhibiting action of adenosine. To evaluate the coupling proteins (Ns and Ni) involved in the stimulation and inhibition of adenylate cyclase more directly, cholera- and pertussis-toxin-dependent ADP ribosylation studies were performed. The incorporation of [32P]ADP ribose in the presence (specific) or absence (nonspecific) of the toxins was markedly decreased in membranes prepared from infected cells. However, in membranes prepared from infected or uninfected cells previously treated with pertussis toxin, there was a significant reduction in specific pertussis-toxin dependent ADP ribosylation. The infection-associated diminution in toxin-dependent ADP ribosylation complements the impaired inhibition of adenylate cyclase data. Collectively, the data further substantiate an infection-associated alteration in the adenylate cyclase complex, probably at the level of the guanine nucleotide binding proteins.     

Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells.

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.     

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

Cyclic AMP concentrations modulate both calcium flux and hydrolysis of phosphatidylinositol phosphates in mouse T lymphocytes

Activation of T cells by lectins or mAb directed at components of the Ag-specific TCR results in hydrolysis of phosphorylated derivatives of phosphatidylinositol and an increase in intracellular free calcium concentration (Cai). We report that cholera toxin, which activates adenylate cyclase by ADP ribosylation of a G protein, also reduces both inositol phosphate (IP) production and the rise in Cai in Con A-stimulated murine T cells. We find that similar dose-dependent inhibitory effects can be induced by each of four other agents that raise cAMP levels in such cells: forskolin, PGE2, 2-chloroadenosine, and isoproterenol. The effects of these agents on IP production are reversible and therefore do not simply reflect cytotoxicity. Activation by PHA and by antibody to the T3-epsilon-chain of the TCR complex are also inhibited by agents that increase intracellular cAMP. Thus, changes in cAMP concentration seem to regulate both IP production and the Ca2+ response, two early components of the mitogen-induced activation process.     

G protein coupling of antigen receptor-stimulated polyphosphoinositide hydrolysis in B cells

Cross-linking of the IgM and IgD Ag-R on mature B lymphocytes provokes the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. We show here that in permeabilized, [3H]inositol-labeled mouse B cells the nonhydrolyzable GTP analogue GTP gamma S induces release of inositol phosphates, including inositol trisphosphate. The response is markedly augmented by the addition of polyclonal anti-Ig or anti-mu or anti-delta mAb. Inositol phosphate release provoked in intact B cells by any of the anti-receptor antibodies was not inhibited by pertussis toxin and only partially inhibited by cholera toxin. The results therefore indicate that both IgM- and IgD-R on B cells are coupled to the polyphosphoinositide-specific phosphodiesterase by one or more G proteins, which have yet to be identified.     

Thyrotropin-releasing hormone stimulation of polyphosphoinositide hydrolysis in GH3 cell membranes is GTP dependent but insensitive to cholera or pertussis toxin

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).     

Inhibition by pertussis toxin of guanyl nucleotides exchange on transducin in bovine rod cell membranes

The effect of pertussis toxin on GTP-binding protein of bovine rod cell outer segments (transducin) was studied. Pertussis toxin was shown to ADP ribosylate either alpha subunit of free transducin or transducin-GDP complex, whereas GTP and its analogue Gpp(NH)p strongly inhibit ADP ribosylation of transducin. Pertussis toxin inhibits rod outer segment membrane GTPase and GTPase of homogeneous transducin by 40% and 70-80%, respectively. Activation of rod cell cyclic nucleotide phosphodiesterase by transducin is reduced after its preincubation with pertussis toxin. In transducin modified by pertussis toxin, 83% of GDP becomes tightly bound and cannot be exchanged with Gpp(NH)p. The stabilization of complex transducin-GDP after ADP ribosylation can explain the inhibitory effect of pertussis toxin on GTP hydrolysis by transducin, and on phosphodiesterase activation by guanyl nucleotides.     

Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

Cyclic AMP can partially restore platelet-derived growth factor-stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ-ras-transformed NIH-3T3 cells

NIH-3T3 cells transformed by the EJ-ras oncogene display reduced platelet-derived growth factor (PDGF)-stimulated phospholipase C activity as measured by inositol 1,4,5-triphosphate (IP3) synthesis and Ca2+ mobilization. The lack of PDGF-stimulated Ca2+ mobilization in EJ-ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ-ras transformed cells with cholera toxin or 8-bromo-cyclic AMP, but not pertussis toxin or the beta-subunit of cholera toxin, results in a slight recovery of PDGF-stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21-mediated inhibition of PDGF-stimulated intracellular events can be partially and transiently reversed by cyclic AMP.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems.

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.     

Evidence for coupling of bradykinin receptors to a guanine-nucleotide binding protein to stimulate arachidonate liberation in the osteoblast-like cell line, MC3T3-E1.

The effect of bradykinin on the activation production of inositol 1,4,5-trisphosphate and prostaglandin E2 (PGE2) was examined in the murine osteoblastic cell line, MC3T3-E1. Bradykinin, at concentrations ranging from 1 to 1000 nM, stimulated the production of inositol 1,4,5-trisphosphate 2.5- to 3-fold within 10 s, and elevated cytosolic-free Ca2+, even in the absence of external Ca2+. This process is mediated through the activation of phospholipase C. Bradykinin at the same concentration also stimulated the production of PGE2 and caused a release of 3H radioactivity from the cells prelabeled with [3H]arachidonic acid, probably via the activation of phospholipase A2. Pretreatment of the cells with pertussis toxin inhibited the stimulation of PGE2 production and 3H radioactivity release, while the elevation in cytosolic Ca2+ and the production of inositol 1,4,5-trisphosphate were not altered by toxin-pretreatment. The addition of an unhydrolyzable analog of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid enhanced the release of 3H radioactivity. The simultaneous presence of bradykinin with GTP gamma S further activated the 3H radioactivity release in the beta-escin-permeabilized cells. These results provide evidence that receptors for bradykinin in the MC3T3-E1 couple stimulating arachidonate release, probably via the activation of phospholipase A2, through a guanine nucleotide binding protein sensitive to pertussis toxin.     

Receptor coupled events in bradykinin action: rapid production of inositol phosphates and regulation of cytosolic free Ca2+ in a neural cell line.

The addition of bradykinin to NG115-401L cells grown on coverslips results in the generation of rapid transient increases in intracellular [Ca2+] and inositol phosphates. Changes in intracellular Ca2+, measured using the fluorescent indicator dye Fura-2, show two components; an initial rapid peak in [Ca2+]i which is essentially independent of extracellular Ca2+, and a sustained plateau dependent on the presence of extracellular Ca2+. Analysis of bradykinin stimulated production of [3H]inositol phosphates, by h.p.l.c., shows a rapid biphasic production of inositol 1,4,5-trisphosphate, inositol tetrakisphosphate and inositol bisphosphates, followed by a sustained rise in inositol 1,3,4-trisphosphate production. Quantitative measurements have indicated the presence of other, more polar, [3H]inositol-labelled metabolites which do not show major changes on bradykinin stimulation. The initial phase of inositol phosphate production parallels the rapid transient increase in intracellular [Ca2+], however, the second phase of inositol phosphate production occurs when intracellular [Ca2+] is declining and implies a complex series of regulatory events following receptor stimulation. Similar time courses of inositol 1,4,5-trisphosphate and Ca2+ signals provides supporting evidence that inositol 1,4,5-trisphosphate is the second messenger coupling bradykinin receptor stimulation to release of Ca2+ from intracellular stores.     

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.     

The effect of cholera toxin on the inhibition of vasopressin-stimulated inositol phospholipid hydrolysis is a cyclic AMP-mediated event at the level of receptor binding.

Incubation of L6 skeletal myoblasts for 16 h with cholera toxin but not with pertussis toxin, led to the inhibition of inositol phosphate generation induced by subsequent exposure to vasopressin. The effects of the toxin on inositol lipid metabolism were accompanied by the total ADP-ribosylation of the available cholera-toxin substrates within the cells. Immunological analysis demonstrated that the two polypeptides modified in vivo by cholera toxin were different forms of Gs alpha (alpha subunit of Gs). No novel cholera-toxin substrate(s) were detected. The cholera-toxin-mediated inhibition of vasopressin-stimulated inositol phosphate generation could be mimicked by both forskolin and dibutyryl cyclic AMP, but not by the separated subunits of the toxin. Receptor-binding studies demonstrated that the inhibition of agonist-stimulated inositol phosphate generation was accompanied by a decrease in cell-surface vasopressin-binding sites, with no effect on the affinity of these for the hormone. We suggest that the effect of cholera toxin and agents which increase intracellular cyclic AMP on vasopressin-stimulated inositol lipid hydrolysis is an effect on receptor number, and that there is no requirement to postulate a role for a novel G-protein, which is a substrate for cholera toxin, in the regulation of inositol phospholipid metabolism.     

Ca2+ ionophore A23187 and thrombin inhibit the pertussis-toxin-induced ADP-ribosylation of the alpha-subunit of the inhibitory guanine-nucleotide-binding protein and other proteins in human platelets

Inhibitory guanine-nucleotide-binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis-toxin-dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor-mediated activation of Gi proteins, leading to their subsequent dissociation into alpha i and beta/gamma subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis-toxin-dependent ADP ribosylation of the alpha subunit of Gi (Gi alpha) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca(2+)-ionophore A23187 or thrombin, but not phorbol 12,13-dibutyrate, decreased the subsequent pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponin-permeabilized platelets in a time-dependent and dose-dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis-toxin-dependent ADP ribosylation of Gi alpha in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis-toxin-dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP-ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Gi alpha, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis-toxin-dependent ADP ribosylation of Gi alpha in saponized platelets; the decrease of pertussis-toxin-dependent ADP ribosylation of Gi alpha after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis-toxin-dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to G1 alpha and involves other proteins. We conclude that the decrease in pertussis-toxin-dependent ADP ribosylation of Gi in thrombin-stimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP-ribosylating activity of pertussis toxin.     

Astrocytes possess prostaglandin F2 alpha receptors coupled to phospholipase C.

We examined the effect of prostaglandin (PG) F2 alpha on phosphoinositide (PI) hydrolysis in rat cultured astrocytes. PGF2 alpha stimulated the formation of [3H]inositol phosphates in [3H]inositol-labeled astrocytes with the ED50 value of 23 nM, whereas PGD2 and PGE2 were much less effective than PGF2 alpha. Transformation of astrocytes was accompanied by an increase in the stimulatory response of PGF2 alpha. Pretreatment of the astrocytes with pertussis toxin and cholera toxin did not affect the PGF2 alpha-evoked PI hydrolysis. In the digitonin-permeabilized astrocytes, PGF2 alpha significantly enhanced the GTP gamma S-evoked PI hydrolysis in the presence of Ca2+. These results indicate that rat cultured astrocytes possess PGF2 alpha receptors coupled to phospholipase C.