Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C.

Previous studies in Chinese-hamster fibroblasts (CCL39 line) indicate that an important signalling pathway involved in thrombin's mitogenicity is the activation of a phosphoinositide-specific phospholipase C, mediated by a pertussis-toxin-sensitive GTP-binding protein (Gp). The present studies examine the effects of thrombin on the adenylate cyclase system and the interactions between the two signal transduction pathways. We report that thrombin exerts two opposite effects on cyclic AMP accumulation stimulated by cholera toxin, forskolin or prostaglandin E1. (1) Low thrombin concentrations (below 0.1 nM) decrease cyclic AMP formation. A similar inhibition is induced by A1F4-, and both thrombin- and A1F4- -induced inhibitions are abolished by pertussis toxin. (2) Increasing thrombin concentration from 0.1 to 10 nM results in a progressive suppression of adenylate cyclase inhibition and in a marked enhancement of cyclic AMP formation in pertussis-toxin-treated cells. A similar stimulation is induced by an active phorbol ester, and thrombin-induced potentiation of adenylate cyclase is suppressed by down-regulation of protein kinase C. Therefore, we conclude that (1) the inhibitory effect of thrombin on adenylate cyclase is the direct consequence of the activation of a pertussis-toxin-sensitive inhibitory GTP-binding protein (Gi) possibly identical with Gp, and (2) the potentiating effect of thrombin on cyclic AMP formation is due to stimulation of protein kinase C, as an indirect consequence of Gp activation. Our results suggest that the target of protein kinase C is an element of the adenylate cyclase-stimulatory GTP-binding protein (Gs) complex. At low thrombin concentrations, activation of phospholipase C is greatly attenuated by increased cyclic AMP, leading to predominance of the Gi-mediated inhibition.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Studies on the mechanisms of signalling and inhibition by pertussis toxin of fibroblast growth factor-stimulated mitogenesis in Balb/c 3T3 cells.

Basic fibroblast growth factor (bFGF) stimulates mitogenesis of Balb/c 3T3 fibroblast cells. This stimulation may be mediated by multiple signal pathways as it is accompanied by the formation of inositol phosphates, activation of PKC (protein kinase C) and a decrease in intracellular cAMP levels. The multiple positive and negative pathways implicated for FGF-induced mitogenesis may interact and each may contribute in varying degrees to the final cellular response. At least two types of G-proteins may be involved in the intracellular signalling pathways of FGF. Pertussis toxin blocks FGF and TPA (12-O-tetradecanoylphorbol-13-acetate) induced. PKC-mediated mitogenesis and also the associated fall in intracellular cAMP levels. However, pertussis toxin has no effect upon FGF-induced inositol phosphates formation. Thus, inhibition of mitogenesis by pertussis toxin may involve pertussis toxin sensitive G-proteins which may affect at least two separate putative signal pathways involving adenylate cyclase and protein kinase C. Pertussis toxin insensitive G-proteins may also be involved in coupling the FGF receptor to phosphoinositidase C.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

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.     

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.     

Tyrosine kinase-activating growth factors potentiate thrombin- and AIF4- -induced phosphoinositide breakdown in hamster fibroblasts. Evidence for positive cross-talk between the two mitogenic signaling pathways

Basic fibroblast growth factor (FGF) and alpha-thrombin can stimulate DNA synthesis in Chinese hamster fibroblasts (CCL39) by two separate signaling pathways (Chambard, J.C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987) Nature 326, 800-803) but can also act synergistically. We have examined whether this synergism might depend upon changes in inositol lipid metabolism. Indeed, FGF, which has no effect on its own on phosphoinositide hydrolysis, potentiates (by up to 2-fold) thrombin-induced formation of inositol phosphates. This enhancing effect is also observed upon direct activation by AIF4- of the GTP-binding protein coupled to phospholipase C, and is best revealed when phospholipase C is weakly stimulated. With low thrombin concentrations or with AIF4-, the formation of inositol phosphates is immediately increased with a marked reduction of the initial lag, whereas at high thrombin concentrations, the stimulation by FGF becomes pronounced only after desensitization of phospholipase C to thrombin. FGF-induced potentiation is not mimicked by calcium ionophores, but is likewise elicited by epidermal growth factor, platelet-derived growth factor, and to a lesser extent by insulin, other growth factors known to activate receptor tyrosine kinases. We therefore propose that the tyrosine kinase-activating growth factors enhance the coupling between GTP-binding protein and phospholipase C, presumably through the phosphorylation of one of these two proteins. Treatment of cells with pertussis toxin attenuates thrombin-induced phospholipase C activity but does not impede the potentiation by FGF. Comparison of the potentiating effects of FGF on inositol phosphate formation and on DNA synthesis suggests than an increased production of second messengers by the inositol lipid pathway in the first hours of stimulation might be, at least in part, responsible for the synergistic actions of FGF and thrombin on DNA synthesis.     

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.     

Inhibitory effects of pertussis toxin on a depolarization-evoked Ca2+ influx in NG108-15 cells

Depolarized stimulation 1.5-fold increased Ca2+ influx which was inhibited by pretreatment with verapamil or LaCl3. Treatment with pertussis toxin, islet-activating protein (IAP), induced a reduction in 50 mM K+-induced Ca2+ influx and stimulated adenylate cyclase (AC) activity in NG108-15 cells. However, addition of dibutyryl cAMP or forskolin treatment elevating cAMP level exerted no effects on a depolarization-induced Ca2+ influx. Dissociated B-oligomer of IAP after treatment with dithiothreitol and ATP increased a depolarization-evoked Ca2+ influx. It is suggested that inhibitory GTP-binding protein (G1) or other IAP substrate proteins could directly be involved in Ca2+ influx via voltage-sensitive Ca2+ channel.     

Enhancement of differentiation of rat adipocyte precursor cells by pertussis toxin

To examine whether GTP-binding protein(s) is (are) involved in adipocyte differentiation, the effect of pertussis toxin (PT) was studied in rat adipocyte precursor cell culture. PT potentiated adipose conversion induced by dexamethasone, insulin, and 1-methyl-3-isobutylxanthine in a dose- and time-dependent fashion. Attenuation of an inhibitory control of adenylate cyclase was not the mechanism of action of PT. The dose-dependent inhibition of PT-catalyzed ADP-ribosylation of the Mr 40,000 protein of the cell membrane by preincubation of the toxin was inversely related to the potentiating effect on differentiation. PT-sensitive G protein(s) may be involved in adipocyte differentiation in a negative fashion.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Withdrawal of GTP-dependent inhibition

12-O-Tetradecanoylphorbol-13-acetate (TPA) enhances the apparent maximal velocity of adenylate cyclase in S49 lymphoma cells, an effect that seems not to result from an increased rate of activation of the catalytic subunit by the stimulatory GTP-binding protein (Gs) (Bell, J. D., Buxton, I. L. O., and Brunton, L. L. (1985) J. Biol. Chem. 260, 2625-2628). In membranes from wild type S49 cells, this enhancing effect of TPA is largely GTP-dependent; TPA enhances forskolin-stimulated adenylate cyclase activity by 35% in the presence of guanine nucleotide but only slightly (approximately 10%) in its absence. TPA causes comparable results in membranes from the cyc- variant that lacks the GTP-binding subunit of Gs. Blockade of the activity of the inhibitory GTP-binding protein (Gi) by high concentrations of Mg2+ (100 mM) or Mn2+ (3 mM) abolishes the effect of TPA to enhance adenylate cyclase activity in wild type membranes. The potentiation by TPA of cAMP accumulation in intact cells is greater than and not additive with the similar effect of pertussis toxin (an agent known to abolish hormonal inhibition of adenylate cyclase). Kinetic experiments indicate that TPA decreases the rate of activation of Gi by guanine nucleotide. We conclude that the resultant withdrawal of tonic inhibition of adenylate cyclase is one mechanism by which phorbol esters enhance guanine nucleotide-dependent cAMP synthesis.     

Neuropeptide Y stimulation of myosin light chain phosphorylation in cultured aortic smooth muscle cells

Neuropeptide Y (NPY) is released from an extensive network of postganglionic sympathetic perivascular neurons. NPY has been shown to affect vascular tone postsynaptically by 1) directly stimulating contraction; 2) inhibiting vasorelaxation; and 3) potentiating contraction elicited by exogenous vasoconstrictors. The molecular mechanisms mediating these effects of NPY are undefined. Therefore, we examined the possibility that NPY could stimulate smooth muscle contraction through myosin light chain phosphorylation in cultured porcine aortic smooth muscle cells. NPY (100 nM) caused a rapid, transient increase in myosin light chain (MLC) phosphorylation, an important regulatory event in the initiation of smooth muscle contraction. NPY-stimulated MLC phosphorylation was prevented by preincubation of cells with pertussis toxin and was independent of extracellular Ca2+. In parallel studies, NPY alone had no detectable effect on cellular cAMP or cGMP content; however, NPY potently inhibited forskolin-stimulated cAMP accumulation (IC50 = 0.03 nM) through a pertussis toxin-sensitive pathway. NPY had no detectable effect on basal phosphoinositide hydrolysis or protein kinase C activation but enhanced angiotensin II-stimulated production of inositol phosphates and activation of protein kinase C. These results indicate that NPY-stimulated MLC phosphorylation can occur in the absence of detectable changes in cAMP content, cGMP content, inositol phosphate production, or protein kinase C activation; however, the interactions between NPY and other vasoactive agents may be mediated by the indirect effects of NPY on adenylate cyclase activity and phosphoinositide hydrolysis.     

Variations in guanine-binding proteins (Gs, Gi) in cultured bovine adrenal cells. Consequences on the effects of phorbol ester and angiotensin II on adrenocorticotropin-induced and cholera-toxin-induced cAMP production

The corticotropin (ACTH) or cholera-toxin-induced cAMP production by cultured bovine adrenal cells increased progressively between days 0 and 7 of culture. Angiotensin II (A-II), which inhibited both basal and ACTH-stimulated adenylate cyclase of crude adrenal membranes, had no effect on ACTH-induced or cholera-toxin-induced cAMP production by fresh isolated cells (day 0) but progressively potentiated the stimulatory action of both effectors from day 0----1 to day 7 of culture. In contrast, phorbol ester had a potentiating effect on fresh isolated cells. Pretreatment of cells with pertussis toxin enhanced the potentiating effect of A-II on cells between 0 and 3 days of culture, but not after 7 days. ADP-ribosylation by cholera toxin (ribosylating alpha s proteins) or pertussis toxin (alpha i proteins), of adrenal membranes prepared from fresh isolated or cultured cells revealed an increase in alpha s and a dramatic decrease in alpha i, the ratios alpha i/alpha s on days 0, 3 and 7 of culture were 4, 0.6 and 0.1 respectively. These results indicate that (a) A-II had a double effect on ACTH-induced or cholera-toxin-induced cAMP production: one inhibitory mediated by Gi, the other stimulatory mediated by protein kinase C activation; this could explain the lack of apparent effect of A-II on fresh cells; (b) the progressive decrease of alpha i might be responsible for the appearance of the potentiating effect of A-II whereas the progressive increase of alpha s could explain the enhanced responsiveness to ACTH or cholera toxin of cultured cells.     

Cholera toxin blocks glucagon-mediated inhibition of the liver plasma membrane (Ca2+-Mg2+)-ATPase

We have previously shown that liver plasma membrane (Ca2+-Mg2+)-ATPase activity is inhibited by glucagon. To investigate the possible involvement of a GTP-binding (G) protein in this regulation, we have examined the effects of pertussis toxin and cholera toxin on inhibition of (Ca2+-Mg2+)-ATPase by glucagon. Treatment of liver plasma membranes with pertussis toxin did not affect the sensitivity of (Ca2+-Mg2+)-ATPase to the hormone. In contrast, treatment of plasma membranes or prior injection of animals with cholera toxin prevented inhibition of the (Ca2+-Mg2+)-ATPase by glucagon. Even though adenylate cyclase activity was increased by cholera toxin treatment, addition of cyclic AMP did not mimic the effect of cholera toxin in blocking glucagon-mediated inhibition of (Ca2+-Mg2+)-ATPase activity. These data suggest that a cholera toxin-sensitive protein, perhaps Gs or a Gs-like protein, is involved in the regulation of liver (Ca2+-Mg2+)-ATPase activity. The results emphasize the possible role of Gs-like proteins in regulation of enzymes other than adenylate cyclase and suggest that the study of (Ca2+-Mg2+)-ATPase may provide a useful enzymatic system to examine such regulation.     

Enhancement of differentiation of cultured adipogenic cells (TA1) by pertussis toxin.

Differentiation of adipocytes is controlled by a variety of hormones and growth factors. To investigate the possible role of GTP-binding proteins (G proteins) in the process of adipose conversion, we studied the effect of pertussis toxin on differentiation of the fibroblast/adipocyte cell line (TA1). Pertussis toxin potentiated dexamethasone- and indomethacin-induced adipocyte differentiation in a time- and dose-dependent fashion. Addition of dibutyryl cAMP or forskolin inhibited adipose conversion, indicating that an abolishment of inhibitory control of adenylate cyclase is not responsible for the action of pertussis toxin. The B oligomer of the toxin did not mimic the effect of the holotoxin. Pertussis toxin catalyzed ADP-ribosylation of 40,000 molecular mass protein of the membrane fraction was dose-dependently inhibited by the pretreatment of the cells with the toxin. These results indicate the possible involvement of pertussis toxin-sensitive G proteins in adipogenesis.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

A novel mechanism for the inhibition of adenylate cyclase via inhibitory GTP-binding proteins. Calmodulin-dependent inhibition of the cyclase catalyst by the beta gamma-subunits of GTP-binding proteins

The adenylate cyclase catalytic protein partially purified from rat brain membranes was activated by the stimulatory GTP-binding protein (Gs), forskolin, and Ca2+-calmodulin. The Ca2+-calmodulin-stimulated activity was markedly, but the Gs- or forskolin-stimulated activity was essentially not, inhibited by low concentrations of the beta gamma-subunits of the inhibitory GTP-binding protein (Gi). The inhibition appeared to be competitive with calmodulin. On the other hand, the association of increasing amounts of beta gamma with the alpha of Gi, which was measured based on the ADP-ribosylation by islet-activating protein, pertussis toxin, was apparently competed by Ca2+-calmodulin. Furthermore, beta gamma bound to calmodulin-Sepharose in the presence of Ca2+, but not in its absence. Thus, the direct interaction of beta gamma with calmodulin is a likely mechanism involved in beta gamma-induced inhibition of the calmodulin-stimulated adenylate cyclase.     

Pertussis Toxin-Catalyzed ADP-Ribosylation: Effects on the Coupling of Inhibitory Receptors to the Adenylate Cyclase System

Abstract

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (G_i). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of G_i. ADP-ribosylation of G_i has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.