SQ-27986 inhibition of platelet aggregation is mediated through activation of platelet prostaglandin D2 receptors

SQ-27986, a oxabicycloheptane derivative, potently inhibits ADP-, collagen- and arachidonic acid-induced platelet aggregation in human platelet-rich plasma. Human platelet aggregation induced by ADP is inhibited by SQ-27986 (EC50 = 22nM), and the inhibitory action of SQ-27986 can be prevented with N-0164, a PGD2 antagonist. By comparison, ADP-induced rat platelet aggregation is unaffected by SQ-27986 (IC50 greater than 80 microM). Washed human platelets treated with SQ-27986 exhibit elevated cAMP levels and activated cAMP-dependent protein kinase. Elevation of platelet cAMP levels (greater than 4 fold basal) and activation of the cAMP-dependent protein kinase (greater than 4 fold) are observed with SQ-27986 concentrations above 100 nM. The SQ-27986-induced elevation of cAMP can be prevented by N-0164. Lysed platelets treated with SQ-27986 showed stimulated adenylate cyclase activity. SQ-27986 competes with [3H]prostaglandin D2 binding to isolated platelet membranes (EC50 for SQ-27986 is 20 nM, which was more potent than cold PGD2 itself). Radiolabeled Iloprost binding is virtually unaffected by SQ-27986 (EC50 greater than 100 microM), indicating that SQ-27986 does not interact with platelet prostacyclin receptors. These studies indicate that SQ-27986 inhibits platelet aggregation by activating platelet adenylate cyclase via stimulation of platelet PGD2 receptors.     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

Stimulation of prostaglandin D2 receptors on human platelets by analogs of prostacyclin.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.     

Inhibition of prostaglandin E1-induced activation of adenylate cyclase in human blood platelet membrane

Activation of human blood platelet adenylate cyclase is initiated through the binding of prostaglandin E1 to the membrane receptors. Incubation of platelet membrane with [3H]prostaglandin E1 at pH 7.5 in the presence of 5 mM MgCl2 showed that the binding of the autacoid was rapid, reversible and highly specific. The binding was linearly proportional to the activation of adenylate cyclase. Although the membrane-bound radioligand could not be removed either by GTP or its stable analogue 5'-guanylylimido diphosphate, 150 nM cyclic AMP displaced about 40% of the bound agonist from the membrane. Scatchard analyses of the binding of the prostanoid to the membrane in the presence or absence of cyclic AMP showed that the nucleotide specifically inhibited the high-affinity binding sites without affecting the low-affinity binding sites. Incubation of the membrane with 150 mM cyclic AMP and varying amounts of prostaglandin E1 (25 nM to 1.0 microM) showed that the percent removal of the membrane-bound autacoid was similar to the percent inhibition of adenylate cyclase at each concentration of the agonist. At a concentration of 25 nM prostaglandin E1, both the binding of the agonist and the activity of adenylate cyclase were maximally inhibited by 40%. With the increase of the agonist concentration in the assay mixture, the inhibitory effects of the nucleotide gradually decreased and at a concentration of 1.0 microM prostaglandin E1 the effect of the nucleotide became negligible. These results show that cyclic AMP inhibits the activation of adenylate cyclase by low concentrations of prostaglandin E1 through the inhibition of the binding of the agonist to high-affinity binding sites.     

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.     

Multiple coupling of neurohormone receptors with cyclic AMP and inositol phosphate production in anterior pituitary cells

Regulation of adenohypophyseal hormone secretions has been shown to involve cyclic AMP production, modulation of phosphatidyl inositol diphosphate breakdown and Ca2+ mobilization. Various neurohormone receptors are positively or negatively coupled to adenylate cyclase activity in anterior pituitary cells. The effects of these neurohormones on adenylate cyclase activity are consistent with the effect on hormone secretions, suggesting that modulation of the enzyme activity is actually involved in the regulation of adenohypophyseal secretions. Thus DA inhibits, whereas VIP stimulates adenylate cyclase activity of the same cell type, which, according to the effect of these neurohormones on prolactin secretion, appear to be lactotrophs. On the other hand, SRIF inhibits, whereas GRF stimulates the adenylate cyclase activity of another cell type, namely somatotrophs, whereas CRF appears to act on a third cell type, corticotrophs. Peripheral hormones have been shown to modulate the sensitivity of anterior pituitary cells to these neurohormones. Estradiol long-term treatment has an anti-dopaminergic effect on prolactin secretion. The steroid also suppresses the dopamine inhibition of adenylate cyclase. This effect appears selective to the DA inhibition, since AII inhibition of the enzyme is only partially reduced, whereas the somatostatin inhibition is markedly increased. Peripheral hormones seem to affect the sensitivity of adenohypophyseal cells not only by modulating the number of receptors for a given neurohormone but also by interfering with the coupling mechanisms of these receptors. AII and DA inhibit the adenylate cyclase activity of lactotroph cells. The prolactin stimulation induced by angiotensin is not consistent with the effect of the peptide on adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioactive amide of prostaglandin E1 and ethanolamine plasmalogen analog of platelet-activating factor inhibits several pathways of human platelet aggregation

The influence of an amide of prostaglandin E1 and ethanolamine plasmalogen platelet-activating factor analog 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-11alpha, 15alpha-dioxy-9-keto-13-prostenoyl)ethanolamine (PGE1-PPAF) on platelet-activating factor (PAF)-, ADP-, and thrombin-induced human platelet aggregation has been studied. It was found that PGE1-PPAF inhibits the PAF-, ADP-, and thrombin-induced platelet aggregation in platelet-rich plasma. 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine inhibited PAF-induced aggregation up to 50% but had no influence on platelet aggregation induced by ADP or thrombin. The ethanolamine plasmalogen analog of PAF 1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolami ne, having a palmitoyl residue instead of PGE1, did not inhibit platelet aggregation induced by PAF, ADP, or thrombin. We propose that inhibition of human platelet aggregation by PGE1-PPAF is mediated by its action on platelet PAF-receptors and the adenylate cyclase system.     

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans.

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.     

Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked responses in fura-2-loaded human platelets.

Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP-evoked Mn2+ influx and the release of Ca2+ from intracellular stores. U46619 decreased the elevated concentration of 3',5'-cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors, like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.     

Stimulation of high-affinity hormone-sensitive GTPase of human platelets by 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor)

1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor) inhibits human platelet adenylate cyclase via the GTP-dependent mechanism. Inhibition of adenylate cyclase correlates with the stimulation of high affinity hormone-sensitive GTPase. The half-maximal effects of PAF on both enzymes are observed at concentrations about 10(-8) M. Phentolamine, an alpha-adrenergic antagonist, does not abolish the PAF-induced inhibition of adenylate cyclase. The obtained data suggest that PAF receptors are coupled with the GTP-binding inhibitory protein.     

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.     

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil

Trapidil (N,N-diethyl-5-methyl[l,2,4]triazolo[l,5-α]pyrimidine-7-amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K_i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β-PGI_i). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.     

Modulation of adenylate cyclase of human platelets by phorbol ester. Impairment of the hormone-sensitive inhibitory pathway

The influence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory NS protein nor on stimulatory hormone receptors (prostaglandin E1) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.     

Antagonism of the prostaglandin endoperoxide imhibition of hormone-stimulated adenylate cyclase by guanosine triphosphate and 5'-guanylyl-imidodiphosphate.

The prostaglandin endoperoxide prostaglandin H2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(nh)p to be maximally effective at 3.3 muM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H2. Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H2-sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H2 stabilizes the fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Desensitization of prostaglandin-activated platelet adenylate cyclase.

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2 washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 x 10(-5) M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10(-7) M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Protein kinase C interferes with Ni-mediated inhibition of human platelet adenylate cyclase

Addition of phorbol ester-activated, partially purified protein kinase C to membranes of human platelets had no effect on forskolin stimulation of the adenylate cyclase and increased stimulation by prostaglandin E1 only at high GTP concentrations by preventing inhibition by GTP. Hormonal inhibition of the platelet adenylate cyclase by epinephrine was eliminated or largely impaired. At low GTP concentrations, epinephrine even caused a small increase in cyclase activity. The data suggest that activated protein kinase C interferes with GTP- and hormone-induced adenylate cyclase inhibition probably by phosphorylating the inhibitory guanine nucleotide-binding regulatory component Ni.