Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Current concepts for a drug-induced inhibition of formation and action of thromboxane A2

Urinary and plasma metabolites of thromboxane A2 (TxA2) indicate an increased TxA2 synthesis in a number of diseases, whereby TxA2 is assumed to contribute to the underlying pathomechanisms by its profound effects on platelet aggregation and smooth muscle contraction. In some clinical situations the increment in TxA2 biosynthesis is accompanied by an increased formation of prostacyclin (PGI2) which is one of the most potent inhibitors of platelet activation and smooth muscle contraction. Therefore, drugs are being developed which suppress the formation or action of TxA2 without interfering with its functional antagonist PGI2. Low doses of acetylsalicyclic acid (ASA) preferentially inhibit cyclooxygenase activity in platelets and the synthesis of TxA2 in vivo. However, neither low doses (approximately 300 mg/day) nor very low doses spare the formation of PGI2 completely. Despite its limited selectivity, very low dose ASA (approximately 40 mg/day) provides an attractive perspective in TxA2 pharmacology. Although thromboxane synthase inhibitors selectively suppress TxA2 biosynthesis PGH2 can accumulate instead of TxA2 and substitute for TxA2 at their common TxA2/PGH2 receptors. Thromboxane synthase inhibitors can only exert platelet-inhibiting and vasodilating effects if PGH2 rapidly isomerizes to functional antagonists like PGI2 that can be formed from platelet-derived PGH2 by the vessel wall. TxA2/PGH2 receptor antagonists provide a specific and effective approach for inhibition of TxA2. These inhibitors do not interfere with the synthesis of PGI2 and other prostanoids but prevent TxA2 and PGH2 from activating platelets and inducing smooth muscle contractions. Most of the available TxA2/PGH2 receptor antagonists produce a competitive antagonism that can be overcome by high agonist concentrations. Since in certain disease states very high local TxA2 concentrations are to be antagonized, non-competitive receptor antagonists may be of particular interest. Some recent TxA2/PGH2 receptor antagonists produce such a non-competitive type of inhibition due to their low dissociation rate constant. As a consequence, agonists like TxA2 or PGH2 only reach a hemiequilibrium state at their receptors, previously occupied by those antagonists. A combination of a thromboxane synthase inhibitor with a TxA2/PGH2 receptor antagonist presents a very high inhibitory potential that utilizes the dual activities of the synthase inhibitor to increase PGI2 formation and of the receptor antagonist to antagonize PGH2 and TxA2. Such combinations or dual inhibitors, combining both moieties in one compound, prolong the skin bleeding time to a greater extent than thromboxane synthase inhibitors and even more than low dose ASA or TxA2/PGH2 receptor antagonists.     

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX).

Prostacyclin (PGX) (57)-9-deoxy-6,9alpha-epoxy-delta5-PGF1alpha has been found to be a potent stimulator of cAMP accumulation in platelets than PGE1. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH2, and a PGH2-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH2.     

Desensitization of turkey erythrocyte adenylate cyclase. Beta-adrenergic receptor phosphorylation is correlated with attenuation of adenylate cyclase activity

Preincubation of turkey erythrocytes with beta-adrenergic agonists leads to an attenuation of the responsiveness of adenylate cyclase to subsequent hormonal stimulation. Recently, our laboratory has shown (Stadel, J. M., Nambi, P., Shorr, R. G. L., Sawyer, D. D., Caron, M. G., and Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3173-3177) using 32Pi incorporation that phosphorylation of the beta-adrenergic receptor accompanies this desensitization process. We now report that, as determined from intracellular [gamma-32P] ATP specific activity measurements, this phosphorylation reaction occurs in a stoichiometric fashion. Under basal conditions there exists 0.75 +/- 0.1 mol of phosphate per mol of receptor whereas under maximally desensitized conditions this ratio increases to 2.34 +/- 0.13 mol/mol. This phosphorylation of the receptor is dose-dependent with respect to isoproterenol and exhibits a dose-response curve coincidental with that for isoproterenol-induced desensitization of adenylate cyclase. The time courses for receptor phosphorylation and adenylate cyclase desensitization are identical. In addition, the rate of resensitization of adenylate cyclase activity is comparable to the rate of return of the phosphate/receptor stoichiometries to control levels. Both the phosphorylation and desensitization reactions are pharmacologically specific as indicated by the high degree of stereoselectivity, rank order of catecholamines, and blockade by the specific beta-adrenergic antagonist, propranolol. Incubation of turkey erythrocytes with cAMP and cAMP analogs maximally activates cAMP-dependent protein kinase but only partially mimics isoproterenol in promoting phosphorylation of the receptor in concordance with their partial effects in inducing desensitization. Conversely, activators or inhibitors of Ca2+/calmodulin kinase or protein kinase C do not affect the isoproterenol-induced desensitization. These results indicate that desensitization of turkey erythrocyte adenylate cyclase is highly correlated with phosphorylation of the beta-adrenergic receptor and that these events are mediated, at least partially, by cAMP.     

Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Schizophrenia and reduced cyclic AMP production: evidence for the role of receptor-linked events

Reduced cyclic AMP (cAMP) production has been found in platelets of schizophrenic patients. cAMP is generated physiologically as a result of a series of steps beginning with receptor activation by a ligand, progressing through activation of the enzyme protein, adenylate cyclase. The deficit of cAMP found in the schizophrenic population may occur at any one, or at multiple steps in this cascade. The present study attempts to discriminate whether impaired adenylate cyclase itself was responsible for the cAMP deficit or whether abnormalities in receptor events or linkage are present in schizophrenics. The production of cAMP following direct stimulation of adenylate cyclase by NaF was contrasted with receptor mediated activation of adenylate cyclase by prostaglandin E1 (PGE1) in disrupted platelet preparations from schizophrenics and normal controls. cAMP formation stimulated by NaF was not different in platelets of schizophrenics as compared to controls, however, platelets of schizophrenics showed reduced response to PGE1 stimulation. The authors interpret these findings as evidence for a membrane associated abnormality of either receptor or receptor-adenylate cyclase linkage in the schizophrenias.     

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.     

Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number

Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.     

Inhibition of prostaglandin biosynthesis by SQ 28,852, a 7-oxabicyclo[2.2.1]heptane analog

7-Oxabicyclo[2.2.1]heptane analogs of prostaglandin (PG) H2 can act as thromboxane (Tx) A2 receptor antagonists or agonists, PGI2 and/or PGD2 receptor agonists, or exhibit a mixture of the above activities. SQ 28,852, a new analog with a hexyloxymethyl omega side chain, is a potent inhibitor of PG synthesis. SQ 28,852 inhibited collagen and arachidonic acid (AA)-induced platelet aggregation and TxB2 and PGE2 formation, but did not block platelet aggregation induced by ADP or the TxA2 mimics, 9,11-azo PGH2, SQ 26,655, and U-46,619. It also blocked conversion of AA to TxB2, PGE2, and 6-keto PGF1 alpha by microsomal preparations of human platelets, bovine seminal vesicles, and bovine aortas, respectively, but did not inhibit the conversion of PGH2 to TxA2 by the platelet microsomal preparation. SQ 28,852 (p.o.) protected mice against the lethal effects of AA (75 mg/kg, i.v.). The I50 values for SQ 28,852, indomethacin and aspirin were 0.025, 0.05 and 15 mg/kg, respectively. Neither SQ 28,852 nor indomethacin protected mice from death caused by 9,11-azo PGH2. SQ 28,852 (0.01 to 1 mg/kg, i.v.) inhibited AA-induced bronchoconstriction in anesthetized guinea pigs for at least 60 min. As an inhibitor of AA-induced bronchoconstriction, SQ 28,852 was 16- and 45-times more potent than indomethacin at 3 and 60 min after i.v. administration, respectively. SQ 28,852 did not inhibit bronchoconstriction induced by histamine or 9,11-azo PGH2, indicating its specificity of action in vivo. SQ 28,852 is the first example of a new class of cyclooxygenase inhibitors whose structure is similar to that of the naturally occurring endoperoxide, PGH2.     

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.     

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.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).     

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.     

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2.

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.     

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.