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.     

Stable 9 beta- or 11 alpha-halogen-15-cyclohexyl-prostaglandins with high affinity to the PGD2-receptor

Various chemically stable prostaglandin analogues were studied for their affinity towards the PGD2-receptor in human platelet membranes in order to define the requirements for specific ligand binding to this receptor. On replacing the 11- or 9-hydroxyl groups of PGF2 alpha by an 11 alpha- or 9 beta-chloro- or fluoro atom, stable prostaglandin analogues were obtained, which showed high affinity towards the PGD2-receptor. The lower side chain consisted of a 15-cyclohexyl group or of the natural 15-n-pentyl group, other substitutents decreased the affinity substantially. The highest PGD2-mimetic activity with a relative affinity of 0.5 to the PGD2-receptor was found in 9-deoxy-9 beta-chloro-16,17,18,19,20-pentanor-15-cyclohexyl-PGF2 alpha (ZK 110 841, compound 16 in Table 1). ZK 110 841 is a chemically stable crystalline substance, which is orally active and which might thus turn out to be an interesting tool for the study of PGD2-receptor interactions. Some other prostaglandin as well as prostacyclin analogues with a 15-cyclohexyl or 15-n-pentyl group exhibited in addition to their known high affinity to the PGE2-receptor of human uterine membranes or the PGI2-receptor of human platelets also affinities to the PGD2-receptor. Generally, the receptor affinities correlate with the activities as stimulators of adenylate cyclase and inhibitors of thrombin induced elevation of cytoplasmic free calcium as well as their ability to inhibit ADP-induced platelet aggregation. The PGI2-character regarding the effector systems prevails in compounds with affinity to both the PGI2- and PGD2-receptor. Compounds which bind to the PGE2- and PGD2-receptor show a flat dose response curve regarding platelet activation suggesting a mixture of pro- and antiaggregatory properties within these molecules.     

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.     

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.     

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.     

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.     

Pharmacological and cardiovascular properties of a hydantoin derivative, BW 245 C, with high affinity and selectivity for PGD2 receptors

Initial experiments demonstrated that the hydantoin prostaglandin derivative, BW 245 C, has potent anti-aggregatory activity on human platelets which may result from its structural similarity with one of the natural prostaglandins. The aim of the present study was to extend this preliminary pharmacological characterization and to determine which, if any, prostaglandin receptor-type is responsible for mediating the biological activity of BW 245 C. A marked species variation was observed in the anti-aggregatory potency of BW 245 C such that in the human (0.36 X PGE1) it was about one hundred times more effective than in the rat (0.003 X PGE1). The relative potencies of PGI2 (ca. 10 X PGE1) and PGE1 were, however, similar in both species. An intravenous bolus injection of 250 micrograms/kg BW 245 C lowered systolic (-23%) and diastolic (-34%) blood pressure in spontaneously hypertensive rats. In radioligand binding studies it showed a high affinity and selectivity for PGD2 platelet receptors, binding to PGI2 or PGE2 receptors was not detectable. Therefore it is concluded that the platelet and cardiovascular actions of BW 245 C are mediated by PGD2 receptors and this accounts for the observed species variation which is a characteristic of this prostaglandin.     

Interleukin 2 modulation of adenylate cyclase. Potential role of protein kinase C

Interleukin 2 (IL 2) stimulated DNA synthesis of murine T lymphocytes (CT6) in a concentration-dependent manner, over a range of 1-1000 units/ml. This proliferative effect of IL 2 was attenuated by simultaneous exposure to prostaglandin E2 (PGE)2. In intact cells, IL 2 inhibited both basal and PGE2-stimulated cAMP production; the amount of cAMP generated was dependent upon the relative concentrations of IL 2 and PGE2. The effect of IL 2 on CT6 cell proliferation and cAMP production was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which, like IL 2, causes a translocation and activation of protein kinase C. While PGE2 stimulated adenylate cyclase activity in membrane preparations, neither IL 2 nor TPA inhibited either basal or stimulated membrane adenylate cyclase activity. However, when CT6 cells were pretreated with IL 2 or TPA and membranes incubated with calcium and ATP, both basal and PGE2-and NaF-stimulated membrane adenylate cyclase activity was inhibited. This inhibition of adenylate cyclase activity was also observed if membranes from untreated cells were incubated with protein kinase C purified from CT6 lymphocytes in the presence of calcium and ATP. The data suggest that the decreased cAMP production which accompanies CT6 cell proliferation results from an inhibition of adenylate cyclase activity mediated by protein kinase C and that these two distinct protein phosphorylating systems interact to modulate the physiological response to IL 2.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells.

Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Effects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells.

Rank order of agonist potency for activation of adenylate cyclase by the naturally occurring prostanoids PGE2, PGF2 alpha, PGD2, the stable PGI2 analogue iloprost, and the TXA2 mimetic U 46619, provides evidence for the existence of a distinct PGE-receptor on guinea-pig duodenal enterocytes. The PGE-receptor is likely to be of the EP2-subtype since the specific EP2-agonist 11-deoxy-PGE1 stimulated adenylate cyclase activity with a 20-fold higher potency than the EP1-agonist 17-phenyltrinor-PGE2 and the EP3-agonists MB 28767 and GR 63799. In addition, sulprostone (acting on both EP1- and EP3-receptors) was ineffective. Since the specific EP1-antagonist SC 19220 did not inhibit PGE2-stimulated adenylate cyclase activity, the involvement of EP1-receptors could be further excluded. The synthetic prostaglandin E-analogues misoprostol and nocloprost stimulated adenylate cyclase almost identically, though they were about 10-fold less potent than the natural PGE2.     

Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.     

Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

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.     

Structural specificity for prostaglandin effects on hepatocyte glycogenolysis.

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.     

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.