Platelet Adenylate Cyclase Activity Research Articles

The local anaesthetic benzyl alcohol attenuates the α2-adrenoceptor-mediated inhibition of human platelet adenylate cyclase activity when stimulated by prostaglandin E1, but not that stimulated by forskolin

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.

The antagonism by BW A868C of PGD2 and BW245C activation of human platelet adenylate cyclase

1. In glycerol-lysed human platelets, prostaglandin D2 (PGD2) and the hydantoin BW245C both activate adenylate cyclase in a biphasic manner. These activations are qualitatively different from those of carbacyclin, iloprost and prostaglandin E2 (PGE2) whose E/[A] curves can be adequately described by rectangular hyperbolae. 2. Prostaglandin E1 (PGE1) had E/[A] curves of slope significantly lower than that expected for a rectangular hyperbolae. 2. Prostaglandin E1 (PGE1) had E/[A] curves of slope significantly lower than that expected for a rectangular hyperbola. 3. The selective PGD2 antagonist BW A868C shifts the first phase of the PGD2 and BW245C E/[A] curves but has no effect on the second phase. 4. Applying a two-receptor model enables a pKB to be derived for BW A868C of 9.11. 5. BW A868C has no effect on carbacyclin, iloprost, prostacyclin, PGE1 and PGE2 at a concentration 1,000 fold that of its KB against PGD2 and BW245C. 6. These results indicate that PGD2 and BW245C are capable of activating adenylate cyclase in human platelets through the DP-receptor and by another mechanism as yet uncharacterized.

塩酸ビフェメランの抗血小板作用

We investigated the erect of bifemelane hydrochloride on human platelet aggregation and platelet cyclic nucleotide metabolism in order to elucidate its mechanism. Bifemelane inhibited platelet aggregation induced by collagen and ADP with an IC50 value of 6.6×10-5M and 1.2×10-4M, respectively. The inhibitive effects of the agent on platelet aggregation increased in the presence of 10-9M PGI2. Bifemelane significantly increased platelet adenylate cyclase activity in concentrations of 10-5M and 10-4M and inhibited cyclic AMP phosphodiesterase activity with an IC50 value of 6.6×10-4M. Cyclic AMP accumulation in intact platelets in response to bifemelane alone significantly increased only in high concentrations of 10-3M. However, a synergistic effect on cyclic AMP accumulation was found between the bifemelane and PGI2. Although a concentration of 10-4M of bifemelane had no effect on guanylate cyclase activity it did attenuate activity at 10-3M. Bifemelane inhibited cyclic GMP phosphodiesterase activity with an IC50 value similar to cyclic AMP phosphodiesterase (4.5×10-4M). Bifemelane up to a concentration of 10-4M, did not alter cyclic GMP accumulation. However, bifemelane in a concentration 10-4M did decrease activity.We conclude that bifemelane hydrochloride inhibits human platelet aggregation by modulating the cyclic AMP metabolism. Synergism between bifemelane and prostacyclin may also contribute to the enhancement of the antiplatelet action of bifemelane in vivo.

Inhibition of prostaglandin E 1-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.

Inhibition of prostaglandin E 1-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.

Vasopressin inhibition of human platelet adenylate cyclase: variable responsiveness between donors and involvement of a G-protein different from G i

There is controversy concerning the inhibitory effect of arginine-vasopressin (AVP) on human platelet adenylate cyclase activity, which putatively involves G i as the G-protein. To clarify this point, the effects of AVP on human platelet membranes were studied by measuring the activities of the high-affinity GTPase, as an index of G-protein involvement, and of adenylate cyclase. AVP stimulated GTPase activity in a dose-dependent fashion (K Act = 1.1 ± 0.2 nM) and caused a parallel adenylate cyclase inhibition (K Act = 1.3 ± 0.7 nM). The extent of these AVP-induced responses varied considerably from one subject to another but they were linearly related, suggesting a causal relationship between the two activities. Moreover, a difference in responsiveness to the inhibitory effects to epinephrine on adenylate cyclase was also observed between donors. Since the AVP- and epinephrine-stimulated GTPase activities were additive at their respective maximal effect, and in view of the lack of linear relationship between AVP- and epinephrine-induced adenylate cyclase inhibition, our results suggest, that in spite of the AVP inhibitory action on platelet adenylate cyclase, the G-protein involved in this effect is different from G i.

Lithium modulation of second messenger signal amplification in man: Inhibition of phosphatidylinositol- specific phospholipase C and adenylate cyclase activity

The activity of phosphatidylinositol-specific phospholipase C was significantly reduced in platelets obtained from 20 euthymic manic-depressive patients on therapeutic lithium doses (mean blood level 0.85 mEq/l) compared to an age- and sex-matched group of 36 control subjects. The activities of prostaglandin E 1-, aluminum/NaF-, and forskolin-stimulated platelet adenylate cyclase activity were also measured in a similar group of 16 lithium-treated and 22 control subjects. A marked reduction in both postreceptor (aluminum/NaF and forskolin) and receptor-stimulated (prostaglandin E 1) platelet adenylate cyclase activity was observed in the lithium-treated group (mean blood level 0.81 mEq/l). These findings support the hypothesis that lithium's therapeutic mode of action in manic-depressive psychosis is mediated by the combined down-regulation of both principal second messenger systems, inositol phosphates and cyclic adenosine monophosphate, by reducing the activity of phosphatidylinositol-specific phospholipase C and adenylate cyclase.

Rapid antidepressant response to alprazolam in depressed patients with high catecholamine output and heterologous desensitization of platelet adenylate cyclase

The present study examined the relationship between 24-hr urinary catecholamine (norepinephrine and epinephrine) output and measures of platelet adenylate cyclase (AC) activity in depressed patients ( n = 17) and control subjects ( n = 10). In both groups, significant inverse correlations were observed when 24-hr urinary catecholamine levels were examined in relation to measures of both receptor-mediated (prostaglandin D 2 and α 2-adrenergic) and postreceptor-mediated(NaF) platelet AC enzyme activities, suggesting that circulating catecholamines may regulate platelet AC by heterologous (agonist-nonspecific) desensitization of the AC enzyme complex. Depressed patients who had favorable antidepressant responses to alprazolam had significantly higher pretreatment urinary catecholamine output and lower receptor-mediated platelet AC enzyme activities than control subjects, whereas the nonresponders did not. After 8 days of treatment with alprazolam, urinary catecholamine levels declined significantly. In responders, receptor-mediated measures of platelet AC activity increased significantly by day 8 to values comparable to those in control subjects; but similar changes were not observed in nonresponders. Prior to treatment, responders showed a strict linear relationship between receptor-mediated (prostaglandin D 2) and postreceptor-mediated (NaF) stimulation of platelet AC activity through the stimulatory guanine nucleotide regulatory protein (Ns), whereas nonresponders did not. This suggests the presence of two distinct coupling

Differences in platelet enzyme activity between alcoholics and nonalcoholics.

Blood platelets are an accessible tissue that reflects the activity of many enzymes found in the brain. To investigate the possible effect on such enzymes of long-term consumption of large quantities of ethanol, we assayed the activities of two enzymes, monoamine oxidase and adenylate cyclase, in platelet membranes of men with alcoholism and controls matched for sex and age. We also compared these two groups in terms of the inhibition of platelet monoamine oxidase activity by ethanol in vitro (400 mM), and in terms of the stimulation of adenylate cyclase activity by various agents. There was no significant difference in monoamine oxidase activity between the alcoholics and the controls. However, the inhibition of monoamine oxidase by ethanol was significantly higher in the platelets of alcoholics. The basal activity of adenylate cyclase was the same in platelets from the alcoholics and the controls, but the platelet adenylate cyclase activity after stimulation with guanine nucleotide, cesium fluoride, or prostaglandin E1 was significantly lower in alcoholics. These differences were not associated with age, race, smoking, or illicit drug use, and there was no significant correlation with the duration of problems with alcohol. The changes were long-lasting; cesium fluoride-stimulated adenylate cyclase activity was lower in alcoholic subjects who had abstained from alcohol for one to four years. Discriminant analysis showed that the use of values for the inhibition of monoamine oxidase activity by ethanol and cesium fluoride-stimulated adenylate cyclase activity correctly classified 75 percent of the alcoholics and 73 percent of the controls. These measures may be of value either as indexes of excessive alcohol consumption or as an indication of a predisposition to alcoholism.

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.

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets.

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.

Alpha 2-adrenoceptor-mediated inhibition of platelet adenylate cyclase activity in heroin addicts in abstinence.

The inhibition of basal and forskolin-stimulated platelet adenylate cyclase activity by (-)adrenaline was studied in ten heroin addicts during spontaneous withdrawal. Both basal and forskolin-stimulated enzyme activities were increased during heroin withdrawal and the inhibitory effect induced by (-)adrenaline was potentiated with parallel shifts to the left of the concentration-effect curves. The number of binding sites for [3H](-)adrenaline in platelet membranes was also increased during withdrawal. Treatment with clonidine markedly attenuated the inhibitory effect induced by (-)adrenaline on platelet adenylate cyclase activity. The results indicate that the heroin withdrawal syndrome induces supersensitivity of the platelet alpha 2-adrenoceptor-adenylate cyclase system.

Effect of lithium in vitro and after chronic treatment on human platelet adenylate cyclase activity: Postreceptor modification of second messenger signal amplification

LiCl in vitro markedly inhibits forskolin-stimulated human platelet adenylate cyclase activity by competing competitively with Mg 2 + for a site on the catalytic subunit. The sensitivity of platelet membrane adenylate cyclase to lithium inhibition for individual manic patients was determined by the Dixon plot procedure: marked individual differences in sensitivity to lithium were observed pretreatment (0.66 m M-3.15 m M LiCl). After 3 weeks of continuous treatment with lithium in vivo a significant decrease in adenylate cyclase affinity for lithium was observed (pretreatment average K i = 1.38 ± 0.92 mM vs. treatment average K i = 2.98 ± 1.35 mM LiCl, n = 10). The clinical implications of these findings relating to chronic lithium exposure are discussed.

Functional uncoupling of the platelet alpha 2-adrenergic receptor-adenylate cyclase complex in the elderly.

Elderly humans demonstrate decreased responsiveness in several hormone-receptor systems, including adrenergic receptors. Studies of the beta-adrenergic receptor (beta-AR) system have shown that reduced beta-adrenergic sensitivity in the elderly may be due to reduced beta-AR affinity for agonists. To determine the mechanisms underlying altered alpha-adrenergic sensitivity in the elderly, we assessed the relationships between age and platelet membrane alpha 2-adrenergic receptor (alpha 2-AR)-binding properties, receptor-linked adenylate cyclase (AC) activity, and the affinity of the alpha 2-AR-AC complex for agonists in 18 young (mean age, 24 yr; range 19-34) and 13 elderly (mean age, 69 yr; range, 63-85) normal subjects. In platelet membrane preparations from elderly compared to young subjects, we found similar antagonist-binding properties and similar activity of the catalytic unit of platelet AC, as indicated by the cAMP response to sodium fluoride stimulation. However, mean epinephrine-mediated inhibition of sodium fluoride-stimulated platelet AC activity was less in the elderly [20 +/- 4% (+/- SEM) vs. 31 +/- 2% inhibition; P less than 0.005). In addition, platelet alpha 2-AR affinity for agonist was lower in the elderly, as indicated by the higher concentration of epinephrine needed to inhibit 50% of specific [3H]yohimbine binding (IC50, 3.2 +/- 0.6 vs. 1.4 +/- 0.3 microM; P less than 0.02). These data provide evidence that platelet membranes from elderly humans have decreased responsiveness to alpha-adrenergic stimulation, which can be attributed to reduced alpha 2-AR-AC affinity for agonists. Similarly to reported age-related alterations in beta-adrenergic receptor function, these results suggest that there is also functional uncoupling of the alpha 2-AR-AC complex in elderly humans.

Plasmin: A possible physiological modulator of the human platelet adenylate cyclase system

Plasmin was recently reported to inhibit platelet aggregation. We report here on the interaction of plasmin with the adenylate cyclase system of human platelets. Human plasmin caused a dose- and time-dependent increase in adenylate cyclase activity when added to a crude platelet membrane preparation. Both basal and prostaglandin E1-stimulated adenylate cyclase activity doubled in presence of plasmin. This stimulatory activity was shared by papain and alpha-chymotrypsin, but not by thrombin which displayed a slightly inhibitory effect. Plasmin not only stimulated platelet adenylate cyclase activity, but also suppressed the GTP-dependent alpha 2-adrenergic inhibition, thereby producing a five- to six-fold increased activity measured in the presence of adrenaline and GTP. These effects of plasmin on the adenylate cyclase system were suppressed by the addition of the protease inhibitor leupeptin, and of soybean trypsin inhibitor, indicating that proteolysis mediated these effects. We also examined the adenylate cyclase activity in membranes prepared from intact platelets incubated with increasing doses of plasmin. Incubation of platelets with plasmin concentrations as low as 0.25 mg/ml resulted in an irreversible increase in membrane adenylate cyclase activity and suppression of the adrenaline-mediated inhibition of enzyme activity. These results suggest that the proteolytic stimulating effect of plasmin on the platelet adenylate cyclase system may account for the inhibition of platelet aggregation.

The presence of free G protein beta/gamma subunits in human neutrophils results in suppression of adenylate cyclase activity.

We have examined the adenylate cyclase of human neutrophil membranes and compared it to that of human platelet membranes. Stimulated activities were at least 20-fold lower in the neutrophil than in the human platelet. The inhibitory hormone epinephrine was able to attenuate markedly the adenylate cyclase activity of human platelets at micromolar concentrations, whereas little inhibition was observed in the human neutrophil at up to 100 microM concentrations. When we examined the ability of exogenous pure beta/gamma subunits to affect adenylate cyclase activity in both systems, we observed dose-dependent inhibition of stimulated adenylate cyclase activities in the platelet, whereas no inhibition of neutrophil adenylate cyclase could be detected. This difference did not appear to be due to differences in the degree of incorporation of beta/gamma into each membrane. The effects of G protein alpha subunits were also examined. In the platelet, unliganded G protein alpha produced an increase in adenylate cyclase activity of limited extent which saturated at relatively low levels of alpha subunit. In the neutrophil, the effect of unliganded G protein alpha did not appear to saturate and produced much larger relative increases in adenylate cyclase activity. Quantitation of the free beta/gamma activity in neutrophil extracts detected free beta/gamma activity even in the absence of G protein activators. We hypothesize the human neutrophil to be a system in which an excess of free beta/gamma subunits is present and which suppresses neutrophil adenylate cyclase activity. This excess of free beta/gamma minimizes any additional effect of exogenous beta/gamma, but can be reversed by addition of proteins which can bind beta/gamma subunits, e.g. G alpha subunits.

Purification of a beta 35 form of the beta gamma complex common to G-proteins from human placental membranes.

The significance of the 36,000-35,000-dalton doublet of proteins referred to as the beta-subunit of the G-proteins remains unresolved. An immunological distinction between the 36,000 (beta 36)- and 35,000 (beta 35)-dalton proteins has been reported (Roof, D. J., Applebury, M. L., and Sternweis, P. C. (1985) J. Biol. Chem. 260, 16242-16249; Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 265-269). The availability of homogeneous preparations of beta 36 and beta 35 will facilitate studies designed to address the significance of the doublet structure. This manuscript presents the preparative purification of the beta 35 protein from a highly enriched source, human placenta. Unlike the beta gamma complex associated with G-proteins from placenta, the beta 35 preparations consist predominantly of the 35,000-dalton protein. The gamma-peptide associated with beta 35 is indistinguishable electrophoretically and immunologically from that associated with the placental G-protein oligomers. The beta 35 preparation and beta-subunit doublet exhibit similar specific activities in inhibiting human platelet adenylate cyclase activity. The preparations have proven useful for the generation of a panel of rabbit polyclonal antisera that recognize beta 35, Gt beta 36, and both beta 36 and beta 35 in the doublet associated with Gs, Gi, Go, and Gp. One antiserum generated to beta 35 recognizes the gamma-peptide associated with these preparations and human placental G-protein oligomers. The antiserum does not recognize Gt gamma. Protease digestion of Gt beta 36 and human placental beta 35 with Staphylococcus aureus V8 protease identified a unique peptide generated from beta 35 which is absent in beta 36 digests.

Effects of heparin on prostacyclin binding and platelet adenylate cyclase activity stimulated by iloprost and forskolin

Heparin is known to impair the antiaggregatory effectiveness of prostacyclin (PGI2) for adenosine diphospate-induced aggregation in citrated platelet rich plasma. Thus porcine mucosal heparin (PMH) from different commercial sources was investigated for its ability to compete with specific platelet prostacyclin binding. In concentrations up to 250 IU/ml PMH itself did not interfere with the binding of 3 × 10−9M 9-3H-PGI2-sodium salt to intact washed platelets. The displacement of specifically bound PGI2 observed by PMH (Ki 60 IU/ml) containing 4-chloro-m-cresol (4-CC) was caused by the preservative (Ki 4-CC 3.0 × 10−4M). Although 60 IU/ml PMH (free of 4-CC) have been shown to inhibit basal 3 × 10−5M forskolin-, and 10−8M Iloprost-stimulated adenylate cyclase in platelet membranes (−63%, −62%, −83% respectively), no effect of PMH on 3H-cyclic-AMP formation has been observed when intact platelets were studied by 3H-adenine prelabeling technique. There is also no evidence that the preincubation of PGI2 (5× 10−7M) with 1000 IU/ml Phi might neutralize the effectiveness of this eicosanoid. In contrast, PGI2 preincubated with PMH (10 min, 37°C) caused a more pronounced increase of platelet 3H-cyclic AMP (+65%) compared with PGI2 incubated in 5 × 10−2M Tris-HCl, pH 7.4. Thus our data provide no evidence that PMH interferes with i) specific platelet PGI2 binding, ii) PGI2-stimulated cyclic AMP synthesis or iii) neutralizes PGI2 by formation of a biologically less active PGI2-PMH complex.

Age related decline in aluminum-activated human platelet adenylate cyclase: Post-receptor changes in cyclic AMP second messenger signal amplification in normal aging and dementia of the Alzheimer type

Low, micromolar concentrations of aluminum (in the presence of NaF) were shown to strongly activate human platelet adenylate cyclase and provided a useful probe for evaluating cyclic AMP second messenger function distal to the receptor: The effect of normal aging and disease state on second messenger activity in man was studied by measurements of the aluminum-activated enzyme. A significant decline in aluminum-stimulated platelet adenylate cyclase activity in older, healthy subjects was observed. An age-associated decline in NaF-stimulated cyclic AMP synthesis was also demonstrated for normal, non-demented subjects. These findings suggest an age-associated lesion at the level of the guanine nucleotide regulatory protein/catalytic subunit of the adenylate cyclase complex. However, for patients with Alzheimer's disease no such decline in platelet adenylate cyclase activity was detected, and increased sensitivity to both aluminum and NaF was demonstrated.

Effects of enprofylline on A 1 and A 2 adenosine receptors

The effects of enprofylline were studied on A 1 adenosine receptors of rat fat cells and on A 2 adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5′-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a K B of 130 μM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A 2 receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N 6-R-(−)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a K B of 32 μM. The K i value for inhibition of [ 3H]PIA binding to fat cell membranes was 45 μM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with K i values of 15, 130 and 110 μM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A 1 and A 2 adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline.