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R J Haslam

Publications and source records attributed to R J Haslam.

At least 19 recordsLinked to original sources
PubMed1991

Synergistic actions of nitrovasodilators and isoprenaline on rat aortic smooth muscle.

Previous studies have established that nitrovasodilators potentiate the inhibition of platelet function by activators of adenylyl cyclase, but uncertainty exists as to whether a comparable effect is seen in vascular smooth muscle. We initially studied the effects of the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), on the relaxation by isoprenaline of rat aortic smooth muscle that had been precontracted by phenylephrine. Concentrations of SNP (0.25 nM) and SIN-1 (30 nM) that relaxed aortic smooth muscle less than 30% alone, caused significant (3-fold) decreases in the IC50 values for isoprenaline. The cAMP phosphodiesterase inhibitors, cilostamide (20 nM) and Ro 20-1724 (10 microM), caused comparable reductions in the IC50 values for isoprenaline. At these concentrations, each of the four compounds also increased the maximum relaxation achieved with isoprenaline. Even more marked synergistic interactions were observed between isoprenaline and either the nitrovasodilators or the cAMP phosphodiesterase inhibitors when these compounds were added simultaneously before contraction of aortic smooth muscle by phenylephrine. Thus, concentrations of SNP (5 nM), SIN-1 (1 microM), cilostamide (1 microM) and Ro 20-1724 (100 microM) that inhibited contraction by less than 30% decreased the IC50 values for isoprenaline by 8- to 10-fold. At the above concentrations, these compounds each caused a supra-additive inhibition of contraction when added with 100 nM isoprenaline. Thus, synergism between nitrovasodilators and isoprenaline, an activator of adenylyl cyclase, could be detected in vascular smooth muscle and was particularly marked when inhibition of contraction was studied. This action of nitrovasodilators resembled that of inhibitors of cAMP phosphodiesterase.

Adenylyl Cyclases↗
PubMed1991

Effects of nitrovasodilators on platelet cyclic nucleotide levels in rabbit blood; role for cyclic AMP in synergistic inhibition of platelet function by SIN-1 and prostaglandin E1.

Nitrovasodilators increase both cyclic GMP and cyclic AMP in isolated platelets (Maurice DH, Haslam RJ. Mol Pharmacol 1990;37:671-81). To determine whether this occurs in blood, platelet cyclic[3H]GMP and cyclic [3H]AMP were measured in prelabeled rabbit platelets resuspended in modified Tyrode's solution or citrated blood. In the former medium, increases in cyclic [3H]nucleotides in response to nitroprusside (NP) and 3-morpholinosydnonimine (SIN-1) were maximal by 1 min; in blood, maximal increases were observed only after 10 min and were much smaller. In blood, SIN-1 was more effective than the same concentration of NP. After 10 min, 100 microM SIN-1 increased platelet cyclic[3H )GMP by 475 +/- 58% and cyclic[3H]AMP by 29 +/- 7% (means +/- SEM, 18 experiments). Supraadditive increases in platelet cyclic [3H]AMP in blood were observed when SIN-1 was combined with prostaglandin E1 (PGE1). Thus, after 10 min, SIN-1 (100 microM), PGE1 (20 nM), and SIN-1 + PGE1 increased cyclic[3H]AMP by 25 +/- 7, 35 +/- 6, and 130 +/- 17%, respectively (four experiments). In the same experiments, release of platelet [14C]serotonin by platelet-activating factor (PAF) was inhibited by 22 +/- 5, 2 +/- 2, and 61 +/- 5%, respectively. Increases in platelet cyclic[3H]GMP with SIN-1 were unaffected by PGE1. These results suggest that although cyclic GMP may mediate the effects of SIN-1 alone on platelet function, cyclic AMP mediates the synergistic action of SIN-1 and PGE1. M&B 22,948 (a selective cyclic GMP phosphodiesterase inhibitor) enhanced the increases in platelet cyclic[3H]GMP and cyclic[3H]AMP caused by SIN-1 and also increased the associated inhibition of [14C]serotonin release. M&B 22,948 also augmented the synergistic increases in cyclic[3H]AMP and inhibition of platelet function caused by SIN-1 + PGE1. The results show that a selected nitrovasodilator (e.g., SIN-1), a prostaglandin and a cyclic GMP phosphodiesterase inhibitor can exert synergistic effects on platelets in blood. This may be relevant to the pharmacologic management of thromboembolic disease.

Alprostadil↗
PubMed1991

Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype.

We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.

Amino Acid Sequence↗
PubMed1990

Nitroprusside enhances isoprenaline-induced increases in cAMP in rat aortic smooth muscle.

Low concentrations of sodium nitroprusside (SNP) and of isoprenaline acted synergistically to inhibit the phenylephrine-induced contraction of rat aortic smooth muscle. In experiments with these concentrations, SNP enhanced the increases in smooth muscle cAMP caused by isoprenaline by 4- to 5-fold, whereas the SNP-induced increases in tissue cGMP were unaffected by isoprenaline. We conclude that cAMP is likely to mediate the synergistic inhibition of the contraction of rat aortic smooth muscle by these compounds.

Animals↗
PubMed1990

Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5'-[gamma-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C.

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.

Animals↗
PubMed1990

Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins.

Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins.

Antibodies↗
PubMed1990

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.

Blood Platelets↗
PubMed1990

Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP.

We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by different concentrations of SNP; this relationship was not affected by addition of M&B 22,948. The results strongly suggest that the increases in platelet cyclic [3H]AMP caused by nitrovasodilators in the presence or absence of activators of adenylate cyclase are mediated by the inhibition by cyclic GMP of cyclic AMP breakdown.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1987

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport.

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.

Anti-Bacterial Agents↗
PubMed1979

Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets.

Human platelets that had been preincubated with 5-hydroxy[(3)H]tryptamine and [(32)P]P(i) were stirred with various agents; the secretion of 5-hydroxy[(3)H]tryptamine from platelet granules and the radioactivity of platelet [(32)P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[(3)H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E(1), which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[(3)H]tryptamine. Prostaglandin E(1) also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca(2+) concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714-722; (1978) Thromb. Res.12, 619-628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E(1) may initiate processes that decrease the Ca(2+) concentration in the cytosol, so inhibiting both the Ca(2+)-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E(1) did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca(2+) concentration in the platelet cytosol. As prostaglandin E(1) did inhibit the release of 5-hydroxy[(3)H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.

Anti-Bacterial Agents↗
PubMed1978

Cyclic nucleotides in platelet function.

Inhibition of adenylate cyclase in intact platelets by addition of compounds such as 2', 5' - dideoxyadenosine prevented the inhibition of platelet aggregation by PGE1 but did not affect the responses of platelets to aggregating agents in the absence of PGE1. This confirms that cyclic AMP mediates the effects of PGE1 but indicates that the level of cyclic AMP in unstimulated platelets is too low to affect the actions of aggregating agents. Studies on the phosphorylation of proteins in intact 32P-labelled platelets showed that PGE1 increased the phosphorylation of a membrane-bound polypeptide (P24) and prevented the increased phosphorylation of other polypeptides (P47 and P20) that occurred on addition of inducers of the release reaction. It is suggested that the cyclic AMP-dependent phosphorylation of P24 stimulates the active transport of Ca(2+) out of the platelet cytosol, so preventing phosphorylation of P47 and P20, reactions which may be involved in the release mechanism. As increases in platelet cyclic GMP could be dissociated from both platelet aggregation and the release reaction, it is proposed that the bidirectional regulation of platelet function is achieved primarily by the opposing actions of increases in the concentrations of Ca(2+) and cyclic AMP.

Blood Platelets↗
PubMed1978

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

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

Adenosine↗
PubMed1978

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

Blood Platelets↗