Structure of the carboxy-terminal PH domain of pleckstrin at 2.1 Angstroms.

Pleckstrin is an important intracellular protein involved in the phosphoinositide-signalling pathways of platelet activation. This protein contains both N- and C-terminal pleckstrin-homology (PH) domains (N-PH and C-PH). The crystal structure of C-PH was solved by molecular replacement and refined at 2.1 Angstroms resolution. Two molecules were observed within the asymmetric unit and it is proposed that the resulting dimer interface could contribute to the previously observed oligomerization of pleckstrin in resting platelets. Structural comparisons between the phosphoinositide-binding loops of the C-PH crystal structure and the PH domains of DAPP1 and TAPP1, the N-terminal PH domain of pleckstrin and a recently described solution structure of C-PH are presented and discussed.

Analyses of proteins involved in vesicular trafficking in platelets of mouse models of Hermansky Pudlak syndrome.

Hermansky Pudlak syndrome (HPS) is an autosomal recessive inherited disorder characterized by defects in synthesis and/or secretion of three related subcellular organelles: melanosomes, platelet-dense granules, and lysosomes. In the mouse, mutant forms of any of 14 separate genes result in an HPS-like phenotype. The mouse pearl and mocha genes encode subunits of the AP3 adaptor protein complex, confirming that HPS mutations involve proteins regulating intracellular vesicular trafficking. Therefore, expression of several additional proteins involved in vesicular transport was examined by immunoblotting of platelet extracts from HPS mutant and control mice. Platelet levels of SCAMPS (secretory carrier membrane proteins), Rab11, Rab31, NSF (N-ethylmaleimide-sensitive fusion protein), syntaxin 2, syntaxin 4, munc18c, and p115/TAP (p115/transcytosis-associated protein) were not significantly altered in several different HPS mutants. However, gunmetal (gm/gm) platelets contained decreased amounts of SNAP-23. The Snap23 gene was mapped to mouse chromosome 5, demonstrating it cannot encode the gm gene, which maps to chromosome 14. It is likely therefore that the gm gene functions upstream of SNAP-23 in vesicular trafficking.

Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets.

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398+/-50 nM and 252+/-16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16+/-0.01 in intact platelet enzyme, to 0.37+/-0.05 in cGI-PDEDelta1 and to 0.49+/-0.04 in cGI-PDEDelta2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [32P]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and [32P]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.

Activation of cGMP-stimulated phosphodiesterase by nitroprusside limits cAMP accumulation in human platelets: effects on platelet aggregation.

cGMP enhances cAMP accumulation in platelets via cGMP-inhibited phosphodiesterase (PDE3) [Maurice and Haslam (1990) Mol. Pharmacol. 37, 671-681]. However, cGMP might also limit cAMP accumulation by activating cGMP-stimulated phosphodiesterase (PDE2). We therefore evaluated the role of PDE2 in human platelets by using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to inhibit this enzyme selectively. IC50 values for the inhibition of platelet PDE2 by EHNA, with 10 microM cAMP as substrate in the absence and in the presence of 1 microM cGMP, were 15 and 3 microM respectively. Changes in platelet cyclic [3H]nucleotides were measured after prelabelling with [3H]adenine and [3H]guanine. Nitroprusside (NP) caused concentration-dependent increases in [3H]cGMP and a biphasic increase in [3H]cAMP, which was maximal at 10 microM (49+/-6%) and smaller at 100 microM (32+/-6%) (means+/-S.E.). In the presence of EHNA (20 microM), which had no effects alone, NP caused much larger increases in platelet [3H]cAMP (125+/-14% at 100 microM). EHNA also enhanced [3H]cGMP accumulation at high NP concentrations. In accord with these results, EHNA markedly potentiated the inhibition of thrombin-induced platelet aggregation by NP. The roles of cAMP and cGMP in this effect were investigated by using 2', 5'-dideoxyadenosine to inhibit adenylate cyclase. This compound decreased the accumulation of [3H]cAMP but not that of [3H]cGMP, and diminished the inhibition of platelet aggregation by NP with EHNA. We conclude that much of the effect of NP with EHNA is mediated by cAMP. Lixazinone (1 microM), a selective inhibitor of PDE3, increased platelet [3H]cAMP by 177+/-15%. This increase in [3H]cAMP was markedly inhibited by NP; EHNA blocked this effect of NP. Parallel studies showed that NP suppressed the inhibition of platelet aggregation by lixazinone. EHNA enhanced the large increases in [3H]cAMP seen with 20 nM prostacyclin (PGI2), but had no effect with 1 nM PGI2. NP and 1 nM PGI2 acted synergistically to increase [3H]cAMP, an effect attributable to the inhibition of PDE3 by cGMP; EHNA greatly potentiated this synergism. In contrast, NP decreased the [3H]cAMP accumulation seen with 20 nM PGI2, an effect that was blocked by EHNA. The results show that, provided that cGMP is present, PDE2 plays a major role in the hydrolysis of low cAMP concentrations and restricts any increases in cAMP concentration and decreases in platelet aggregation caused by the inhibition of PDE3. At high cAMP, PDE2 plays the major role in cAMP breakdown, whether cGMP is present or not.

Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins.

Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane-associated Ca2+- and GTP-binding proteins and occurs independently of PKC activation.

Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C.

The major substrate of protein kinase C(PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100-120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100-120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.

Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.

Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors.

Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC50 = 19 +/- 3 nM), lixazinone (EC50 = 122 +/- 8 nM), milrinone (EC50 = 5320 +/- 970 nM) and siguazodan (EC50 = 18880 +/- 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III.

Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

Synergistic inhibitory effects of atriopeptin II and isoproterenol on contraction of rat aortic smooth muscle: roles of cGMP and cAMP.

Atriopeptin II and isoproterenol acted synergistically to inhibit the phenylephrine-induced contraction of aortic smooth muscle from Wistar-Kyoto (WKY) rats. Thus, a weakly inhibitory concentration of atriopeptin II (10 nM) caused a 5-fold decrease in the IC50 of isoproterenol from 169 nM to 32 nM, whereas a low concentration of isoproterenol (100 nM) increased the maximum inhibition attributable to atriopeptin II from 43% to 74%. Atriopeptin II (10 nM) increased the cGMP found in aortic smooth muscle and approximately doubled the accumulation of cAMP caused by isoproterenol. The results suggest that cGMP, formed by the action of atriopeptin II on receptor guanylyl cyclase (GC-A), may inhibit aortic cyclic nucleotide phosphodiesterase type III (PDE III) and that an increased accumulation of cAMP then mediates the observed synergism.

Measurement of both cyclic [3H]AMP and cyclic [3H]GMP in cultured vascular smooth muscle cells labeled with [3H]hypoxanthine: use in studies of cardiovascular drugs.

We describe a method for prelabeling cultured vascular smooth muscle cells that permits rapid and accurate measurements of changes in the amounts of cyclic AMP and of cyclic GMP in 5 x 10(5) cells. This procedure utilizes [3H]hypoxanthine to radiolabel both the adenine and guanine nucleotide pools and simple column chromatographic steps to isolate and separate the 3H-labeled cyclic nucleotides. The application of the method to studies of the actions of cardiovascular drugs on vascular smooth muscle cells is illustrated by measurements of the effects of isoproterenol, nitroprusside, and inhibitors of cyclic AMP phosphodiesterases on the cyclic nucleotide levels in these cells. If required, the mass amounts of cyclic AMP and cyclic GMP present could be determined by measurement of the specific radioactivities of the precursor [3H]ATP and [3H]GTP, respectively. The cyclic nucleotide values calculated by the latter method were almost identical to those obtained with larger numbers of cells using commercially available radioimmunoassays, thus validating the prelabeling assays. The method described should be applicable to any type of cultured cell that can utilize [3H]hypoxanthine to replenish its ATP and GTP pools.

Photoaffinity labelling of cyclic GMP-binding proteins in human platelets.

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.

GTP gamma S and phorbol ester act synergistically to stimulate both Ca(2+)-independent secretion and phospholipase D activity in permeabilized human platelets. Inhibition by BAPTA and analogues.

We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca(2+)-independent exocytosis in platelets. Although GTP gamma S, and to a lesser extent phorbol 12-myristate 13-acetate (PMA), caused some secretion of 5-HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5-HT was closely correlated to the stimulus-induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonate-labelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose-dependent inhibitions of both GTP gamma S-induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.

Evidence that activation of phospholipase D can mediate secretion from permeabilized platelets.

Studies on electropermeabilized human platelets indicated that any two of three distinct factors must be present for marked secretion of dense or alpha-granule constituents to occur. These factors are Ca2+, activation of protein kinase C (PKC) and activation of an unidentified GTP-binding protein ('GE'). Thus, in the absence of Ca2+, phorbol ester and GTP[S] acted synergistically to promote secretion, whereas in the presence of Ca2+, either activation of PKC or addition of GTP[S] was sufficient. In all cases, secretion correlated with the activation of phospholipase D (PLD), as detected by the formation of [3H]phosphatidic acid (PA) in the absence of ethanol or of [3H]phosphatidylethanol (PEt) in the presence of ethanol. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca2+ and were inhibited by phorbol ester. Ethanol partially inhibited secretion in the absence of Ca2+. BAPTA, a known inhibitor of Ca(2+)-independent secretion in permeabilized cells, caused parallel inhibitions of secretion and PLD activity. GTP[S] enhanced PKC activity, as indicated by pleckstrin phosphorylation, apparently by stimulating the formation of PA in the absence of Ca2+, as well as of DAG in the presence of Ca2+. PA and stable analogues, including PEt, stimulated the Ca(2+)-independent phosphorylation of pleckstrin and other proteins in platelet supernatant fraction. The results suggest that PA formed by activation of PLD may mediate secretion from permeabilized platelets by PKC-dependent and independent mechanisms. However, in intact platelets stimulated by thrombin, PLD accounted for only 10-20% of the total PA formed and can only play a major role in secretion if this PA fraction is distinct from that formed by the combined actions of PLC and DAG kinase.

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.

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.

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.

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.