Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and thrombin in permeabilized human platelets.

It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 micrograms/ml saponin allows ADP-ribosylation by pertussis toxin of the alpha i-subunit of Gi (Ni), a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate- (GTP[gamma S]-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3H]inositol. Phospholipase C activity was measured by [3H]polyphosphoinositide decreases and formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the alpha i-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Gi-protein was not directly related to activation or inhibition of platelet phospholipase C by GTP [gamma S]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C.

Properties and distribution of phosphatidylinositol-specific phospholipase C in human and horse platelets.

Phospholipase C has been studied in homogenates, total particulate and soluble fractions of horse and human platelets. This enzyme, assayed with exogenous L-3-phosphatidyl[14C]inositol, is predominantly localized in the soluble fraction and its distribution parallels that of lactate dehydrogenase. A small percentage of activity present in the particulate fraction seems to be due to contamination with soluble enzyme. Enzyme from horse and human platelets appears identical, having a Km of 0.10-0.15 mM, acid pH optimum (pH 5.5) and showing Ca2+-dependency and weak inhibition by deoxycholate. Analysis of the reaction products shows the formation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate in almost equal amounts. Platelet stimulation with thrombin does not seem to induce association of the cytosolic activity to the membranes. The cytosolic activity is not affected by pretreatment of the intact platelets with prostacyclin or thrombin. Degradation of phosphatidylinositol present in a membrane fraction isolated from platelets by cytosolic phospholipase C requires addition of deoxycholate. Our information suggests that the degradation of phosphatidylinositol in stimulated platelets is mainly achieved by exposure of the substrate to the cytosolic enzyme and by an increase of the free Ca2+ concentration needed for optimal phospholipase C activity.

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin.

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.

Dihomogammalinolenic acid, but not eicosapentaenoic acid, activates washed human platelets.

Dihomogammalinolenic acid (2.5-20 microM) added to suspensions of washed human platelets induces platelet shape change and the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of phospholipase C. It also stimulates the phosphorylation of a 40 kDa protein, indicating the activation of protein kinase C. Dihomogammalinolenic acid is converted mainly to 12-hydroxyheptadecadienoic acid and to a smaller extent to prostaglandin E1 and thromboxane B1. Small quantities of the lipoxygenase product 12-hydroxyeicosatrienoic acid are also observed. Indomethacin, by blocking platelet cyclooxygenase, prevents the activation of phospholipase C, protein kinase C, and platelet shape change induced by dihomogammalinolenic acid. Compound UK 38485, a specific thromboxane synthetase inhibitor, does not block platelet activation induced by dihomogammalinolenic acid. The results indicate that endoperoxides derived from dihomogammalinolenic acid, such as prostaglandin G1 or prostaglandin H1, may be responsible for the stimulation of phospholipase C and protein kinase C, and for the induction of platelet shape change. Eicosapentaenoic acid does not activate platelets and is poorly metabolized by platelet cyclooxygenase and lipoxygenase. Eicosapentaenoic acid is a better inhibitor of platelet activation induced by various agonists in washed platelets than dihomogammalinolenic acid. Eicosapentaenoic acid and dihomogammalinolenic acid are, however, equally effective in inhibiting aggregation induced by collagen in platelet-rich plasma. We suggest that eicosapentaenoic acid might be a better antithrombotic agent than dihomogammalinolenic acid.

Effect of adrenergic agonists on phosphatidylinositol labelling in heart and aorta.

(1) The metabolism of phosphatidylinositol has been investigated in heart fragments and aorta slices incubated with adrenergic agonists. (2) Noradrenaline and isoprenaline had no stimulatory effect on 32Pi incorporation into phosphatidylinositol in cat and guinea-pig hearts. (3) Incorporation of 32Pi into phosphatidylinositol was enhanced by noradrenaline and methoxamine in cat aorta. (4) This information is consistent with the idea that the enhanced phosphatidylinositol turnover produced during adrenergic stimulation is mediated through alpha-adrenergic receptors and not through beta-adrenoceptors.

Preparation of 32P-labeled inositides and their degradation by soluble kidney enzymes.

A method is described for the preparation of radioactive inositol lipids for studies of their enzymic degradation. Kidney cytosol fractions have been used to produce diesteratic cleavage. High voltage electrophoresis at pH 4.3 is used to separate D-myoinositol 1 : 2-cyclic phosphate and D-myoinositol 1-phosphate from hydrolysis of phosphatidylinositol. Radioactivity co-migrating with myoinositol diphosphate and triphosphate is separated by electrophoresis at pH 1.5 following enzymatic hydrolysis of phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Relative activities for hydrolysis of the various inositides suggest the presence of more than one phosphodiesterase.

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.

Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction.

Insulin-like growth factor-I (IGF-1) stimulates the production of 3-phosphoinositides and increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by antiphosphotyrosine antibodies, a small portion of which are also associated with the IGF-1 receptor. In vitro reconstitution experiments showed that p85 associates with high affinity to the IGF-1 receptor and this interaction is mediated through the p85 SH2 groups. Moreover, in vitro, p85 is a substrate for the IGF-1 receptor tyrosine kinase activity. In this study, we analyzed the in vivo association of p85 with tyrosyl- phosphorylated proteins and its tyrosyl phosphorylation state, in response to IGF-1. After stimulation with IGF-1, the major tyrosylphosphorylated protein that was associated with p85 was a 185-kilodalton protein, identified as IRS-1. Only a small fraction of p85 was associated with the IGF-1 receptor. In contrast, the PDGF receptor was the major protein associated with p85 upon stimulation. Neither ligand stimulated the tyrosyl phosphorylation of p85 in vivo. In order to determine whether the SH2 domains of p85 were involved in its association with p185 in vivo after IGF-1 stimulation, different SH2-constructs of p85 were expressed in COS-1 cells. After stimulation with IGF-1, the expressed SH2 proteins were immunoprecipitated with specific antibodies, and associated p185 was detected on Western blots. These results show that both the p85 N-SH2 and N+C-SH2 associate with IRS-1 after IGF-1 stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Didecanoylglycerol and phorbol 12,13-dibutyrate enhance anterior pituitary hormone secretion in vitro.

Experiments were designed to evaluate the role of activators of protein kinase C, such as 1,2-diacylglycerol and phorbol esters, on the release of all the anterior pituitary (AP) hormones in vitro. Dispersed rat AP cells were incubated in the presence of 1,2-didecanoylglycerol (DiC10), a synthetic diacylglycerol, or phorbol 12,13-dibutyrate (PDBu), a tumor-promoting phorbol ester, at different concentrations and for varying periods of time. ACTH and beta-endorphin (beta-End) secretion were enhanced by DiC10 in a concentration-dependent manner, with a minimal effective concentration of 5 microM. PDBu at 5 nM produced a significant release of both ACTH and beta-End. The effect of DiC10 and PDBu was time dependent, with maximal responses occurring at 15-30 min for DiC10 and 30-60 min for PDBu. Release of GH was also enhanced significantly by DiC10 and PDBu, with minimal effective concentrations of 1 microM and 1 nM, respectively. Maximal release of GH was already attained within 15 min with DiC10 or 60 min with PDBu. In additional experiments, the effects of DiC10 and PDBu on secretion of LH, FSH, PRL, and TSH were evaluated. The results indicate that 5-25 microM DiC10 produced a concentration-dependent release of each of those hormones, and that 5 microM was the minimal effective concentration in every case. Nearly maximal stimulation was achieved within 15 min for each hormone. PDBu (50 nM) significantly enhanced LH, FSH, PRL, and TSH release within 30 min. Although qualitatively all hormones were similarly stimulated, both with respect to time and concentration, some quantitative differences were observed. ACTH and beta-End release were enhanced 100% by DiC10 and 300% by PDBu, whereas the increase in other hormones was of a lesser magnitude. The present study indicates that two specific stimulators of protein kinase C, diacylglycerol and phorbol ester, can enhance secretion of all AP hormones in a concentration- and time-dependent manner. This suggests that formation of endogenous 1,2-diacylglycerol may represent a physiological intracellular messenger in the events leading to AP peptide hormone release.

Insulin like growth factor-I induces limited association of phosphatidylinositol 3-kinase to its receptor.

Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell line that overexpresses human IGF-I receptors, led to an increase in the amount of a phosphatidylinositol kinase that could be immunoprecipitated by anti-IGF-I receptor or anti-phosphotyrosine antibodies. The identity of the lipid produced in phosphatidylinositol kinase assays of anti-IGF-I receptor or anti-phosphotyrosine immunoprecipitates indicated that IGF-I selectively increased the amount of immunoprecipitated phosphatidylinositol 3-kinase activity. The amount of immunoprecipitated phosphatidylinositol 3-kinase activity that was increased by IGF-I followed a time course that paralleled the stimulation of IGF-I receptor beta-subunit autophosphorylation. The amount of phosphatidylinositol 3-kinase activity detected in anti-IGF-I receptor immunoprecipitates represented only 2% of that which was immunoprecipitated by anti-phosphotyrosine antibody. Furthermore, phosphatidylinositol 3-kinase activity which was recovered with anti-phosphotyrosine antibody was present in both cytosol and particulate cell fractions at approximately similar levels. Taken together, these results suggest that the stimulation of the IGF-I receptor tyrosine kinase leads to an increase in the amount of phosphatidyl inositol 3-kinase activity immunoprecipitated by antiphosphotyrosine and anti-IGF-I receptor antibodies and to a limited association with the IGF-I receptor itself, even though these cells express very high levels of IGF-I receptors. That the majority of phosphatidylinositol 3-kinase activity does not tightly associate with the IGF-I receptor after IGF-I stimulation suggests that it may be associated with other tyrosine phosphorylated proteins. Alternatively, the kinase itself may become phosphorylated on tyrosine and dissociate from the IGF-I receptor. In this manner, an increase of phosphatidylinositol 3-kinase activity by IGF-I deviates from the activation of phosphatidylinositol 3-kinase by platelet-derived growth factor receptor in that a tight association with the receptor is not produced after stimulation.

The signal transduction induced by thrombin in human platelets.

The stimulation of human platelets by thrombin leads to the activation of phospholipases C and A2, protein kinases, formation of 3-inositol phospholipids and mobilization of Ca2+. These biochemical reactions closely parallel platelet shape change, granular secretion and aggregation. The membrane-bound transducers for the thrombin receptor seem to be the heterotrimeric G protein Gi2 and the ras-related G protein rap 1-b. Phosphorylation of rap 1-b by the action of the cyclic AMP-dependent protein kinase seems to uncouple the thrombin receptor from phospholipases. This causes inhibition of the formation of second messenger molecules and the onset of physiological responses.

Incorporation of synthetic 1,2-diacylglycerol into platelet phosphatidylinositol is increased by cyclic AMP.

1,2-Didecanoylglycerol (diC10) is taken up by human platelets and sequentially converted to 1,2-didecanoylphosphatidic acid (PA10) and 1,2-didecanoylphosphatidylinositol (PI10). Agents that increase cyclic AMP in platelets, such as prostacyclin and forskolin, sequentially convert diC10 to PA10 and PI10. They decrease formation of PA10 with a parallel accumulation of PI10. This might reflect an inhibition of phosphatidylinositol kinase.

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists.

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.

Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro.

The cornea has one of the highest acetylcholine (ACh) concentrations of any tissue but the function of the ACh has remained enigmatic. During studies on corneal arachidonic acid metabolism, we observed that ACh stimulates formation of labeled phosphatidic acid in rat corneas whose phospholipids were prelabeled with [14C]arachidonate. ACh did not affect the metabolism of free [14C]arachidonate. [14C]Arachidonyl-phosphatidic acid formation was doubled after 10 min of incubation in the presence of ACh concentrations of 10(-4) M or greater. The stimulation by ACh could be completely blocked by atropine and scopolamine and partially blocked by d-tubocurarine. These studies suggest that intact rat cornea has muscarinic cholinergic receptors and that the enzymes of the inositol phospholipids pathway are present since phosphatidic acid is an obligatory intermediate in that cycle of reactions.

Maturation of megakaryoblastic cells is accompanied by upregulation of G(s)alpha-L subtype and increased cAMP accumulation.

In platelets and megakaryoblasts Gs, the trimeric G-protein that stimulates adenylyl cyclase, is present in a short, 45 kDa, and a long, 52 kDa isoform termed G(s)alpha-S and G(s)alpha-L, respectively. To assess the relative contribution of these isoforms in the cellular synthesis of cAMP, the ratio G(s)alpha-S/G(s)alpha-L was changed in the megakaryoblastic cell line DAMI by inducing cell maturation with recombinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cytometric analysis confirmed that this treatment induced a moderate (TPO) and extensive (PMA) increase in nuclear ploidy and expression of the glycoproteins-IIIa and -Ib. Northern blot analysis revealed downregulation of total Gs-mRNA after treatment of DAMI-cells with TPO and PMA. Western blot analysis showed significant (P < 0.05) upregulation of Gs-L with respective amounts of 27 +/- 4% of total Gs in untreated cells, 35 +/- 1% in TPO- and 41 +/- 3% in PMA-treated DAMI cells (n = 3-4). DAMI cells contained 6 +/- 1 pmol cAMP/10(6) cells, which was not changed by treatment with TPO or PMA. In untreated cells this level increased to 70 +/- 9 pmol cAMP/10(6) cells after 10 min stimulation with 1 micromol/l of the stable prostacyclin analog iloprost. The same stimulation with iloprost resulted in 165 +/- 32 pmol cAMP/10(6) in TPO-treated cells and in 588 +/- 100 pmol cAMP/10(6) in cells treated with PMA. Thus, a shift from G(s)alpha-S to G(s)alpha-L during megakaryoblast maturation strongly potentiates the production of cAMP. A similar shift may occur during normal megakaryocyte maturation and may explain the extreme sensitivity to prostacyclin of platelets, which contain G(s)alpha-S and G(s)alpha-L in approximately equal amounts.

Regulation of hormone-induced Ca2+ mobilization in the human platelets.

alpha-Thrombin, gamma-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca2+ stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca2+ level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on alpha 2-adrenergic receptors, was by itself ineffective at mobilizing Ca2+ stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca2+ from cellular stores. This appeared to be due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. Our current work is to characterize this protein that may be a member of the Gi, GTP-binding protein family.

Sustained proteolysis is required for human platelet activation by thrombin.

A maximally effective dose of indomethacin does not prevent serotonin release and aggregation in human platelets stimulated with thrombin. Thrombin induces rapid activation of inositol phospholipids-specific phospholipase C, which is reflected by the degradation of inositides and the phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Thrombin also activates protein kinase C and myosin light chain kinase as indicated by phosphorylation of the 40,000 and 20,000 dalton proteins, respectively. Leupeptin, a protease inhibitor that does not inhibit thrombin's proteolytic activity or its binding to platelet surface, is able to reverse platelet activation by thrombin when it is administered after the addition of the agonist and indomethacin. The results suggest a proteolytic-mediated pathway in transmembrane signalling involved in platelet activation by thrombin.

Adhesion of human platelets to collagen in the presence of prostacyclin, indomethacin and compound BW 755C.

Prostacyclin (1 ng to 2 micrograms per ml), which effectively inhibits platelet secretion and aggregation, does not affect adhesion of a proportion of platelets (10-38%) to collagen (50-100 micrograms/ml). Adhesion is not detectable by changes of light transmission (as measured in the optical aggregometer) and is not affected by inhibitors of cyclooxygenase and lipoxygenase enzymes such as indomethacin and compound BW 755C. This adhesion is independent of the collagen concentration (50-400 micrograms/ml) and the incubation time (5-20 min). This suggests that adhesion to collagen is related to a specific platelet population. Adhesion in the presence of prostacyclin, indomethacin and BW 755C occurs in parallel with the formation of a limited amount of phosphatidic acid. Under those conditions it is also possible to observe some phosphorylation of a 40,000 dalton protein which is a substrate for protein kinase C activity. Phosphorylation of the 20,000 dalton protein, or myosin light chain, is less evident. Chlorpromazine (25-100 micrograms/ml) inhibited the adhesion of platelets to collagen, but propanolol (0.5-4 microM) was inactive. The adhesion of platelets to collagen in these experiments parallels the formation of a fraction of phosphatidic acid and 40,000 dalton protein phosphorylation, which are independent of the increased levels of platelet cyclic-AMP induced by high concentrations of prostacyclin. It is also independent of the formation of cyclooxygenase or lipoxygenase products.

Metabolism of inositides and the activation of platelets.

Platelet activation is associated with the active metabolism of inositide lipids. Phosphodiesteratic cleavage of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate is a consequence of receptor-coupled mechanisms. Degradation of phosphatidylinositol-4,5-biphosphate is Ca2+-insensitive while that of phosphatidylinositol requires Ca2+. The phosphodiesteratic breakdown of these inositides induces the formation of 1,2-diacylglycerol which is rapidly phosphorylated to phosphatidic acid. These biochemical changes might be related to fundamental mechanisms of amplication involved in the process of platelet activation. Phosphatidic acid constitutes an ubiquitous marker for the action of a wide variety of platelet stimuli.

The role of phospholipase C in platelet responses.

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.