Ceramide-induced vasorelaxation: An inhibitory action on protein kinase C.

Experiments were designed to examine the role of sphingosine, PP2A phosphatases, and protein kinase C (PKC) inhibition in mediating the vasodilatory effects of ceramide in rat thoracic aorta. Sphingosine did not cause vasorelaxation, and oleoylethanol-amine, a ceramidase inhibitor, did not affect sphingomyelinase-induced relaxation. Okadaic acid potentiated the relaxation response to ceramide. These observations rule out involvement of sphingosine and PP2A phosphatases in mediating ceramide-induced relaxation. Sphingomyelinase attenuated contractile and single-cell intracellular calcium responses to phorbol ester. Chelerythrine incubation potentiated the relaxation response to ceramide. These observations support a role for PKC inhibition in mediating the vasodilatory effects of ceramide.     

Protein kinase C inhibits TRH-stimulated phosphoinositide hydrolysis in GH3 cells

The GH3 pituitary tumor line expresses TRH receptors that stimulate phosphoinositide hydrolysis and hormone secretion. After protein kinase C was identified in GH3 cells by direct labeling with [3H]phorbol dibutyrate (PDB), the response to phorbol ester and TRH pretreatment on subsequent TRH-stimulated inositol phosphate (IP) accumulation was found to be inhibitory. Both phorbol myristate acetate (PMA) and PDB were effective in this regard at low nM concentrations within a few minutes, whereas phorbols that do not stimulate protein kinase C were without effect. Furthermore, the mono-, bis- and tris-phosphate forms of IP were all reduced by an average of 30-40% after 5 min of PMA. TRH concentration-response studies indicated a clear change in TRH efficacy induced by PMA. Finally, preincubation with TRH itself was also capable of reducing the subsequent response to TRH. Because TRH receptor action is thought to activate protein kinase C by producing diacylglycerol, these data indicate a negative feedback system via protein kinase C operative during continuous exposure to TRH in GH3 cells.     

Pharmacological modification of mechanical and electrical responses of frog heart to thrombin

Summary The pharmacological modification of the thrombin effect on the mechanical and electrical responses of frog heart was examined in the Straub heart preparation and in single ventricular cells.Associated with the positive inotropic action thrombin increases voltage and duration of action potentials of isolated frog ventricular cells. As found by the patch-clamp technique in the cell-attached mode, thrombin stimulates single l-type Ca²⁺ channels, presumably mediated by a second messenger. The enhancement of contractility by thrombin depends on the proteolytic activity of the enzyme because enzymatically inactivated thrombin has no effect on frog hearts.The positive inotropic effect of thrombin as well as its stimulation of Ca²⁺ channel currents were inhibited by the proein kinase C blocker 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). However, phorbol 12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, was ineffective in stimulating the inotropic action of thrombin. The inhibition of the thrombin-induced enhancement of contractility by indometacin indicates an involvement of arachidonic acid in the action of thrombin on frog heart. Send offprint requests to B. Nilius at the above address     

Regulation of human basophil function by phosphatase inhibitors.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.     

Protein phosphatase-protein kinase interplay modulates alpha 1b-adrenoceptor phosphorylation: effects of okadaic acid

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.     

Inhibition of platelet activation by tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.     

Effects of bosentan (Ro 47-0203), an ETA-, ETB-receptor antagonist, on regional haemodynamic responses to endothelins in conscious rats.

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.     

Enhancement of cyclic AMP modulated salivary amylase secretion by protein kinase C activators

The effect of protein kinase C activators on isoproterenol-induced amylase secretion were investigated in isolated rat parotid cells. Pretreatment with phorbol dibutyrate potentiated isoproterenol-induced amylase secretion. This effect of phorbol dibutyrate was mimicked by dioctanoylglycerol or carbachol. Phorbol dibutyrate also potentiated secretion evoked by the adenylate cyclase activator forskolin and by dibutyryl cAMP. Neither phorbol dibutyrate nor carbachol enhanced isoproterenol-induced cAMP accumulation. The present study reveals a coordinate interaction between cAMP and protein kinase C at a step in the secretory mechanism distal to cAMP generation.     

YD-3, a novel inhibitor of protease-induced platelet activation

1. In the present study, the antiplatelet effects and mechanisms of a new synthetic compound YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole] were examined. 2. YD-3 inhibited the aggregation of washed rabbit platelets caused by thrombin (IC(50)=28.3 microM), but had no or little inhibitory effect on that induced by arachidonic acid, collagen, platelet-activating factor (PAF) or U46619. YD-3 also suppressed generation of inositol phosphates caused by thrombin. On the other hand, thrombin-induced fibrin formation was not affected by YD-3, indicating YD-3 does not inhibit the proteolytic activity of thrombin. 3. In washed human platelets, however, YD-3 had only mild inhibitory effect on the low concentration (0.05 u ml(-1)) of thrombin-induced human platelet aggregation, and did not affect that induced by higher concentrations (> or =0.1 u ml(-1)) of thrombin or SFLLRN, the protease-activated receptor 1 (PAR1) agonist peptide. By contrast, YD-3 inhibited both human and rabbit platelet aggregation elicited by trypsin with IC(50) values of 38.1 microM and 5.7 microM, respectively. 4. YD-3, at 100 microM, had no effect on ristocetin-induced glycoprotein Ib (GPIb)-dependent aggregation of human platelets. In addition, platelets treated with chymotrypsin, which cleaves GPIb, enhanced rather than attenuated the inhibition of YD-3 on thrombin-induced human platelet aggregation. These data indicate that GPIb plays no role in the antiplatelet effect of YD-3. 5. In SFLLRN-desensitized human platelets, high concentration of thrombin (1 u ml(-1)) could still elicit intracellular Ca(2+) mobilization, and the rise of [Ca(2+)](i) was prevented by either leupeptin or YD-3. 6. Our results suggest that YD-3 inhibits a non-PAR1 thrombin receptor which mediates the major effect of thrombin in rabbit platelets, but in human platelets, this receptor function becomes significant only when the function of PAR1 has been blocked or attenuated.     

Cyclohexane diester analogues of phorbol ester as potential activators of protein kinase C

Phospholipid-dependent, Ca2(+)-sensitive protein kinase (protein kinase C) is activated by the plant product phorbol ester at nanomolar concentrations and also in vivo at micromolar concentrations by diacylglycerols. We designed and synthesized cyclohexane diester analogues of the phorbol ester C ring as potential high-affinity activators of protein kinase C. We proposed that the necessary pharmacophore of phorbol ester could be mimicked by diesters of appropriately substituted cyclohexanediols. A series of 1,2-cyclohexanediol diesters with different substituents at position 4 was synthesized. These substituents were designed to mimic the 6,7-double bond and C-20 hydroxy of phorbol ester. Competitive binding vs [3H]phorbol dibutyrate determined that these compounds have an affinity for protein kinase C of 1 mM or more, and thus they do not bind to nor are they activators of this enzyme.     

Inhibition by gomisin C (a lignan from Schizandra chinensis) of the respiratory burst of rat neutrophils.

1. The possible mechanisms of action of the inhibitory effect of gomisin C on the respiratory burst of rat neutrophils in vitro was investigated. 2. The peptide formyl-Met-Leu-Phe (FMLP) induced superoxide anion (O2-) formation and O2 consumption, which was inhibited by gomisin C in a concentration-dependent manner (IC50 21.5 +/- 4.2 micrograms ml-1 for O2- formation). Gomisin C also suppressed O2- formation and consumption at low concentrations of phorbol myristate acetate (PMA) with an IC50 value of 26.9 +/- 2.1 micrograms ml-1 for O2- formation. However, gomisin C did not affect the responses induced by a high concentration of PMA. 3. Gomisin C had no effect on O2- generation and uric acid formation in the xanthine-xanthine oxidase system, and failed to alter O2- generation during dihydroxyfumaric acid (DHF) autoxidation, indicating that it does not scavenge superoxide. 4. Like trifluoperazine (TFP), gomisin C attenuated the activity of PMA-activated neutrophil particulate NADPH oxidase in a concentration-dependent manner. 5. Gomisin C reduced the elevations of cytosolic free Ca2+ in neutrophils stimulated by FMLP in the presence or absence of EDTA. Cyclopiazonic acid (CPA) induced the release of Ca2+ from intracellular stores and this was also reduced by gomisin C. However, the Ca2+ influx pathway activated by CPA was not affected by gomisin C. 6. The cellular cyclic AMP level was markedly increased by forskolin, but not by gomisin C. Moreover, the inositol phosphate levels in FMLP-activated neutrophils were not affected by gomisin C. 7. These results show that the inhibitory action of gomisin C on the respiratory burst is not mediated by changes in cellular cyclic AMP or in inositol phosphates, or by scavenging O2- released from neutrophils, but may be mediated partly by the suppression of NADPH oxidase and partly by the decrease of cytosolic Ca2+ released from an agonist-sensitive intracellular store.     

Protein kinase C-mediated phosphorylation and desensitization of human alpha(1b)-adrenoceptors

Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.     

Okadaic acid increases the phosphorylation state of alpha1A-adrenoceptors and induces receptor desensitization

Okadaic acid, a protein phosphatase inhibitor, and phorbol myristate acetate, an activator of protein kinase C, increased the phosphorylation state of alpha1A-adrenergic receptors. The effects of these agents were of similar magnitude but that of okadaic acid developed more slowly. Wortmannin (inhibitor of phosphoinositide 3-kinase), but not staurosporine (inhibitor of protein kinase C), abolished the effect of okadaic acid on the alpha1A-adrenoceptor phosphorylation state. The effect of phorbol myristate acetate on this parameter was blocked by staurosporine and only partially inhibited by wortmannin. Okadaic acid markedly increased the co-immunoprecipitation of both the catalytic and regulatory subunits of phosphatidylinositol 3-kinase and of Akt/protein kinase B with the adrenoceptor and only marginally increases receptor association with protein kinase C epsilon. Okadaic acid induced desensitization of alpha1A-adrenoceptors as evidenced by a decreased ability of noradrenaline to increase intracellular calcium. Such desensitization was fully reverted by wortmannin. Our data indicate that inhibition of serine/threonine protein phosphatases increases the phosphorylation state of alpha1A-adrenergic receptor and alters the adrenoceptor function.     

Identification of protein kinase C phosphorylation sites involved in phorbol ester-induced desensitization of the histamine H1 receptor

The histamine H1 receptor (H1R)-mediated signaling cascade is inhibited by phorbol ester-induced protein kinase C (PKC) activation. Cloning studies of the H1Rs have shown that several potential PKC phosphorylation sites are located in the third intracellular loop of H1R. To elucidate the molecular mechanism of PKC-mediated desensitization, we identified amino acid residues that are involved in the desensitization of the H1R. Two amino acid residues (Ser396, Ser398) were determined to be PKC phosphorylation sites by in vitro phosphorylation studies using a series of synthetic peptides. Treatment with phorbol ester decreased histamine-induced accumulation of inositol phosphates in Chinese hamster ovary cells expressing the H1R with a rightward shift in the EC50 value, which implies the uncoupling of the receptor from the G protein. Site-directed mutagenesis studies showed that substitution of alanine for Ser398 but not for Ser396 markedly attenuated the effect of phorbol ester, which suggests that the Ser398 residue was primarily involved in PKC-mediated desensitization.     

Fluoride stimulates in vitro vascular prostacyclin synthesis: interrelationship of G proteins and protein kinase C

The role of G proteins in mediating adrenoceptor-prostacyclin synthesis coupling was investigated using the G protein activator, sodium fluoride. Sodium fluoride (NaF) stimulated in vitro rat aortic prostacyclin (PGI2) synthesis (EC50 = 5 x 10(-3) mol.l-1), an action inhibited completely by the presence of EDTA (10(-2) mol.l-1). The NaF-PGI2 dose-response curve was moved to the left by the presence of adrenaline, phorbol 12,13-dibutyrate (PDBU) and the Ca2+ ionophore A23187 in the incubation media. NaF-stimulated (5 x 10(-3) mol.l-1) PGI2 synthesis was inhibited by the Ca2+ channel blockers, verapamil and nifedipine, the protein kinase C inhibitor, H7, and lanthanum. Prazosin and yohimbine were without effect on NaF action, but partially inhibited adrenaline-potentiated NaF-stimulated PGI2 synthesis. Cyclic adenosine-3',5'-monophosphate (cAMP) and dibutyryl cAMP were without effect on de novo or NaF-, adrenaline-, PDBU- or A23187-stimulated PGI2 synthesis. Since fluoride is known to stimulate adenyl cyclase and phospholipase C, these data suggest that: (1) NaF stimulates in vitro rat aortic PGI2 synthesis by initiating Ca2+ influx; (2) this Ca2+ influx is mediated by protein kinase C, probably through G protein activation of phospholipase C and the generation of the protein kinase C activator, diacyl glycerol; and (3) adenyl cyclase and protein kinase A are not involved in NaF-stimulated PGI2 synthesis by the rat aorta.     

The role of the diacylglycerol-protein kinase C system in mediating adrenoceptor-prostacyclin synthesis coupling in the rat aorta

The role of the diacylglycerol-protein kinase C system in mediating adrenoceptor-stimulated prostacyclin (PGI2) synthesis in the isolated rat aorta was investigated using the diacylglycerol-mimetic phorbol 12,13-dibutyrate (PDBU) and the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperizine (H7). PDBU stimulated rat aortic PGI2 synthesis in a dose-dependent manner, an action potentiated by adrenaline and the calcium ionophore A23187. EDTA (10(-2) mol X l-1) completely inhibited PDBU-stimulated PGI2 synthesis. The calcium channel blockers, verapamil and nifedipine, inhibited PDBU-stimulated PGI2 synthesis in a dose-dependent manner, as did the protein kinase C inhibitor H7. The adrenoceptor antagonists phentolamine, prazosin and yohimbine were without effect on PDBU-stimulated PGI2 synthesis. H7 also inhibited adrenaline-stimulated, but not trauma-, A23187- or arachidonate-stimulated PGI2 synthesis. These experiments constitute evidence that adrenoceptor-PGI2 synthesis coupling is mediated by diacylglycerol-protein kinase C initiation of calcium influx in the rat aorta.     

Modulation of the accumulation of inositol phosphates and the mobilization of calcium in aortic myocytes

In vascular smooth muscle cells the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of C-kinase, inhibited the accumulation of inositol phosphates and the mobilization of calcium produced by several agonists. In the same way, TPA inhibited the fluoride-induced activation of phosphoinositide metabolism. These results suggest a C-kinase action at a post-receptor level. Moreover, the fluoride-induced accumulation of inositol phosphates shows the presence of one or more guanine nucleotide-binding proteins (G-proteins) in the regulation of receptor-phospholipase C coupling. This was confirmed by the use of N-ethylmaleimide and pertussis toxin. These results support the view that, in addition to the induction of sustained contractions, C-kinase can activate negative feedback mechanisms in aortic myocytes.     

Okadaic acid enhances human T cell activation and phosphorylation of an internal substrate induced by phorbol myristate acetate.

Okadaic acid is a potent tumor promoter and an inhibitor of serine/threonine-specific protein phosphatases. We studied the effect of okadaic acid in human T cell activation and phosphorylation of internal substrates. Okadaic acid at up to 4 nM enhanced phorbol myristate acetate (PMA)-induced proliferation and CD25 (IL-2 receptor, p55) expression, although it showed no activation by itself. Okadaic acid induced hyperphosphorylation of a 60 kDa protein in T cells as well as non-T cells, as reported in fibroblasts and keratinocytes. Preincubation with 4 nM okadaic acid enhanced PMA induced phosphorylation of the 80 kDa protein, an internal substrate of protein kinase C in T cells. These results suggest that okadaic acid inhibited dephosphorylation of protein kinase C specific substrates, and as a result, enhanced T cell activation mediated by protein kinase C pathway.     

N-ethylmaleimide inhibition of thrombin-induced platelet aggregation.

The data presented in this report show that N-ethylmaleimide (NEM) is a powerful inhibitor of thrombin-induced platelet aggregation. NEM increased guanosine 3', 5'-cyclic monophosphate (cGMP) and adenosine 3', 5'-cyclic monophosphate (cAMP) levels in intact cells. The inhibition of cAMP high-affinity phosphodiesterase and cGMP phosphodiesterase was implicated in the elevation of the cyclic nucleotides. NEM dose dependently blocked the thrombin-stimulated, but not the phorbol myristate acetate-dependent phosphorylation of the protein kinase C substrate pleckstrin. Myosin light chain phosphorylation was also inhibited by NEM. In addition, the sulphydryl reagent inhibited Ca2+ mobilisation induced by thrombin. The data indicate that phospholipase C activation by thrombin is interrupted by NEM at the level of receptor-mediated signal transduction.