In this paper we examine the effect of the vasodilator peptide bradykinin on endothelial cell regulation of phosphoinositide (PI) turnover. The data show that the activation of PI turnover by bradykinin in bovine pulmonary artery endothelial cells is insensitive to pertussis toxin, which ADP ribosylates a membrane protein of mol wt 40,000. However, this effect of bradykinin can be potentiated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S), an activator of G proteins, and depressed by guanosine 5'-O-(2-thio)diphosphate (GDP beta S), an inhibitor of G proteins. After endothelial cells were preincubated for 1 h with GTP gamma S, there was a three- to fourfold increase in PI turnover. Preincubation of cells with GDP beta S did not affect the basal level of PI turnover, but completely prevented activation of PI turnover by bradykinin. 4 beta-Phorbol-12 beta-myristate-13 alpha-acetate can block the bradykinin-stimulated inositol monophosphate formation in cultured endothelial cells. The effects of bradykinin on PI turnover were blocked by B2 antagonists but not by B1 antagonists. Taken together, these results indicate that in endothelial cells the bradykinin B2 receptor is coupled to phospholipase C via a G protein (or proteins) that is not a substrate for pertussis toxin (neither Gi nor Go).

Endothelial cells (ECs) were isolated from bovine pulmonary artery and maintained in long-term culture. On reaching confluency, ECs formed a characteristic "cobblestone" monolayer. One hour after addition of 1 nM platelet-activating factor (PAF) to the growth medium, ECs underwent dramatic changes in shape from their normal polygonal morphology to more elongated spindle-shaped forms. More pronounced effects were evident in the presence of 0.1 nM phorbol-12-myristate-13-acetate (PMA), a potent activator of C kinase. It was found that at concentrations from 10(-11)-10(-7) M, PAF stimulates the phosphoinositide turnover in EC. The half-maximal activation in the release of inositol phosphates was at 10(-9) M. This finding suggested that an increase in intracellular Ca2+ concentration and activation of protein kinase C were involved in the mechanism of action of PAF on EC. The metabolic responses of EC were evaluated by measuring the activity of beta-adrenergic receptor-coupled adenylate cyclase (AC) in a crude membrane fraction and by assay of prostacyclin and thromboxane released by cultured EC. AC from control membranes was activated by isoproterenol in a dose-dependent manner (EC50 = 30 nM) from 0.8-5.5 pmol cAMP/min/mg protein. If the membranes were isolated after preincubation of ECs with 1 nM PAF or 0.1 nM PMA, the AC activity was decreased by 70 and 90%, respectively; in both cases, affinity for isoproterenol was lowered threefold (EC50 = 100 nM). Our data suggest that PAF interaction with EC leads to an apparent beta-adrenergic receptor desensitization that probably acts via a phosphorylation mechanism involving C kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Ca2 ' -dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1 -1.0 pM). At low Ca2+ (0.08-0.3 pM) adenylate cyclase was stimulated (K, = 0.10 pM), whereas at higher Ca2+ (> 0.3 pM) the enzyme was inhibited to 70-80% control (Ki = 0.8 pM). Membrane fractions, prepared by washing in the presence of LaC1, to remove endogenous calmodulin ( z 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 pM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin ( K , = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 pM). Prostaglandin El (PGE1) did not alter K i or K, values for Ca". Calmodulin did not alter the ECSo for PGE, stimulation of adenylate cyclase but increased the V,,, (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3 +-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.Calcium inhibits the activity of adenylate cyclase from a variety of tissues [l]. However, in some tissues such as brain [2], heart [3] and smooth muscle [4], calcium affects adenylate cyclase in a biphasic manner; low calcium concentrations stimulate, while higher concentrations inhibit, enzymic activity. The stimulatory action of Ca2+ is mediated by calmodulin [2-41. In platelets, Ca2+ effects on adenylate cyclase have not been well studied, and available reports [5, 61 have shown that Ca2 + produces only concentration-dependent inhibition of cyclase activity.In the present paper we confirm that Ca2 +-dependent inhibition of adenylate cyclase activity in platelet membranes occurs at micromolar concentrations of Ca". We show for the first that submicromolar concentrations of Ca2+ activate human platelet adenylate cyclase and that this stimulation is mediated by calmodulin.

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