Selective inactivation by endogenous protein kinase C of human platelet high-affinity GTPase coupled with PAF receptors.

Incubation of human platelets with protein kinase C activator 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) abolished stimulation of membrane high-affinity GTPase by platelet-activating factor (PAF). GTPase stimulation by epinephrine decreased by 30%, while the prostaglandin E1 (PGE1) effect was unchanged. Basal GTPase activity (22.

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine.

Verapamil (ED50 = 3 x 10(-6) M) and nicardipine (ED50 = 10(-6) M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration [( Ca2+]i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl2, PAF stimulated the inflow of Ba2+ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and 45Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca2+ entry.

Evidence for the receptor-operated calcium channels in human platelet plasma membrane.

Loading of human platelets with quin2 considerably increases platelet activating factor (PAF)-induced 45Ca2+ uptake. 45Ca2+ binding in the absence of agonists remains unchanged. The data show that PAF stimulates calcium ion influx into platelets, since quin2 provides substantial additional buffer capacity for Ca2+ in the cytoplasm.

[Blockade of thrombocyte calcium channels by cardiotropic compounds].

The effect of calcium antagonists (verapamil, nicardipine, nifedipine), nitrates (glycerin trinitrate, isosorbide dinitrate and sodium nitroprusside) and of antiarrhythmic drugs (ethmozin, ethacizin) on the increase of platelet Ca2+ concentration brought about by aggregation inductors was studied. All the analyzed substances produced an inhibitory effect on the induction of cellular Ca2+ level increase due to the action of platelet aggregation factor, ADP, vasopressin and endoperoxide PGH2 stable analogue. The degree of this inhibitory effect of calcium antagonists and nitrates was independent of the nature of the stimulator used.

Interaction of stable prostaglandin endoperoxide analogs U46619 and U44069 with human platelet membranes: coupling of receptors with high-affinity GTPase and adenylate cyclase.

It has been demonstrated using a membrane preparation of human platelets that stable analogs of PGH2, U46619 and U44069, control the activity of adenylate cyclase and a high-affinity hormone-sensitive GTPase. At 10(-8)-10(-6) M, the analogs inhibit the basal activity of adenylate cyclase by 20-25%. With a further rise in U46619 and U44069 concentrations up to 10(-5)-10(-4) M, the inhibition is abolished and adenylate cyclase activity is stimulated in a dose-dependent fashion.

Stimulation of high-affinity hormone-sensitive GTPase of human platelets by 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor).

1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor) inhibits human platelet adenylate cyclase via the GTP-dependent mechanism. Inhibition of adenylate cyclase correlates with the stimulation of high affinity hormone-sensitive GTPase. The half-maximal effects of PAF on both enzymes are observed at concentrations about 10(-8) M.

Separation and reconstitution of regulatory and catalytic components of heart adenylate cyclase.

Ion-exchange chromatography of rabbit heart adenylate cyclase solubilized with lubrol PX results in two peaks of activity, AC I and AC II, differing in their sensitivity to guanylylimido-diphosphate [Gpp(NH)p], NaF and cholera toxin. AC I is activated 4- to 5-fold by Gpp(NH)p, 10- to 20-fold by NaF and 3- to 4-fold by cholera toxin. AC II is insensitive to Gpp(NH)p and cholera toxin, but is activated 2-fold by the fluoride.

The regulatory effect of ATP and its non-hydrolyzed analogs on heart adenylate cyclase.

The effect of ATP on rabbit heart adenylate cyclase was investigated. The activation of adenylate cyclase by isoproterenol, guanyl nucleotides and NaF increases at a rise in ATP concentration. A similar regulatory effect is exerted by nonhydrolyzed analogs of ATP - adenyl-5'-ilimidodiphosphate and adenosine-5'-(alpha, beta-methylene) triphosphate.

[Interrelation of the regulatory effects of adenosine, ATP and fluoride on heart adenylate cyclase].

Adenosine inhibits membrane adenylate cyclase from rabbit heart and considerably alters its regulatory properties. In the presence of 1 mM adenosine the enzyme activation by isoproterenol and guanyl-5'-ylimidodiphosphate is sharply decreased, while that by fluoride is practically completely eliminated. The values of the apparent rate constants for the enzyme inhibition by adenosine with respect to the basal activity and the activity in the presence of NaF are equal to 1.