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 (22.4 +/- 1.1 pmol Pi/min per mg protein) was not affected by PMA. Therefore, a study was performed of the effect of endogenous protein kinase C activation on adenylate cyclase regulation by agonists. PMA pretreatment completely suppressed PAF inhibition of basal adenylate cyclase activity but hardly influenced the inhibition by PAF of forskolin-stimulated activity. Adenylate cyclase inhibition by epinephrine in the presence of propranolol was not suppressed completely after platelet incubation with PMA. Epinephrine effects on basal and forskolin-stimulated activities decreased equally. Platelet pretreatment with PMA increased PGE1-stimulated activity by abolishing the inhibitory effect of high GTP concentrations. These studies indicate that protein kinase C selectively inhibits PAF effects, presumably by inactivating a GTP-binding protein coupled with PAF receptors.

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. Nicardipine suppresses also Ca2+ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF. The blockers reduce the [Ca2+]i increase induced by ADP, vasopressin, and PGH2 analogue U46619.

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. PAF and prostaglandin endoperoxide H2 analogue, U46619, induce Ba2+ and Mn2+ entry. The entrance of these cations and the rise in [Ca2+]i are both blocked by cAMP elevation and activation of protein kinase C by phorbol ester which indicates a single mechanism for agonist-induced transport of all three cations into platelets. It is assumed that agonists stimulate Ca2+ entry through the receptor-operated channels.

[Blockade of thrombocyte calcium channels by cardiotropic compounds]. / Blokirovanie kal'tsevykh kanalov trombotsitov kardiotropnymi soedineniiami.

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. Calcium-blocking action of calcium antagonists and nitrates was due to suppression of the entry of Ca2+ into the cells. The action of nitrates on platelets was accompanied by an acceleration of cGMP and cAMP synthesis. Unlike nitrates, antiarrhythmic drugs did not influence the intracellular level of cyclic nucleotides. On the basis of the data obtained it is suggested that calcium-blocking action of different types of compounds can be mediated by different intracellular mechanisms.

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. In the presence of PGE1, only inhibitory action of U46619 was observed at all the concentrations tested. The inhibitory action of the analogs on adenylate cyclase correlates with the activation of the high-affinity hormone-sensitive GTPase. It is concluded that U46619 and U44069 inhibit human platelet adenylate cyclase via specific receptors coupled to the GTP-binding inhibitory protein.

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. Phentolamine, an alpha-adrenergic antagonist, does not abolish the PAF-induced inhibition of adenylate cyclase. The obtained data suggest that PAF receptors are coupled with the GTP-binding inhibitory protein.

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 differences in the regulatory properties of AC I and AC II are probably due to the unequal distribution of the N-protein, the regulatory component of adenylate cyclase, in the preparations. Addition of the N-protein, obtained by thermoinactivation of AC I, to AC II results in restoration of the enzyme sensitivity to Gpp(NH)p as well as in an increase of the stimulating effect of NaF. It is shown that the lag-period observed during adenylate cyclase stimulation by Gpp(NH)p is due to the slow transition of the N-protein into an activated conformation. This Gpp(NH)p-activated N-protein interacts with the catalytic component of adenylate cyclase without any lag-period. Addition of GTP to the Gpp(NH)p-activated soluble adenylate cyclase complex leads to a decrease of the enzymatic activity. This process presumably occurs via substitution of Gpp(NH)p for GTP in the N-protein with subsequent hydrolysis of GTP and not by exchange of the N-protein-Gpp(NH)p complex for the N-protein-GTP complex within the catalytic component of adenylate cyclase.

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. Consequently the regulatory influence of ATP is not due to chemical modification of the enzyme or to phosphorylation of endogenous GDP. Earlier we demonstrated that regulation of heart adenylate cyclase by ATP is not mediated by the adenosine binding center (Biokhimiya USSR (1982), 47, 455-464). It is assumed that the regulatory effect of ATP is accomplished by a specific binding site for this nucleotide.

[Interrelation of the regulatory effects of adenosine, ATP and fluoride on heart adenylate cyclase]. / Vzaimosviaz' mezhdu reguliatornymi éffektami adenozina, ATP i ftorida na adenilattsiklazu serdtsa.

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 1.6 . 10(-4) and 5.9 . 10(-5) M, respectively. The inhibiting effect of adenosine is rapid and irreversible and is not changed in the presence of 2 mM theophylline and during adenylate cyclase solubilization. It is assumed that the enzyme inhibition is mediated by the P-sites. An increase in ATP concentration causes an increase in the enzyme activation by fluoride. At low ATP concentrations (10(-5) - 2 . 10(-5) M) adenosine inhibits the activation by fluoride practically completely, while at increasing ATP concentrations the inhibition of the fluoride-stimulated activity of adenylate cyclase is decreased. The effect of adenosine is not competitive with respect to ATP. The increased activation of adenylate cyclase by fluoride in the presence of ATP can be due to the fact that the enzyme affinity for the substrate is decreased in the presence of NaF. Adenosine, which acts presumably at the allosteric site, causes an additional decrease of the fluoride-stimulated adenylate cyclase affinity for ATP. It is also assumed that within the adenylate cyclase complex there probably exists a regulatory site, whose binding to ATP increases the activation of adenylate cyclase by fluoride; the adenosine binding to the P-sites inhibits this activation.