Modulation of adenylate cyclase of human platelets by phorbol ester

Abstract

The influence of the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), a direct activator of the Ca2 +-activated, phospholipid-dependent protein kinase (protein kinase C), was studied on regulation of human platelet adenylate cyclase. Intact platelets were pretreated with the phorbol ester and, thereafter, membranes were prepared and the regulation of the hormone-sensitive adenylate cyclase in these membranes was studied. The following data were obtained: The TPA treatment applied had apparently no effect on the activity of the catalytic moiety of the platelet adenylate cyclase nor on the stimulatory N, protein nor on stimulatory hormone receptors (prostaglandin El) and the mutual interactions of these components of the stimulatory hormone-sensitive pathway. However, the TPA treatment of intact platelets largely impaired the GTP-dependent, hormone-sensitive inhibitory pathway to the adenylate cyclase, involving the inhibitory Ni protein. The pretreatment led to a large reduction or loss of adenylate cyclase inhibition by GTP itself and by the inhibitory agonists, epinephrine and thrombin, inhibiting the untreated enzyme via separate receptors by an Ni-mediated process. In contrast, platelet adenylate cyclase inhibition not involving the Ni protein was not affected by the TPA treatment. The observed effects of TPA were very rapid in onset and were not shared by a derivative of TPA which did not activate protein kinase C. The data obtained suggest than protein kinase C activated by the phorbol ester interferes with the platelet adenylate cyclase system, leading to a specific alteration of the Ni-protein-mediated signal transduction to the adenylate cyclase.The adenylate cyclase of human platelet membranes is regulated by several stimulatory and inhibitory hormone-like factors affecting the catalytic activity upon receptor interaction through an action at the stimulatory (Ns, G,) and inhibitory (Ni, Gi) guanine-nucleotide-binding coupling component, respectively. For example, various prostaglandins such as prostaglandin El (PGE1), I2 and D2 cause an N,-mediated stimulation of adenylate cyclase following receptor interaction. This enzyme stimulation observed in membrane preparations is also reflected in an increase in total platelet cyclic AMP levels [l]. On the other hand, the platelet adenylate cyclase is also influenced by various inhibitory hormonal factors causing enzyme inhibition mediated by the inhibitory Ni protein. In membrane preparations, such a receptor-induced, Ni-mediated adenylate cyclase inhibition has been described for epinephrine and norepinephrine (acting via a2 adrenoceptors), for ADP, vasopressin (via VI receptors), platelet-activating factor and, very recently, also for thrombin [2-61. However, this enzyme inhibition observed in membrane preparations is not reflected in each case in a respective fall in total platelet cyclic AMP levels. For example,

Activation of protein kinase C alters the interaction of α2‐adrenoceptors and the inhibitory GTP‐binding protein (Gi) in human platelets

“…Epinephrine induced a dose-dependent inhibition of forskolin-stimulated adenylate cyclase activity (fig.2); the effect of epinephrine was blocked by 10gM yohimbine indicating the involvement of az-adrenoceptors (not shown). In agreement with the data of Jakobs et al [6], we observed that the effect of epinephrine was marked- ly reduced in membranes from TPA-treated platelets as compared to the controls, i.e. IOOpM epinephrine induced a 35-40070 inhibition of adenylate cyclase activity in control membranes whereas in membranes from TPA-treated platelets the same concentration of epinephrine induced only a 5-100/o inhibition ( fig.2).…”

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi

“…Similarly, we [5] and others [6] have shown that incubation of pure preparations of Gi and Go with a purified human insulin receptor preparation leads to the phosphorylation of ce-Gi. It has been suggested that phosphorylation of Gi by protein kinase C may lead to its inactivation as incubation of platelets with tumour promoting phorbol esters, which activate protein kinase C, blocked the ability of CeE-adrenoceptors to inhibit adenylate cyclase activity [7].…”

Positive inotropic and chronotropic effects of trypsin and some other proteolytic enzymes in the guinea‐pig heart

“…Hashimoto et al (1985) found, with rat liver, that the release ofprotein kinase C in the activated form occurs through limited proteolysis of the plasma membrane by trypsin. Jakobs et al (1985) found, with the human platelet, an impairment of the hormone-sensitive inhibitory pathway, dependent on the inhibitory guanine nucleotide binding regulatory protein (Ni), by 12-0-tetradecanoylphorbor 13-acetate (TPA), an activator of protein kinase C. In their experiments, the adenylate cyclase inhibition not involving the Ni protein and the operation of the stimulatory pathway remained unchanged. Katada et al (1985) found the phosphorylation of Ni protein by TPA in association with the impairment of the inhibitory pathway.…”