Signalling components involved in the coupling of alpha 1-adrenoceptors to phospholipase D in neonatal rat cardiac myocytes.

Abstract

Activation of phospholipase D (PLD) is assumed to be one major pathway by which alpha(1)-adrenoceptors (alpha(1)ARs) induce hypertrophic responses in cardiac myocytes. Heterotrimeric G proteins, protein kinase C (PKC) isoforms, protein tyrosine kinases, monomeric GTPases of the ADP-ribosylation factor (ARF) and Rho families, and as important cofactor phosphatidylinositol 4,5-bisphosphate (PIP(2)) seem to participate in the G protein-coupled receptor dependent regulation of PLD. We therefore studied the role of these components in the coupling of alpha(1)ARs to PLD in neonatal rat cardiac myocytes (NRCM). Stimulation of alpha(1)ARs, most likely of the alpha(1A) subtype, by noradrenaline increased PLD activity three- to fourfold concomitant with the stimulation of phospholipase C (PLC). In contrast, the partial agonist phenylephrine stimulated PLC, but failed to increase PLD activity. The PLC and PLD responses were pertussis toxin insensitive and treatment of the cells with the G(q)-activating toxin of Pasteurella multocida stimulated both phospholipases about fourfold. Over-expression of the G(q)-and G(i)-type-specific regulator of G protein signalling RGS4 blunted alpha(1)AR-induced PLC and PLD stimulation. Ro 31-8220, known to inhibit Ca(2+)-dependent and -independent PKC isoforms, strongly inhibited PLD activity, whereas Gö 6976, known to inhibit preferentially Ca(2+)-dependent PKC isozymes, was without effect. The ARF signalling inhibitor brefeldin A, protein tyrosine kinase inhibitors and the Rho-inactivating toxin B of Clostridium difficile blunted alpha(1)AR-induced PLD stimulation and largely reduced the cellular PIP(2) content. In membranes of toxin B-treated NCRM, PLD activity was similarly reduced, but was fully restored by addition of exogenous PIP(2). We conclude, that alpha(1A)ARs stimulate PLD activity via a G(q/11)-PLCbeta-novel PKC isoform-dependent pathway in NRCM. ARF and Rho GTPases as well as protein tyrosine kinases contribute to PLD stimulation in NRCM, most likely by regulating the supply of PIP(2).