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

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).

Distinct signaling pathways mediate cardiomyocyte phospholipase D stimulation by endothelin-1 and thrombin.

Several G protein-coupled receptors which stimulate phospholipase C (PLC) also activate phospholipase D (PLD) in cardiomyocytes. Here, we characterized PLD activation in neonatal rat cardiomyocytes by the PLC-stimulatory thrombin receptor PAR1, in comparison to the endothelin-1 receptor ET(A)R, which induces PLD stimulation by activation of protein kinase C (PKC) delta and epsilon. Similar to ET(A)R, activation of PAR1 induced PLD stimulation, which, however, was insensitive to PKC inhibition. Furthermore, in contrast to ET(A)R, PLD stimulation by PAR1 was suppressed by overexpression of regulators of G protein signaling specific for G(12)-type G proteins and treatment with brefeldin A, an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor (ARF) GTPases. On the other hand, inactivation of Rho GTPases by Clostridium difficile toxin B and treatment with general tyrosine kinase inhibitors suppressed PAR1- and ET(A)R- as well as phorbol ester-induced PLD stimulation and was associated with a fall in the cellular level of phosphatidylinositol 4,5-bisphosphate (PIP(2)). We conclude that, in contrast to ET(A)R-PLD coupling, PAR1-induced cardiomyocyte PLD stimulation is PKC-independent and mediated by G(12)-type G proteins and ARF GTPases, while Rho and tyrosine kinases regulate PLD stimulation by either receptor, apparently by controlling the cellular level of PIP(2), a common regulator of PLD activity.