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Endocrinology Vol. 141, No. 6 2200-2208
Copyright © 2000 by The Endocrine Society

ARTICLES

The Activation of Phospholipase D by Endothelin-1, Angiotensin II, and Platelet-Derived Growth Factor in Vascular Smooth Muscle A10 Cells Is Mediated by Small G Proteins of the ADP-Ribosylation Factor Family1

Kuntala Shome, Mark A. Rizzo, Chandrasekaran Vasudevan, Bradley Andresen and Guillermo Romero2

Department of Pharmacology of the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Guillermo Romero, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. E-mail: ggr+{at}pitt.edu

We show here that A10 cells express the phospholipase D (PLD) isoforms PLD1b and PLD2. The activation of PLD in these cells by angiotensin II (AngII), endothelin-1 (ET-1), and platelet-derived growth factor (PDGF) was found to be sensitive to inhibitors of the activation of ADP-ribosylation factor (ARF) but not to blockers of Rho protein function. PDGF, AngII, and ET-1 induced the binding of ARF proteins to cell membranes in a permeabilized cell assay. Cells permeabilized and depleted of ARF were no longer sensitive to stimulation with AngII, ET-1, or PDGF, but the addition of recombinant myristoylated human ARF1 restored agonist-dependent PLD activity. Expression of dominant negative ARF mutants blocked receptor-dependent activation of PLD. PLD activity was also potently stimulated by treatment with phorbol esters, but this activity was only partially inhibited by brefeldin A or by the overexpression of ARF dominant negative mutants. Transient expression of catalytically inactive mutants of PLD2, but not PLD1, inhibited significantly PDGF- and AngII-dependent PLD activity. We conclude: 1) the activation of PLD by cell surface receptors occurs primarily by an ARF-dependent mechanism in A10 cells, whereas the activation of PLD by protein kinase C-dependent pathways is only partially dependent on the regulation of ARF proteins; and 2) cell surface receptors, such as AngII and PDGF, signal primarily via PLD2 in A10 cells.




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