Angiotensin II regulates vascular smooth muscle cell pH, contraction, and growth via tyrosine kinase-dependent signaling pathways.

Abstract

Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II-stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2+/-0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4+/-0.1). Ang II (10(-7) mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II-elicited responses. Tyrphostin A-23 significantly attenuated Ang II-stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II-stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II-mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype-mediated contraction and growth.