Role of c-Src in the regulation of vascular contraction and Ca2+ signalingby angiotensin II in human vascular smooth muscle cells

Objective Tyrosine kinases, typically associated with growth-signaling path ways, also play a role in Ang II-stimulated vascular contraction. However t he specific kinases involved are unclear. We hypothesize here that c- Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascu lar smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contra ction induced by Ang II, Methods Cultured VSMCs from resistance arteries of healthy subjects were st udied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-defici ent VSMCs from small arteries of c-Src knockout mice (Src-/- mVSMCs) were a lso investigated. Intracellular free Ca2+ concentration ([Ca2+](i)), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioim munoassay respectively. Contraction was examined in intact rat small arteri es. Results Ang II rapidly increased VSMC c-Src activity, with peak responses o btained at 1 min. Ang II induced a biphasic [Ca2+](i) response (E-max = 636 +/- 123 nmol/l). The initial [Ca2+](i) transient, mediated primarily by Ca 2+ mobilization, was dose-dependently attenuated by the selective Src inhib itor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+](i) re sponses were blunted in cells electroporated with anti-c-Src antibodies and in c Src-/-mVSMCs, Src inhibition decreased Ang II-induced generation of I P3 in human VSMCs. Ang II dose-dependently increased vascular contraction ( E-max = 40 +/- 6.5%). These responses were attenuated by PP2 (E-max = 7.8 /- 0.08%) but not by PP3 (E-max = 35 +/- 4.5%). Conclusions Our findings identify c-Src as an important regulator of VSMC [ Ca2+](i) signaling and implicate a novel contractile role for this non-rece ptor tyrosine kinase in Ang Ii-stimulated vascular smooth muscle. I Hyperte ns 19:441-449 (C) 2001 Lippincott Williams & Wilkins.