Inhibition of c-Src with PP2 attenuated Ang II-induced vascular contraction (Emax = 7.8% vs 35% with inactive analogue PP3) and Ca2+ signaling in vascular smooth muscle cells.
OBJECTIVE: Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II. METHODS: Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration (Ca2+i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries. RESULTS: Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic Ca2+i response (Emax = 636 +/- 123 nmol/l). The initial Ca2+i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited Ca2+i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src-/-mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax = 40 +/- 6.5%). These responses were attenuated by PP2 (Emax = 7.8 +/- 0.08%) but not by PP3 (Emax = 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-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.
Touyz et al. (Thu,) conducted a other in Vascular contraction and Ca2+ signaling. c-Src inhibition (PP2, anti-c-Src antibody, or genetic knockout) vs. Inactive analogue (PP3) or control cells was evaluated on Intracellular free Ca2+ concentration, c-Src activity, IP3 production, and vascular contraction. Inhibition of c-Src with PP2 attenuated Ang II-induced vascular contraction (Emax = 7.8% vs 35% with inactive analogue PP3) and Ca2+ signaling in vascular smooth muscle cells.