Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human 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
R M
RM
MRC Multidisciplinary Research Group on Hypertension, Sungkyun Kwan University School of Medicine, Seoul, Korea. touyzr@ircm.qc.ca
Wu
X H
XH
He
G
G
Park
J B
JB
Chen
X
X
Vacher
J
J
Rajapurohitam
V
V
Schiffrin
E L
EL
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000081247:CSK Tyrosine-Protein Kinase; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology*; D002478:Cells, Cultured; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014661:Vasoconstriction / Q000187:drug effects* / Q000502:physiology*; D019061:src-Family Kinases
D000906:Antibodies; D004791:Enzyme Inhibitors; D000804:Angiotensin II; D007334:Insulin-Like Growth Factor I; D015544:Inositol 1,4,5-Trisphosphate; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human