Angiotensin II regulates vascular smooth muscle cell pH, contraction, and growth via tyrosine kinase-dependent signaling pathways.
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.
Touyz
R M
RM
Medical Research Council Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, Quebec, Canada.
Schiffrin
E L
EL
D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002396:Catechols / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011921:Rats, Inbred WKY; D011945:Receptors, Angiotensin / Q000378:metabolism; D015398:Signal Transduction; D013936:Thymidine / Q000378:metabolism; D020032:Tyrphostins; D014661:Vasoconstriction / Q000502:physiology*
D002396:Catechols; D004791:Enzyme Inhibitors; D009570:Nitriles; D011945:Receptors, Angiotensin; D020032:Tyrphostins; D000804:Angiotensin II; D006859:Hydrogen; D011505:Protein-Tyrosine Kinases; D007930:Leucine; C060641:tyrphostin A23; D002118:Calcium; D013936:Thymidine