Role of extracellular signal-regulated kinases in angiotensin II-Stimulated contraction of smooth muscle cells from human resistance arteries

Backgronnd-We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. Methods and Results-VSMCs derived from resistance arteries (<300 mu m in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+](i), pH(i), and contractile responses. [Ca2+](i) and pH(i) were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT(1) and AT(2)) were assessed with [Sar(1),Ile(8)]Ang II (a nonselective subtype antagonist), losartan (a selective AT(1) antagonist), and PD123319 (a selective AT(2) antagonist). Ang II dose-dependently increased [Ca2+](i) (pD(2)=8.4+/-0.36, E-max=541+/-55 nmol/L), pH(i) (pD(2)=9.4+/-0.29, E-max=7.19+/-0.01), and contraction (pD(2)=9.2+/-0.21, E-max=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar(1),Ile(8)]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. Conclusions-These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT(2) subtype are coupled to signaling cascades involving Ca2+ and pH(i) pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.