Angiotensin II Activates Extracellular Signal-Regulated Kinase Independently of Receptor Tyrosine Kinases in Renal Smooth Muscle Cells: Implications for Blood Pressure Regulation

ABSTRACT Angiotensin II can cause hypertension through enhanced vasoconstriction of renal vasculature. One proposed mechanism for reduction of angiotensin II-induced hypertension is through inhibition of the mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade. MEK/ERK has been shown to phosphorylate the regulatory subunit of myosin light chain at identical positions as myosin light chain kinase. There are multiple mechanisms proposed regarding angiotensin II-mediated ERK activation. We hypothesized that renal microvascular smooth muscle cells (RVSMCs) signal through a unique pathway compared with thoracic aorta smooth muscle cells (TASMCs), which is involved in blood pressure regulation. Use of epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptor-specific inhibitors 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and 6,7-dimethoxy-3-phenylquinoxaline (AG1296), respectively, demonstrates that angiotensin II activates ERK in TASMCs, but not RVSMCs, through transactivation of EGF and PDGF receptors. In addition, inhibition of Src with its specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2) abolishes angiotensin II-, but not EGF-or PDGF-, mediated phosphorylation of ERK in RVSMCs, yet it has no effect in TASMCs. The physiological significance of transactivation was examined in vivo using anesthetized Wistar-Kyoto rats with 15 mg/kg 2Ј-amino-3Ј-methoxyflavone (PD98059), an MEK inhibitor, as well as 20 mg/kg AG1478 and 1.5 mg/kg AG1296 in an acute model of angiotensin II-mediated increase in blood pressure. None of the inhibitors had an effect on basal blood pressure, and only PD98059 reduced angiotensin II-mediated increase in blood pressure. Moreover, in RVSMCs, but not TASMCs, angiotensin II localizes phosphorylated ERK to actin filaments. In conclusion, angiotensin II signals through a unique mechanism in the renal vascular bed that may contribute to hypertension. Extracellular signal-regulated kinases ERK1 and ERK2 (herein referred to as ERK) are involved in smooth muscle cell contraction C.S.E. and L.B.K. contributed equally to this work and share equal authorship as first authors. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.126300. ABBREVIATIONS: ERK, extracellular signal-regulated kinase; MLC20, 20-kDa myosin light chain regulatory subunit; GPCR, G protein-coupled receptor; PLD, phospholipase D; TASMC, thoracic aorta smooth muscle cell; Ang II, angiotensin II; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; RVSMC, renal microvascular smooth muscle cell; PE, polyethylene; DMSO, dimethyl sulfoxide; PD98059, 2Ј-amino-3Ј-methoxyflavone; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; AG1296, 6,7-dimethoxy-3-phenylquinoxaline; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ANOVA, analysis of variance; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene