Mitogen-activated protein extracellular signal-regulated kinase inhibitionattenuates angiotensin II mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+](i), and pH(i)) and in contr action in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selec tive mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mes enteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+](i ), pH(i), and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+](i) and pH(i) wer e measured by fura-2 and BCECF methodology, respectively, and contraction w as determined by photomicroscopy. Ang II-stimulated ERK activity was measur ed by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody a nd by an MAPK enzyme assay. Ang II increased [Ca2+](i) and pH(i) and contra cted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+](i), but not basal pH(i), was higher in SHR compared with WKY. [Ca2+](i) and pH(i) effects of Ang II were enhanced (P<0 .05) in SHR compared with WKY (maximum Ang II-induced response [E-max] of [ Ca2+](i), 576+/-24 versus 413+/-43 nmol/L; E-max of pH(i), 7.33+/-0.01 vers us 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contrac tion and attenuated the augmented Ang II-elicited contractile responses in SHR (E-max,19.3+/-3% reduction in cell length relative to basal length). An g II-stimulated [Ca2+](i) (E-max, 294+/-55 nmol/L) and pH(i) (E-max, 7.27+/ -0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In con clusion; Ang II-stimulated signal transduction and associated VSMC contract ion are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang Il effects in SHR. These data suggest that ERK-dependen t signaling pathways influence contraction and that they play a role in vas cular hyperresponsiveness in SHR.