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Am J Physiol Heart Circ Physiol 287: H1495-H1500, 2004; doi:10.1152/ajpheart.01006.2003
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Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Liming Jin, Zhekang Ying, and R. Clinton Webb

Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000

Submitted 23 October 2003 ; accepted in final form 26 May 2004

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H2O2 are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 µM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca2+-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca2+ sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca2+-independent PKC.

myosin light chain phosphatase; smooth muscle contraction; antioxidants



Address for reprint requests and other correspondence: L. Jin, Dept. of Physiology, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-3000 (E-mail: ljin{at}mcg.edu)




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