Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Abstract

. Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am J Physiol Heart Circ Physiol 287: H1495-H1500; 2004; 10.1152/ajpheart.01006.2003.-Evidence indicates that both the Rho/ Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H 2O2 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 M) significantly reduced X/XOinduced contraction. A Rho kinase inhibitor, (ϩ)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca 2ϩ -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 Ca 2ϩ sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca 2ϩ -independent PKC. myosin light chain phosphatase; smooth muscle contraction; antioxidants CONTRACTION OF SMOOTH MUSCLE is regulated by both Ca 2ϩ -dependent and Ca 2ϩ -independent (Ca 2ϩ sensitization) mechanisms. A rise in intracellular Ca 2ϩ levels leads to myosin light chain (MLC) kinase activation, resulting in an increase in MLC phosphorylation. Importantly, MLC phosphorylation can also be increased through inhibition of MLC phosphatase, which augments smooth muscle force generation without a change in intracellular Ca 2ϩ (38).In recent years, studies have revealed that the small GTPase Rho and its downstream target Rho kinase mediate the Ca 2ϩ sensitization of smooth muscle contraction (37). The exchange of bound GDP for GTP activates Rho and stimulates its translocation from cytosol to membrane. Rho-GTP phosphorylates Rho kinase, which inhibits MLC phosphatase activity by phosphorylation of the MLC phosphatase target subunit (MYPT1). A decrease in MLC phosphatase activity increases phosphorylation of myosin and therefore contributes to smooth muscle contraction at low levels of intracellular Ca 2ϩ concentration. Strong evidence suggests that increased Rho/Rho kinase-dependent Ca 2ϩ sensitization contributes to hypertension and inhibition of Rho or Rho kinase reduces blood pressure (33, 42). Some heterotrimetric G protein-coupled receptor (GPCR) agonists, including angiotensin II (ANG II) and endothelin (ET)-1, induce smooth mus...

TNF-α Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms

“…Nimnual et al (26) demonstrated a down-regulation of Rho by Rac-induced ROS in HeLa cells. However, another study reported an increase in Rho activation by ROS in rat aortic rings (27). A recent article reported that H 2 O 2 is able to regulate both phosphatase and protein kinase activities (29), which could readily affect downstream RhoGTPase signaling.…”

Endothelial dysfunction in diabetic erectile dysfunction

“…101 In addition, RhoA/ Rho-kinase suppresses eNOS gene expression and enzyme activity in the penis 13 and erectile 105 suggesting an additional means of interaction between eNOS-mediated relaxant and RhoA/Rho-kinase-mediated contractile pathways in the penis. Moreover, as shown in nonpenile vascular beds, ROS activate the RhoA/Rho-kinase pathway by promoting increased migration of RhoA to the plasma membrane and its activation, 106 thus stimulating vasoconstriction. Furthermore, Rhokinase stimulates NAD(P)H oxidase with consequent increase in the production of ROS, 107 further contributing to endothelial dysfunction.…”

Endothelial dysfunction and hypertension in aging

“…[106][107][108] Endothelial function and hypertension in aging Y Higashi et al Several investigators have shown an interaction between the RhoA/ ROCk pathway and ROS. 109,110 Indeed, ROS induced by hyperglycemia enhances ROCK activity, leading to atherothrombogenesis through an increase in expression of plasminogen activator inhibitor-1 in vascular endothelial cells. 111 It is well known that cigarette smoking decreases NO bioavailability through the production of ROS.…”