Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB and ROCK-1 Expression in Human and Rabbit Ductus Arteriosus by Increasing Mitochondria-Derived Reactive Oxygen Species

Abstract

Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O2-sensitive K+ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. The effect of increased PO2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O2 constriction. Heterogeneity exists in the sensitivity of constrictors (PO2=endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca2+-ATPase. In term DA, an increase in PO2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a PO2-dependent, rotenone-sensitive increase in mitochondrion-derived reactive O2 species. O2 effects on Rho-kinase are mimicked by exogenous H2O2. In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O2 constriction and lack of PO2-dependent upregulation of Rho-kinase expression. O2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.