Background--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.

Methods and Results--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.

Conclusions--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.