Role of Rho kinase and Na⁺/H⁺ exchange in hypoxia-induced pulmonary arterial smooth muscle cell proliferation and migration

Abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are hallmark characteristics of vascular remodeling in pulmonary hypertension induced by chronic hypoxia. In this study, we investigated the role of the Na⁺/H⁺ exchanger (NHE) and alterations in intracellular pH (pH_i) homeostasis in meditating increased proliferation and migration in PASMCs isolated from resistance-sized pulmonary arteries from chronically hypoxic rats or from normoxic rats that were exposed to hypoxia ex vivo (1% or 4% O₂, 24-96 h). We found that PASMCs exposed to either in vivo or ex vivo hypoxia exhibited greater proliferative and migratory capacity, elevated pH_i, and enhanced NHE activity. The NHE inhibitor, ethyl isopropyl amiloride (EIPA), normalized pH_i in hypoxic PASMCs and reduced migration by 73% and 45% in cells exposed to in vivo and in vitro hypoxia, respectively. Similarly, EIPA reduced proliferation by 97% and 78% in cells exposed to in vivo and in vitro hypoxia, respectively. We previously demonstrated that NHE isoform 1 (NHE1) is the predominant isoform expressed in PASMCs. The development of hypoxia-induced pulmonary hypertension and alterations in PASMC pH_i homeostasis were prevented in mice deficient for NHE1. We found that short-term (24 h) ex vivo hypoxic exposure did not alter the expression of NHE1, so we tested the role of Rho kinase (ROCK) as a possible means of increasing NHE activity. In the presence of the ROCK inhibitor, Y-27632, we found that pH_i and NHE activity were normalized and migration and proliferation were reduced in PASMCs exposed to either in vivo (by 68% for migration and 22% for proliferation) or ex vivo (by 43% for migration and 17% for proliferation) hypoxia. From these results, we conclude that during hypoxia, activation of ROCK enhances NHE activity and promotes PASMC migration and proliferation.