Rho kinase and Na ⁺ /H ⁺ exchanger mediate endothelin-1-induced pulmonary arterial smooth muscle cell proliferation and migration

Excessive production of endothelin-1 (ET-1) has been observed in almost all forms of pulmonary hypertension. ET-1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pH _i ), typically due to activation of Na ⁺ /H ⁺ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET-1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET-1-induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH-sensitive dye, BCECF-AM, to measure changes in resting pH _i and NHE activity induced by exposure to exogenous ET-1 (10 ⁻⁸  mol/L) for 24 h. Cell migration and proliferation in response to ET-1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET-1 had no effect on NHE1 expression, but increased pH _i , NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET-1-induced changes in cell function (proliferation and migration). Our results indicate that ET-1 modulates PASMC migration and proliferation via changes in pH _i homeostasis through a pathway involving ROCK.