Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

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Abstract
Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can 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. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pHi), typically due to activation of Na+/H+ exchange (NHE). In the systemic vasculature, ET-1 increases pHi, Na+/H+ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pHi and smooth muscle growth in the lung; however, the effect of ET-1 on basal pHi and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pHi and NHE activity induced by increasing concentrations of ET-1 (10−10 to 10−8 M). We found that application of exogenous ET-1 increased pHi and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pHi or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pHi.

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