title: Endothelin-1 augments Na⁺/H⁺ exchange activity in murine pulmonary arterial smooth muscle cells via Rho kinase.
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 (pH(i)), typically due to activation of Na(+)/H(+) exchange (NHE). In the systemic vasculature, ET-1 increases pH(i), 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 pH(i) and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH(i) 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 pH(i) 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 pH(i) 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 pH(i) 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 pH(i).
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Clark
authors.initials: C
authors.lastname: Undem
authors.forename: Eon J
authors.initials: EJ
authors.lastname: Rios
authors.forename: Julie
authors.initials: J
authors.lastname: Maylor
authors.forename: Larissa A
authors.initials: LA
authors.lastname: Shimoda
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D019332:Endothelin-1; D005452:Fluoresceins; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase