Ca 2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α 2β 1 isozyme of Na +/K +-ATPase by ouabain

Abstract

Aims We sought to determine the mechanisms of an increase in Ca 2+ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na +/K +-ATPase inhibition by ouabain. Main methods The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na + and Ca 2+ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings We identified the α 2β 1 and α 1β 1 isozymes of Na +/K +-ATPase in caveolae vesicles, and only the α 1β 1 isozyme in noncaveolae fraction of the plasma membrane. The α 2-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na +/H +-exchange inhibitor) and tetrodotoxin (voltage-gated Na +-channel inhibitor) pretreatment prevented ouabain induced increase in Na + and Ca 2+ levels. Ouabain induced increase in Ca 2+ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na +/Ca 2+-exchange inhibitor) and verapamil (L-type Ca 2+-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca 2+ level in the caveolae vesicles, indicating that apart from Na +/Ca +-exchanger and L-type Ca 2+-channels, “slip-mode conductance” of Na + channels could also be involved in this scenario. Significance Inhibition of α 2 isoform of Na +/K +-ATPase by ouabain plays a crucial role in modulating the Ca 2+ influx regulatory components in the caveolae microdomain for marked increase in (Ca 2+) i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.