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 alpha(2)beta(1) and alpha(1)beta(1) isozymes of Na(+)/K(+)-ATPase in caveolae vesicles, and only the alpha(1)beta(1) isozyme in noncaveolae fraction of the plasma membrane. The alpha(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 alpha(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.