Ca2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of alpha2beta1 isozyme of Na+/K+-ATPase by ouabain.
OBJECTIVE
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.


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.


RESULTS
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.


CONCLUSIONS
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.
Ghosh
Biswarup
B
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Kar
Pulak
P
Mandal
Amritlal
A
Dey
Kuntal
K
Chakraborti
Tapati
T
Chakraborti
Sajal
S
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D021941:Caveolae / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007527:Isoenzymes; D046529:Microscopy, Electron, Transmission; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D010042:Ouabain / Q000276:immunology; D011651:Pulmonary Artery / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D015220:Calcium Channels; D007527:Isoenzymes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium