Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhib ition of Na+/K+-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regul atory volume decrease) in hyposmotic conditions (100 mOsm). It is also know n that submitting chick embryo cardiomyocytes to a hyperosmotic solution in duces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH(i)), intracellular Ca2+ ([Ca2+](i)) and Na+/K+-ATPase inhibition during hyposmot ic swelling. Changes in intracellular pH and Ca2+ were monitored using BCEC F and fura-2, respectively. The addition of ouabain (100 mu M) under both i sosmotic and hyposmotic stimuli resulted in a large increase in [Ca2+](i) ( 200%). A decrease in pH(i) (from 7.3 +/- 0.09 to 6.4 +/- 0.08, n = 6; p < 0 .05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca2+. Inh ibition of Na+/H2+ exchange with amiloride (1 mM) had no effect on the ouab ain-induced acidification. Preventing the mitochondrial accumulation of Ca2 + using CCCP (10 mu M) resulted in a blockade of the progressive acidificat ion normally induced by ouabain. The inhibition of mitochondrial membrane K +/H+ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca2+ influx via Na+/Ca2+ exchange, which under hypo smotic conditions activates the K+ and Ca2+ mitochondrial exchange systems (K+/H+ and Ca2+/H+).