Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Abstract

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K(+) and Cl(-) concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K(+) but had significantly elevated intracellular Cl(-) Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K(+) and Cl(-) In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K(+) release were dependent on Cl(-) implying a direct or indirect coupling of K(+) to Cl(-) transport in volume regulation. RVD was partially blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na(+)/H(+) exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na(+)/H(+) and Cl(-)/HCO3 (-) exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K(+) channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C(-)-dependent K(+) release and the resultant regulatory volume decrease in osmotically-swollen cells.