Cell swelling increases intracellular calcium in Necturus erythrocytes

This study examined the role of Ca(2+) in regulatory volume decrease by Necturus erythrocytes. Hypotonic shock (50% tonicity) stimulated an increase in cytosolic free Ca(2+), detected using epi-fluorescence microscopy and the fluorescent Ca(2+) indicator fluo-4-AM (10 microM). A similar increase in fluorescence did not occur under isosmotic conditions, unless cells were exposed to the Ca(2+) ionophore A23187 (0.5 microM). In addition, a low Ca(2+) medium (amphibian Ringer solution with 5 mM EGTA), hexokinase (2.5 U/ml, an ATP scavenger), suramin (100 microM, a P2 receptor antagonist) and gadolinium (10 microM, a stretch-activated channel blocker) each inhibited the swelling-induced increase in Ca(2+). Consistent with these studies, a low Ca(2+) Ringer solution increased osmotic fragility, whereas volume recovery following hypotonic shock (measured with a Coulter counter) was potentiated with A23187 (0.5 microM). By contrast, a low Ca(2+) extracellular medium or buffering intracellular Ca(2+) with BAPTA-AM (100 microM) reduced the rate of volume recovery following hypotonic challenge. Finally, a low Ca(2+) extracellular Ringer solution inhibited whole-cell currents that are activated during cell swelling (measured with the whole-cell patch clamp technique). Our results are most consistent with hypotonic shock causing an increase in cytosolic free Ca(2+), thereby stimulating subsequent volume decrease.