Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+](i)) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tau(vr)) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+](i) initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+](i) transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 mu M cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 mu M ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+](i) transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 mu M ryanodine; 2.10 mu M CPA and 100 mu M ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+](i) transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.