Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca²⁺-induced Ca²⁺ Release

Abstract.

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 (τ _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 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm 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 μm ryanodine; 2.10 μm CPA and 100 μm 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.