The relationship between relative cell volume and time-dependent chang
es in intracellular Ca2+ concentration ([Ca2+](i)) during exposure to
hypotonicity was characterized in SV-40 transformed rabbit corneal epi
thelial 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 3
0 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 ryano
dine (inhibitor of Ca2+ release from intracellular stores). To determi
ne the temporal relationship between an increased plasmalemma Ca2+ inf
lux and the emptying of intracellular Ca2+ stores during the [Ca2+](i)
transients, Mn2+ quenching of fura2-fluorescence was quantified. In t
he presence of CPA, hypotonic challenge increased plasmalemma Mn2+ per
meability 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 dependenc
e of the components of the [Ca2+](i) transient required for a regulato
ry volume decrease (RVD). The results show that ryanodine sensitive Ca
2+ release from an intracellular store leads to a subsequent increase
in plasmalemma Ca2+ influx, and that both are required for cells to un
dergo RVD.