Hypo-osmotic shock of tobacco cells stimulates Ca²⁺ fluxes deriving first from external and then internal Ca²⁺ stores

Hypo-osmotic shock of aequorin-transformed tobacco cells induces a biphasic cytosolic Ca²⁺ influx. Because both phases of Ca²⁺ entry are readily blocked by Ca²⁺ channel inhibitors, we conclude that the Ca²⁺ transients are mediated by Ca²⁺ channels. Evidence that the first but not second Ca²⁺ transient derives from external Ca²⁺ stores is that the first but not second influx is (i) eliminated by membrane-impermeable Ca²⁺ chelators, (ii) enlarged by supplementation of the medium with excess Ca²⁺, and (iii) reduced by the addition of competitive cations such as Mg²⁺ and Mn²⁺. Furthermore, entry of ⁴⁵Ca during osmotic shock is prevented by inhibitors of the first but not second phase of Ca²⁺ entry. Evidence that the second wave of Ca²⁺ influx stems from release of intracellular Ca²⁺ is based on the above data plus observations that probable modulators of intracellular Ca²⁺ channels selectively block this phase of Ca²⁺ influx. Finally, a mechanism of communication between the two Ca²⁺ release pathways has become apparent, since perturbations that elevate or reduce the first Ca²⁺ transient lead to a compensating diminution/elevation of the second and vice versa. These data thus suggest that osmotic shock leads to the sequential opening of extracellular followed by intracellular Ca²⁺ stores and that these Ca²⁺ release pathways are internally compensated.