Ca ²⁺-induced Ca ²⁺ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

Abstract

The presence and physiological role of Ca ²⁺-induced Ca ²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca ²⁺] ([Ca ²⁺] _c). Using targeted aequorin, we have directly monitored [Ca ²⁺] changes inside the ER ([Ca ²⁺] _ER) in bovine adrenal chromaffin cells. Ca ²⁺ entry induced by cell depolarization triggered a transient Ca ²⁺ release from the ER that was highly dependent on [Ca ²⁺] _ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca ²⁺ release was quantal in nature due to modulation by [Ca ²⁺] _ER. Whereas caffeine released essentially all the Ca ²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP ₃)- producing agonists released only 60–80%. Both InsP ₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca ²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca ²⁺] _c measurements showed that the wave of [Ca ²⁺] _c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca ²⁺ pool that can release Ca ²⁺ both via InsP ₃ receptors or CICR.