Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca 2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca 2+ sources: (a) Ca 2+ surrounding single cells was transiently displaced by applying agonist with or without Ca 2+ from an ejection pipette. (b) Intracellular stores of Ca 2+ were depleted by soaking cells in Ca 2+ ‐free plus 1 m M EGTA solution before transient exposure to agonist plus Ca 2+ . Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca 2+ concentration ([Ca 2+ ] i ) was measured by indo‐1 fluorescence. KCl (35 m M ) and nicotine (10 µ M ) caused an immediate increase in [Ca 2+ ] i and secretion in cells with or without internal Ca 2+ stores, but only when applied with Ca 2+ in the ejection pipette. Caffeine (10 m M ) and muscarine (30 µ M ) evoked exocytosis whether or not Ca 2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca 2+ stores. Pretreatment with ryanodine (0.1 µ M ) inhibited caffeine‐ but not muscarine‐stimulated responses. Elevated [Ca 2+ ] i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine‐evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine‐evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ∼20‐s refractory period was found between repeated caffeine‐evoked exocytotic bursts even though [Ca 2+ ] i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca 2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca 2+ . These results and conclusions are consistent with our original observations in the perfused adrenal gland.