EXOCYTOSIS COUPLED TO MOBILIZATION OF INTRACELLULAR CALCIUM BY MUSCARINE AND CAFFEINE IN RAT CHROMAFFIN CELLS

We used cultured rat chromaffin cells to test the hypothesis that Ca2 entry but not release from internal stores is utilized for exocytosis . Two protocols were used to identify internal versus external Ca2+ so urces: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) In tracellular stores of Ca2+ were depleted by soaking cells in Ca2+-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2 +. Exocytosis from individual cells was measured by microelectrochemic al detection, and the intracellular Ca2+ concentration ([Ca2+](i)) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 mu M) c aused an immediate increase in [Ca2+](i) and secretion in cells with o r without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 mu M) evoked exo cytosis whether or not Ca2+ was included in the pipette, but neither p roduced responses in cells depleted of internal Ca2+ stores. Pretreatm ent with ryanodine (0.1 mu M) inhibited caffeine- but not muscarine-st imulated responses. Elevated [Ca2+](i) and exocytosis exhibited long l atency to onset after stimulation by caffeine (2.9 +/- 0.38 s) or musc arine (2.2 +/- 0.25 s). However, the duration of caffeine-evoked exocy tosis (7.1 +/- 0.8 s) was significantly shorter than that evoked by mu scarine (33.1 +/- 3.5 s). The duration of caffeine-evoked exocytosis w as not affected by changing the application period between 0.5 and 30 s. An similar to 20-s refractory period was found between repeated caf feine-evoked exocytotic bursts even though [Ca2+](i) continued to be e levated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffein e mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetyl choline action depends primarily on influx of external Ca2+. These res ults and conclusions are consistent with our original observations in the perfused adrenal gland.