Real-time studies of zymogen granule exocytosis in intact rat pancreatic acinar cells

1. An adequate understanding of secretion requires the measurement of exocytosis on the same time scale as that used for second messenger dynamics. To investigate the kinetics of ACh-evoked secretion in pancreatic acinar cells, exocytosis of zymogen granules was quantified by continuous, time-differential analysis of digital images. The validity of this method was confirmed by simultaneous fluorescence imaging of quinacrine-loaded zymogen granules. 2. Basal rates of exocytosis were low (0.2 events min(-1)). ACh stimulated a biphasic increase in secretory activity, maximal rates exceeding 20 events min(-1) after 10 s of ACh application (10 muM). Over the next 15 s the rate of exocytosis fell to less than 4 events min(-1); then began a second phase of secretion that peaked 15 s later at -11 events min(-1), but subsequently declined in the continued presence of agonist. 3. Measurements of fura-2 fluorescence demonstrated a biphasic increase in intracellular [Ca2+] ([Ca2+](i)). Comparison of the [Ca2+](i) records and time-differential analysis revealed that the fall in exocytotic rate following the initial burst occurred despite the fact that [Ca2+](i) remained high. 4. The second phase of secretion depended on both [Ca2+](i), and [ACh]. At 10 muM ACh there was a decrease in the steepness of the relationship between [Ca2+](i) and exocytosis that led to an enhancement of the slow secretory phase. 5. We propose that acinar cells contain two pools of secretory vesicles: a small pool of granules that is exocytosed rapidly but is quickly depleted, and a reserve pool of granules that can be recruited by ACh in a process that is modulated by second messengers other than calcium.