Pepsinogen secretion: coupling of exocytosis visualized by video microscopy and [Ca2+]iin single cells

Abstract

Conventional in vitro studies of pepsinogen secretion have measured secretion into the bulk medium and have demonstrated the critical role of Ca2+ in the process. The present study was undertaken to obtain further details of the process of secretion and its relation to Ca2+changes over very short time periods. The relation between Ca2+ mobilization and exocytosis in an isolated individual peptic cell of the bullfrog was investigated by a method to measure both intracellular Ca2+([Ca2+]i), using a fluorescent Ca2+indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 × 10−7 M) and bethanechol (3.2 × 10−4 M) caused a rapid increase in [Ca2+]i(initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak, [Ca2+]iwas maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated [Ca2+]iand the related exocytosis were eliminated by the depletion of extracellular Ca2+. Low concentrations of bombesin (3.2 × 10−10 M) and bethanechol (3.2 × 10−7 M) caused sustained low-amplitude Ca2+oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that 1) cellular response differs between high and low concentrations of stimulus, 2) there is a close relation between [Ca2+]iand exocytosis, 3) exocytosis follows elevation of [Ca2+]iby 14–45 s ( n = 6), and 4) there is a significant positive correlation between the peak [Ca2+]iand the number of exocytoses.