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The role of Ca2+ in the time-dependent pepsinogen secretion of frog oesophageal peptic glands stimulated by bombesin.

Time- and dose-related stimulation of pepsinogen secretion by bombesin was studied in perifused dispersed peptic glands from the oesophagus of the American bullfrog Rana catesbeiana. The dose response to bombesin was monophasic between 10(-10) and 10(-7) M, with an EC50 of 10(-9) M. Time-dependent secretion was closely monitored at 1-2 min intervals. Though there was overlap, we could discriminate an early response at approximately 2 min (phase I) and a delayed or sustained response at greater than or equal to 2 min (phase II) on the basis of responses in the presence and absence of external Ca2+. Phase I was relatively independent of external [Ca2+] and coincided with 45Ca efflux following a dose-dependent increase in cytosolic [Ca2+], measured by Fura-2AM. Phase II was sustained at approximately 80% of control at an external [Ca2+] of 1-5 microM, but was eliminated by adding 0.5-1 mM EGTA. Bombesin caused a sustained Ca2+ influx and, when this was prevented by EGTA, the response to successive stimulations by bombesin and by acetylcholine was greatly attenuated. The phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, which stimulates secretion at high concentrations, was used as background at a threshold concentration of 10(-7) M, which did not by itself stimulate secretion. At this concentration, 12-O-tetradecanoyl phorbol 13-acetate potentiated the responses to bombesin and to acetylcholine. These results define the different Ca2+ dependencies of the immediate and sustained secretory responses to bombesin, but indicate a complex relationship of stimulation responses to Ca2+ homeostasis in various agonist-sensitive Ca2+ pools.