Abstract
Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca2+-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 µM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca2+]i-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca2+]i increases.
Highlights
Rab proteins are small G-proteins that coordinate membrane transport in both exocytotic and endocytotic pathways [1,2,3,4,5]
Intracellular localization of zymogen granules First we examined, using two-photon excitation imaging, the intracellular localization of ZGs within the fura-2 FF-loaded pancreatic acinar cells of KO and WT mice
Line-mode intensity analysis along the lines shown from the apical to the basal region revealed that in both KO and WT acinar cells, the mean fluorescence-intensity profile was under 700 arbitrary units in the low-intensity area, but above 1,000 in the high-intensity area (Fig. 1B, D)
Summary
Rab proteins are small G-proteins that coordinate membrane transport in both exocytotic and endocytotic pathways [1,2,3,4,5] They include the subfamilies Rab and Rab, which have been implicated in Ca2+-dependent exocytosis [2,6,7,8,9,10]. In several exocrine glands (viz., salivary glands, pancreatic exocrine glands, gastric chief cells, the Paneth cells of the jejunum, and Brunner’s glands of the duodenum) in KO mice, Noc deletion led to accumulations of secretory granules and to impaired agonist-induced amylase secretion from pancreatic acini [13]. It remains unclear how Noc deletion might impair zymogen granule (ZG) exocytosis in pancreatic exocrine glands, agonist-induced regulated exocytosis
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