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First published online 9 June 2004
doi: 10.1242/jcs.01179
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Research Article |
1 Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, 1211 Geneva 4, Switzerland
2 Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
* Author for correspondence (e-mail: claes.wollheim{at}medecine.unige.ch)
Accepted 24 February 2004
Synaptotagmin (Syt) is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic ß-cell is an example of a Ca2+-dependent secretory process. Previous studies have yielded conflicting results as to which Syt isoform is present on the secretory granules in the native ß-cell. Here we show by western blotting and RT-PCR analysis, the presence of both Syt V and Syt IX in rat pancreatic islets and in the clonal ß-cell line INS-1E. The subcellular distribution of the two Syt isoforms was assessed by confocal microscopy and by sedimentation in a continuous sucrose density gradient in INS-1E cells. These experiments show that both proteins colocalize with insulin-containing secretory granules but are absent from synaptic-like microvesicles. Further immunofluorescence studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-secreting 
-cells, whereas Syt IX is associated with insulin granules in ß-cells. Transient overexpression of Syt V and Syt IX did not alter exocytosis in INS-1E cells. Finally, reduction of the expression of both Syt isoforms by RNA interference did not change basal secretion. Remarkably, hormone release in response to glucose was selectively and strongly reduced, indicating that Syt V and Syt IX are directly involved in the Ca2+-dependent stimulation of exocytosis.
Key words: RNA interference, ß-cell, Insulin secretion, INS-1E cells, Pancreatic islet, hGH secretion
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