Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion |
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Authors: | Claes B Wollheim Susanne Ullrich Paolo Meda Lucia Vallar |
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Institution: | (1) Institut de Biochimie Clinique, Centre Médical Universitaire, University of Geneva, 1211 Geneva 4, Switzerland;(2) Institut d'Histologie et d'Embryologie, Centre Médical Universitaire, University of Geneva, 1211 Geneva 4, Switzerland;(3) Department of Medical Pharmacology, University of Milano, Milano, Italy |
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Abstract: | The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP3
inositol trisphosphate
- Ptd-InsP2
phosphatidylinositol 4,5-bisphosphate
- GTPS
guanosine 5-(3-O-thio)triphosphate
- GDPS
guanosine 5-(2-O-thio)diphosphate
- Gpp(NH)p
guanyl-5-yl imidodiphosphate
- TPA
12-O-tetradecanoylphorbol-13-acetate
- OAG
1-oleoyl-2-acetylglycerol
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- EGTA
(ethylenebis(oxyethylenenitrilo)tetraacetic acid
- DAG
diacylglycerol
- Ca2+]i
cytosolic free Ca2+ concentration |
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Keywords: | calcium exocytosis insulin secretion permeabilized cells |
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