title: Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization.
abstract: The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.
authors.affiliation: Department of Medicine, University of Geneva, Switzerland.
authors.forename: G D
authors.initials: GD
authors.lastname: Li
authors.forename: D
authors.initials: D
authors.lastname: Milani
authors.forename: M J
authors.initials: MJ
authors.lastname: Dunne
authors.forename: W F
authors.initials: WF
authors.lastname: Pralong
authors.forename: J M
authors.initials: JM
authors.lastname: Theler
authors.forename: O H
authors.initials: OH
authors.lastname: Petersen
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007328:Insulin; D001127:Arginine Vasopressin; D008345:Manganese; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium