title: Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mechanisms.
abstract: 1. In non-excitable cells, the depletion of intracellular Ca2+ stores triggers Ca2+ influx by a process called capacitative Ca2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 microM) influences Ca2+ influx in electrically excitable pancreatic beta-cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse beta-cells or in whole islets loaded with fura-2. 2. The membrane was first held hyperpolarized by diazoxide, an opener of ATP-sensitive K+ (KATP) channels, in the presence of 4.8 mM K+. Alternating between Ca(2+)-free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (approximately 14 nM) in clusters of beta-cells. A larger rise (approximately 65 nM), resistant to the blockade of voltage-dependent Ca2+ channels by D600, occurred when extracellular Ca2+ was readmitted after emptying intracellular Ca2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca2+ entry in beta-cells. 3. When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K+ in the presence of diazoxide, [Ca2+]i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca2+]i, but had little (at 10 mM K+) or no effect on the plateau level. This confirms that the capacitative Ca2+ entry is small. 4. In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mM glucose (in 4.8 mM K+) induced [Ca2+]i oscillations by promoting Ca2+ influx through voltage-dependent Ca2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca2+]i. Similar results were obtained from whole islets perifused with a medium containing > or = 6 mM glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca2+ entry, probably because of a potentiation of Ca2+ influx through voltage-dependent Ca2+ channels. 5. This potentiating effect of thapsigargin did not result from an acceleration of cell metabolism since the drug did not affect glucose-induced changes in NAD(P)H fluorescence. It is also unlikely to involve the inhibition of KATP channels because thapsigargin steadily elevated [Ca2+]i in cells in which [Ca2+]i oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6. In conclusion, the emptying of intracellular Ca2+ stores in beta-cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose-induced Ca2+ influx through voltage-dependent Ca2+ channels.
authors.affiliation: Unité d'Endocrinologie et Métabolisme, University of Louvain School of Medicine, Brussels, Belgium.
authors.forename: Y
authors.initials: Y
authors.lastname: Miura
authors.forename: J C
authors.initials: JC
authors.lastname: Henquin
authors.forename: P
authors.initials: P
authors.lastname: Gilon
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005711:Gallopamil / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D011725:Pyridines / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D011725:Pyridines; D005711:Gallopamil; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D005947:Glucose; D000109:Acetylcholine; D002118:Calcium