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Emptying of Intracellular Ca2+ Stores Stimulates Ca2+ Entry in Mouse Pancreatic β-Cells by Both Direct and Indirect Mechanisms

Authors

  • Yoshikazu Miura,

    1. Unité d'Endocrinologie et Métabolisme, University of Louvain School of Medicine, UCL 55.30, B-1200 Brussels, Belgium
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  • Jean-Claude Henquin,

    Corresponding author
    1. Unité d'Endocrinologie et Métabolisme, University of Louvain School of Medicine, UCL 55.30, B-1200 Brussels, Belgium
    • To whom correspondence should be addressed.

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  • Patrick Gilon

    1. Unité d'Endocrinologie et Métabolisme, University of Louvain School of Medicine, UCL 55.30, B-1200 Brussels, Belgium
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Abstract

  • 1In 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 μM) influences Ca2+ influx in electrically excitable pancreatic β-cells. The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in clusters of mouse β-cells or in whole islets loaded with fura-2.
  • 2The 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 Ca2+-free medium and medium containing 2.5 mM Ca2+ caused a minor rise in [Ca2+]i (∼14 nM) in clusters of β-cells. A larger rise (∼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 β-cells.
  • 3When 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.
  • 4In 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 ≥ 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.
  • 5This 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.
  • 6In conclusion, the emptying of intracellular Ca2+ stores in β-cells induces a small capacitative Ca2+ entry and activates a depolarizing current which potentiates glucose-induced Ca2+ influx through voltage-dependent Ca2+ channels.

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