Emptying of Intracellular Ca 2+ Stores Stimulates Ca 2+ Entry in Mouse Pancreatic β‐Cells by Both Direct and Indirect Mechanisms

1 In non‐excitable cells, the depletion of intracellular Ca 2+ stores triggers Ca 2+ influx by a process called capacitative Ca 2+ entry. In the present study, we have investigated how the emptying of these stores by thapsigargin (1 μM) influences Ca 2+ influx in electrically excitable pancreatic β‐cells. The cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) was monitored in clusters of mouse β‐cells or in whole islets loaded with fura‐2. 2 The membrane was first held hyperpolarized by diazoxide, an opener of ATP‐sensitive K + (K ATP ) channels, in the presence of 4.8 mM K + . Alternating between Ca 2+ ‐free medium and medium containing 2.5 mM Ca 2+ caused a minor rise in [Ca 2+ ] i (∼14 nM) in clusters of β‐cells. A larger rise (∼65 nM), resistant to the blockade of voltage‐dependent Ca 2+ channels by D600, occurred when extracellular Ca 2+ was readmitted after emptying intracellular Ca 2+ stores with thapsigargin or acetylcholine. Thus there exists a small capacitative Ca 2+ entry in β‐cells. 3 When the membrane potential was clamped at depolarized levels with 10, 20 or 45 mM K + in the presence of diazoxide, [Ca 2+ ] i increased to different plateau levels ranging between 100 and 900 nM. Thapsigargin consistently caused a further transient rise in [Ca 2+ ] i , but had little (at 10 mM K + ) or no effect on the plateau level. This confirms that the capacitative Ca 2+ 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 [Ca 2+ ] i oscillations by promoting Ca 2+ influx through voltage‐dependent Ca 2+ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca 2+ ] 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 Ca 2+ entry, probably because of a potentiation of Ca 2+ influx through voltage‐dependent Ca 2+ 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 K ATP channels because thapsigargin steadily elevated [Ca 2+ ] i in cells in which [Ca 2+ ] i oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6 In conclusion, the emptying of intracellular Ca 2+ stores in β‐cells induces a small capacitative Ca 2+ entry and activates a depolarizing current which potentiates glucose‐induced Ca 2+ influx through voltage‐dependent Ca 2+ channels.