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

Abstract

Ca¥ plays a major role in stimulus-response coupling. In electrically non-excitable cells, the activation of receptors linked to phospholipid hydrolysis evokes a biphasic increase in the concentration of cytoplasmic free Ca¥ ([Ca¥]é). The first phase is due to the stimulation by inositol 1,4,5_tris-phosphate (IP3) of Ca¥ release from intracellular Ca¥ stores. The second phase results from an entry of Ca¥ across the plasma membrane (Berridge, 1993 [Ca¥]i, but had little (at 10 mÒ K¤) or no effect on the plateau level. This confirms that the capacitative Ca¥ entry is small. 4. In clusters of cells whose membrane potential was not clamped with diazoxide, 15 mÒ glucose (in 4·8 mÒ K¤) induced [Ca¥]é oscillations by promoting Ca¥ influx through voltagedependent Ca¥ channels. The application of thapsigargin accelerated these oscillations and increased their amplitude, sometimes causing a sustained elevation of [Ca¥]i. Similar results were obtained from whole islets perifused with a medium containing ü 6 mÒ glucose. The effect of thapsigargin was always much larger than expected from the capacitative Ca¥ entry, probably because of a potentiation of Ca¥ influx through voltage-dependent Ca¥ 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 [Ca¥]é in cells in which [Ca¥]é oscillations persisted in the presence of a maximally effective concentration of tolbutamide. 6. In conclusion, the emptying of intracellular Ca¥ stores in â-cells induces a small capacitative Ca¥ entry and activates a depolarizing current which potentiates glucose-induced Ca¥ influx through voltage-dependent Ca¥ channels.

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1−/− mice

“…The CPA-induced Ca 2+ influx was resistant to D600 but sensitive to extracellular Ca 2+ (Fig. 6), which is consistent with the activation of store-operated channels as described previously for wild-type beta cells [41]. We suggest that this store-operated depolarising current is sufficient to promote membrane depolarisation and thus increase the frequency of V m oscillations.…”

Oscillatory control of insulin secretion

“…The importance of PLC activity for insulin secretion is underlined by the fact that the enzyme is activated not only after exposure of islets and β-cells to various G-protein coupled receptor stimuli, such as acetylcholine/carbachol (Best and Malaisse, 1983;Hellman and Gylfe, 1986a;Best et al, 1987;Biden et al, 1987;Gilon and Henquin, 2001) and ATP (Gylfe and Hellman, 1987;Blachier and Malaisse, 1988), but also after exposure to glucose (Axen et al, 1983;Best and Malaisse, 1983;Laychock, 1983;Montague et al, 1985) and depolarizing agents (Laychock, 1983;Mathias et al, 1985;Best et al, 1987;Biden et al, 1987;Zawalich and Zawalich, 1988 (Prentki et al, 1988;Gylfe, 1991;Hellman et al, 1992;Theler et al, 1992;Miura et al, 1996) and these oscillations are characterized by a much shorter period than the glucose-induced, slow oscillations described above. Most of the [Ca 2+ ] i -elevating effect is due to IP 3 -mediated Ca 2+ mobilization from the ER, but the emptying of the stores also triggers Ca 2+ entry through store-operated channels in the plasma membrane (Liu and Gylfe, 1997;Miura et al, 1997;Dyachok and Gylfe, 2001). Although the store-operated influx of Ca 2+ only causes a small elevation of [Ca 2+ ] i , this pathway may also contribute by its depolarizing effect.…”

Uptake and Release of Ca2+ by the Endoplasmic Reticulum Contribute to the Oscillations of the Cytosolic Ca2+ Concentration Triggered by Ca2+ Influx in the Electrically Excitable Pancreatic B-cell

“…It has recently been demonstrated that the magnitude of the current activated by intracellular pool emptying correlates with the extent of store depletion and that it can be activated even by small decreases in [Ca 2ϩ ] ER (55). In pancreatic B-cells, intracellular Ca 2ϩ pool depletion activates a small Ca 2ϩ entry and induces a depolarizing current, possibly carried by Na ϩ , that potentiates the activation of voltage-dependent Ca 2ϩ channels (17,56). This was evidenced in the present study by the observation that in the presence of 2.5 mM Ca 2ϩ in the medium, TG transformed oscillations of the membrane potential into a sustained depolarization.…”

“…Pancreatic islets were isolated aseptically after collagenase digestion of the pancreas, and when needed, they were dispersed into cells as described previously (17). Cells were allowed to attach to 22-mm circular coverslips and cultured for 2-3 days.…”

“…Previous experiments have shown that intracellular Ca 2ϩ stores of whole islets are efficiently emptied by thapsigargin (TG), a specific inhibitor of the SERCA pump (19), but that this emptying requires preincubation of the islets with the drug (17). In islets pretreated with 1 M TG, the amplitude of [Ca 2ϩ ] c oscillations was much larger than in control islets, and the descending phase of each [Ca 2ϩ ] c oscillation was surprisingly very fast with no slow second phase (Fig.…”