The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K+-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5'-triphosphate (ATP) in the pipette solution but was Ca2+ independent. Na+ and Cl- substitution studies suggested that the swelling-activated current is Cl- selective with a halide permeability sequence of Br > Cl > I. The conductance was reversibly inhibited by the anion channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.

Changes in relative cell volume in response to hypotonic solutions and glucose were studied in single isolated rat pancreatic β‐cells using a video‐imaging technique, β‐cell electrical activity was recorded under similar conditions using the perforated patch technique. Exposure of β‐cells to hypotonic solutions (10 and 33% hypotonicity) caused an immediate increase in cell volume to relative values of 1.09 and 1.33, respectively. This was followed by a gradual regulatory volume decrease. Raising the concentration of glucose from 4 to 20 mm or 12 mm (with substitution of mannitol) increased β‐cell volume by 12 and 10%, respectively. This effect of glucose persisted when Co2+ was added to inhibit insulin release. Glucose‐induced volume increases were sustained for the duration of exposure to elevated hexose concentration. The addition of 16 mm 3‐O‐methylglucose, which is transported into the β‐cell but not metabolized, produced only a transient 5% increase in β‐cell volume. Exposure of β‐cells to a 15% hypotonic solution resulted in a transient depolarization and electrical activity. Raising the glucose concentration to 20 or 12 mm caused a sustained depolarization and generation of electrical activity. However, the addition of 16 mm 3‐O‐methylglucose had no effect on β‐cell membrane potential. The glucose‐induced increase in volume and induction of electrical activity, when measured in single β‐cells simultaneously, showed comparable kinetics. The secretion of insulin from intact pancreatic islets was stimulated by exposure to hypotonic solutions (10–33% hypotonicity). A 15% hypotonic solution stimulated insulin release to a peak value comparable to that elicited by raising the glucose concentration from 4 to 20 mm. Whereas hypotonic solutions caused a transient stimulation of insulin release, the effect of glucose was sustained. It is suggested that glucose increases the volume in rat pancreatic β‐cells by a mechanism dependent upon metabolism of the sugar. The extent of cell swelling evoked by raised glucose concentrations is sufficient to depolarize the cells and induce electrical and secretory activity and may involve activation of a volume‐sensitive anion conductance.

SUMMARYThe perforated patch technique was used to study the effects of hypotonic extracellular solutions on membrane potential and whole-cell currents in intact rat pancreatic ,l-cells. A 30 % reduction in osmolarity resulted in activation of an outwardly rectifying CF-selective conductance in rat f-cells. This conductance was inhibited by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Exposure to a hypotonic medium also led to a transient stimulation of electrical activity accompanied by cell swelling and a gradual return towards control volume. These effects were also associated with the generation of an inward current at a holding potential of -70 mV, and a stimulation of insulin release from intact perifused islets. All of the above effects were inhibited by DIDS. It is suggested that the stimulation of insulin release by hypotonic solutions results from activation of a volume-sensitive anion conductance generating an inward current and leading to a subsequent depolarization of the ,-cell.

A major aspect of stimulation of β-cell function by glucose is the induction of electrical activity. The ionic events that underlie β-cell electrical activity are understood in some detail. At sub-stimulatory glucose concentrations, the β-cell is electrically 'silent'. Increasing the glucose concentration to stimulatory levels results in a gradual depolarisation of the membrane potential to a threshold potential where 'spikes' or action potentials are generated. These action potentials represent the gating of voltage-sensitive Ca²(+) channels, leading to Ca²(+) entry into the cell, thus triggering the release of insulin. The stimulatory actions of glucose on the β-cell depend on the metabolism of the hexose. A major question concerns the molecular mechanism(s) whereby β-cell plasma membrane potential is regulated by changes in glucose metabolism in the cell. This article provides a brief summary of the evidence suggesting that, in addition to metabolically-regulated K(ATP) channels, β-cells are equipped with a volume-regulated anion channel that is activated by glucose concentrations within the range effective in modulating electrical activity and insulin release.

A rise in glucose concentration depolarizes the β-cell membrane potential leading to electrical activity and insulin release. It is generally believed that closure of K ATP channels underlies the depolarizing action of glucose, though work from several laboratories has indicated the existence of an additional anionic mechanism. It has been proposed that glucose activates a volume-regulated anion channel, generating an inward current due to Cl -efflux. This mechanism requires that intracellular

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Preincubation of rat pancreatic islets with 3H-inositol, and subsequent exposure, in the presence of LiCl, to either glucose or carbamylcholine resulted in a rapid stimulation of 3H-inositol 1,4,5-triphosphate and 3H-myo-inositol 1,4-bisphosphate formation, the level of which reached a plateau after about 5 min of stimulation. Both stimuli also caused an approximately linear accumulation of 3H-myo-inositol 1-phosphate. The amounts of 3H-inositol phosphates formed were dependent on the concentration of LiCl. Studies of 32P-labeling of islet ATP, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 4-phosphate revealed that these approached isotopic equilibrium after about 240-min incubation, whereas 32P-labeling of phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine proceeded at a lower rate. Carbamylcholine provoked an immediate fall in 32P-PtdIns(4,5)P2 and, to a lesser extent, 32P-phosphatidylinositol 4-phosphate. Glucose caused a similar response although, in this case, the most marked decline was in a more polar 32P-labeled lipid. Cholecystokinin-pancreozymin was also found to induce 32P-PtdIns(4,5)P2 hydrolysis, although the ionophore A23187 was without effect. Both carbamylcholine and glucose induced an increase in 32P-phosphatidic acid. The results provide two independent pieces of evidence suggesting that phospholipase C-mediated hydrolysis of polyphosphoinositides occurs as an early response in rat islets to either nutrient or neurotransmitter secretagogues.

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