Lena Norlund

Evidence for co‐transport of sodium, potassium and chloride in mouse pancreatic islets.

1. The presence of a loop diuretic‐sensitive co‐transport system for Na+, K+ and Cl‐ was tested in isolated pancreatic islets. 2. Substitution of Cl‐ with the impermeant anion isethionate or addition of frusemide both reduced the ouabain‐resistant islets uptake of 86Rb+ (K+ marker) without affecting the ouabain‐sensitive uptake or equilibrium content of 86Rb+. The effects of Cl‐ substitution and frusemide were overlapping. 3. D‐Glucose reduced the ouabain‐resistant islets uptake of 86Rb+. This effect was additive to the effect of Cl‐ substitution or frusemide. 4. Substitution of Cl‐ with isethionate or addition of frusemide both reduced the efflux of 86Rb+ from the islets. These effects were additive to the reduction of 86Rb+ efflux induced by D‐glucose. 5. Substitution of K+ or Na+ with choline reduced the equilibrium content of 36Cl‐ in the pancreatic islets. 6. These data are compatible with the operation in the pancreatic beta‐cells of a loop diuretic‐sensitive co‐transport system for Na+, K+ and Cl‐, that may serve as an inwardly directed Cl‐ pump.

Glucose reduces both Rb+ influx and efflux in pancreatic islet cells

Microdissected, β‐cell‐rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D‐Glucose (20 mM) induced a biphasic reduction in 86R+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose‐dependently reduced by ouabain with maximum inhibition at 1 mM. D‐Glucose (20 mM) did not affect the ouabain‐sensitive 86Rb+ influx but markedly reduced (48%) the ouabain‐resistant isotope influx. The results suggest that D‐glucose does not affect the Na+/K+ pump in pancreatic β‐cells and that the glucose‐sensitive K+‐transporting modalities (K+ channels) in the β‐cells can mediate both inward and outward K+ flux.

Ultrastructure and membrane permeability of cultured pancreaticβ-cells exposed to alloxan or 6-hydroxydopamine

Stereological techniques on electron microscopy micrographs were used to evaluate the morphological changes of cultured isletβ cells that had been exposed to alloxan or 6-hydroxydopamine. Trypan Blue exclusion by cells cultured for 3 days indicated that the cells were 100% viable. Electron microscopy revealed that nearly all of the surviving cultured cells wereβ cells. Exposure to 5 mmol/l alloxan or 1–5 mmol/l 6-hydroxydopamine for 10 or 30 min caused a general swelling of the cultured cells with a concomitant swelling of mitochondria and nuclei. The size of the secretory granules was not affected by the drugs. Only 3–10% of the cells excluded Trypan Blue after exposure to 5 mmol/l alloxan or 6-hydroxydopamine. The data conform with the hypothesis that a primary action of alloxan and 6-hydroxydopamine is at the plasma membrane level ofβ cells.

Effect of tetracaine and glibenclamide on 45Ca2+ handling by isolated pancreatic islets.

1 The 45Ca2+ uptake in β‐cell‐rich ob/ob‐islets was measured using the La3+ wash technique. 2 Tetracaine (1 mM) markedly enhanced the 45Ca2+ net uptake (120 min) in the presence of 3 mM glucose, and at 7 and 20 mM glucose there were clear tendencies to dose‐dependent increases with 0.1 to 1 mM tetracaine. Glibenclamide 1 μM to 0.2 mM, stimulated the 45Ca2+ net uptake in the presence of 3 mM glucose and 0.1 mM to 0.2 mM glibenclamide potentiated the uptake in the presence of 7 mM glucose. When the drugs were added for only a 10 min incubation period, glibenclamide, 1 μM to 0.2 mM, but not tetracaine (10 μM to 1 mM) increased the short‐term uptake of 45Ca2+. After preincubation with either of the drugs, neither tetracaine (10 μM to 1 mM) nor glibenclamide (10 nM to 0.2 mM) had any effect on the short‐term 45Ca2+ uptake. 3 In islets incubated with 45Ca2+ and tetracaine and washed without La3+ the apparent net uptake of 45Ca2+ was reduced by 0.5 to 1 mM tetracaine both at 3 and 20 mM glucose. 4 Tetracaine (0.5 mM) stimulated the 45Ca2+ efflux in the presence of 3 mM glucose. 5 The results show that both drugs affected the Ca2+ handling. It is suggested that glibenclamide mainly increases Ca2+ influx by voltage‐dependent pathways, whereas tetracaine, at certain concentrations, mobilizes Ca2+ from intracellular stores in the islet cells.

Effects of glibenclamide and tetracaine on 86Rb+ fluxes in mouse pancreatic β-cells

SummaryPotassium transport was measured in β-cell-rich islets from ob/ob-mice using the K+-analogue 86Rb+. Both tetracaine (0.1 mM) and glibenclamide (0.1 μM) reduced the oubain-resistant 86Rb+ influx but did not significantly affect the oubain-sensitive portion (Na+/K+ pump). Tetracaine (0.5–1 mM) or glibenclamide (0.2 mM) decreased the 86Rb+ equilibrium content and glibenclamide (1 μM) transiently reduced the 86Rb+ efflux rate but 0.1 mM tetracaine had only a slight effect on this flux rate. The results suggest that a change in ouabain-resistant (passive) K+ fluxes, but not the Na+/K+ pump, is involved in stimulation of insulin secretion by glibenclamide and tetracaine. Both drugs may exert similar effects on the β-cell plasma membrane.

Morphometry of Rambourg-Positive and Rambourg-Negative β-Cell Granules After Culture With Low and High Glucose Concentrations

Dispersed islet cells from noninbred ob/ob mice were cultured for 3 days with 3 or 20 mM D-glucose and silver stained according to Rambourg et al. Two tinctorial subsets of dark and light intracellular granules were analyzed by morphometry at the ultrastructural level. The two types of granules were similar in size and shape. However, with 3 mM glucose the dark granule cores were surrounded by larger vesicles than the light granules. With 20 mM glucose, both types of granule vesicles and cores became smaller and dark-granule cores became more rounded, compared with cultures with 3 mM glucose. The higher glucose concentration also induced a marked decrease in the number (−84%) and volume density (−90%) of dark granules. In contrast, the number of light granules increased (+60%) with maintenance of their volume density. We suggest that the dark Rambourg-positive and the light Rambourg-negative β-cell granules are functionally distinct subsets. The dark granules are probably engaged in insulin discharge. We discuss the unclear role of the light granules with a view to previously postulated heterogeneities of the insulin granule pool and their significance for exocytosis and intracellular hormone degradation.

A technique for the isolation of highly viable pancreatic B-cells fromob/ob mice

SummaryA method has been developed to prepare free islet cells in suspension from adultob/ob-mice. About 200 collagenase-isolated pancreatic islets were pooled in 4 ml of calcium-free Krebs-Ringer-HEPES buffer supplemented with 1 mM EGTA and 10 µg/ml DNAase. The islets were gently shaken in a water-bath for 10 min at 30°C. Then, the cell suspension was filtered through a nylon screen and centrifuged through ice-cold, dense albumin. The isolated cells, of which more than 99% were B-cells, appeared well preserved both in light- and electron-microscopy. Out of the isolated cells, 7.1±0.5% took up Evans Blue and were thus considered non-viable.