Use of a High Voltage Technique to Determine the Molecular Requirements for Exocytosis in Islet Cells

Abstract

SUMMARY Pancreatic islet cells were subjected to high voltage discharges, which induced pore formation in the plasma membrane. This technique was used to determine the molecular requirements of intracellular sites involved in the control of insulin release in β-cells. Islets, preloaded with 86Rb+ and then shocked, released 92% of the radioisotope within 1 min as compared with only a 55% loss from nonshocked islets. Exposure of the islets to 14C-urea and 3H-sucrose at 0 to 10 min after exposure to high voltage discharges indicated that 68% of the intracellular space was occupied by sucrose, whereas sucrose was excluded from nonshocked islet cells. The pores in the plasma membrane resealed about 30 min after their initial formation, as was indicated by the cellular exclusion of sucrose. The Ca++ concentrations yielding half-maximal and maximal secretory responses from shocked islets were 0.05 × 10−6 M and 0.35 × 10−6 M, respectively; the presence of 16.7 mM glucose did not alter these values. In intact islets, a variation of extracellular Ca++ (only in the presence of 16.7 mM glucose) generated a dose-response curve yielding half-maximal and maximal secretory responses at 2.0 × 10−3 M and 4.0 × 10−3 M, respectively. The total amount of insulin released from shocked islets was three times that released from nonshocked islets during a 15 min incubation period. The addition of 1 or 5 mM ATP during an initial shock and incubation period did not augment the secretory response to 0.05 × 10−6 M Ca++, but the presence of ATP was necessary, or the islets would not respond to 0.35 × 106 M Ca++ during a subsequent shock and incubation period. The presence of 1.0 mM 3-phosphoglycerate or phosphoenolpyruvate augmented the secretory response to 0.05 × 10−6 MCa++ only in the presence of 1.0 mM ATP. Glucose-6- phosphate or fructose-1,6-diphosphate had no influence on the secretory response to Ca++ in the presence of ATP. An increase in Mg++ from 1.0 to 10 mM reduced the secretory response to 0.35 × 10−6 M Ca++ by 63%. Islets, subjected to the high voltage discharges and allowed 30 min to reseal, exhibited a normal secretory response to 16.7 mM glucose. The results indicate that the high voltage technique induces reversible pore formation in β-cells to introduce ions and solutes into the intracellular environment so that the factors controlling exocytosis can be determined.