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

Abstract

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 /3-cells. Islets, preloaded with 86 Rb + 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 14 C-urea and 3 H-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 x 10~6 M and 0.35 x 1O~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 x 10~3 M and 4.0 x 1O~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 x 10 6 M Ca ++ , but the presence of ATP was necessary, or the islets would not respond to 0.35 x 1 0 6 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 x 10~6 M Ca ++ 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 x 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 /3-cells to introduce ions and solutes into the intracellular environment so that the factors controlling exocytosis can be determined. DIABETES 29:911-918, November 1980.

Adenosine 3′,5′‐cyclic monophosphate‐mediated enhancement of calcium‐evoked prolactin release from electrically permeabilised 7315c tumour cells

“…Permeabilised 7315c cells exhibited increased prolactin secretion when the intracellular calcium concentration was increased between 0.1 to 10pM with an EC50 of 5.8.M. These data are entirely consistent with those obtained from several cell types using various permeabilisation techniques (Pace et al, 1980;Knight & Baker, 1982;Dunn & Holz, 1983;Wilson & Kirshner, 1983;Knight & Scrutton, 1980;. In addition, the calcium concentration range of 0.1-10M is similar to that recently found in electrically permeabilised GH3 cells (Ronning & Martin, 1986) and our own results in dispersed intermediate lobe pituitary cells (Yamamoto et al, 1987).…”

Efflux of 86Rb from rat and mouse pancreatic islets: the role of membrane depolarization

“…This would explain the inability of calcium removal or a local anaesthetic to affect the response. The current strengths used in our experiments are far below (i.e., more than two orders of magnitude less) those required to produce dielectric breakdown of cell membranes in pancreatic islets (Pace et al, 1980) and more comparable to those shown by microelectrode studies to depolarize the a-cell membrane (Cook et al, 1981). Although the precise nature of the membrane changes induced by direct electrical stimulation in our experiments must await a more extensive investigation it should be pointed out that these experiments demonstrate for the first time the feasibility of measuring 86Rb efflux from single islets, a technique which has a number of advantages over the usual methods for studying islet…”

l-Cysteine Inhibits Insulin Release From the Pancreatic β-Cell

“…However, we consider that L-cysteine and NaHS may also act on the ␤-cell secretory machinery in addition to inhibiting glucose metabolism, because these substances also inhibited insulin release from SLO-permeabilized islets under the conditions where ATP was exogenously supplied. Moreover, it was reported that glucose metabolism is nullified in permeabilized islets (33), and metabolic inhibitors examined so far in this study failed to decrease insulin release from SLO-treated islets.…”