The widespread role of somatostatin (SRIF) as a mediator of function in the brain and gut has stimulated interest in it mechanism of action. We have examined the mode of action of SRIF in stimulus-secretion coupling in the pancreatic islet beta-cell to determine whether SRIF antagonizes the glucose-induced decrease in K+ permeability (PK). The influence of SRIF on 86Rb fluxes and insulin release in cultured rat islet cells, and also the electrical events recorded from cultured islets and microdissected mouse islets, was examined. In cultured islets, 100 ng/ml SRIF in the presence of 16.7 mM glucose inhibited the incidence of spike activity and evoked hyperpolarization. This effect was counteracted by 0.1 mM quinine and 20 mM tetraethylammonium (TEA), drugs that inhibit the Ca2+-sensitive or voltage-sensitive increase in PK, respectively. These agents also counteracted the inhibitory influence of SRIF on glucose-induced insulin release in cultured islets. SRIF disrupted the typical glucose-induced oscillatory pattern of electrical activity (burst activity) during continuous microelectrode recordings in mouse beta-cells, resulting in a transient 5mV hyperpolarization and a decrease in the frequency of generation of burst activity. The presence of 20 mm TEA prevented the influence of SRIF on the electrical activity. SRIF had no effect on the accumulation of 86Rb into islet cells obtained in the presence of 16.7 mM glucose. However, SRIF enhanced the rate of 86Rb efflux from cells exposed to glucose. SRIF-induced enhancement of 86Rb efflux was antagonized by TEA or quinine. These results indicate that SRIF may activate PK as its primary mode of action, an event that may be sufficient to reduce the accumulation of intracellular Ca2+ thereby disrupting glucose-induced stimulus-secretion coupling.

We have examined the influence of changes in pH on the oscillatory pattern of electrical activity (EA) in the beta-cell by altering medium pH (pHo) and using permeable weak buffers to alter intracellular pH (pHi). A decrease in pH in the presence of glucose elicited depolarization to the active phase and constant spike activity, whereas an increase in pH elicited a decrease in spike activity or silent hyperpolarization. On inhibition of HCO3:Cl antiport by addition of DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), probenecid, or withdrawal of medium HCO-3, there was an increase in the duration of the active phase. A similar result was obtained on the inhibition of Na:H antiport by the addition of amiloride or the reduction of medium [Na+]. The influence of H+ and glucose has been proposed to decrease K+ permeability (PK). However, the influence of pH on 86Rb+ efflux was most effective at subthreshold or 4.2 mM glucose; only a moderate decrease in PK occurred at 8.3 mM glucose, and no effect was obtained at 16.7 mM glucose. Alteration of pHi, and not pHo, induces similar effects on glucose-induced electrical and secretory events. There is a clear dissociation between the influence of inhibitors of the Na:H and HCO3:Cl antiporters on the electrical and secretory events. DIDS and amiloride increased glucose-induced EA, but markedly inhibited the secretory response to glucose. It is evident that pH modulates the electrical events and cationic fluxes and ultimately influences the transduction of information to the mechanisms controlling the secretory process in the beta-cell.

Regulation of intracellular pH is an essential function and may be especially significant in the B-cell in which the influence of glucose on electrical activity is modulated by alterations in pH. Two possible regulatory processes have been examined: Na/H and HCO3/Cl exchange, by using inhibitors, an ionophore, and changes of ionic concentrations. In the presence of 11.1 mM glucose we found that DIDS, an inhibitor of anion exchange, elicited a dose-response increase in the relative duration of the active phase with an ED50 of 99 microM. Probenecid (0.5 mM), an inhibitor of anion fluxes, also augmented the electrical activity (EA) due to glucose. Withdrawal of HCO-3 elicited constant spike activity followed by a resumption of burst activity with a greater duration of the active phase compared to control. These data are consistent with predicted cellular acidification. However, reduction of Cl-o by isethionate substitution produced no marked effect on EA. In contrast, SO-4- substitution for Cl- resulted in variable effects characterized by constant spike activity or a decrease in the duration of the active and silent phases along with silent hyperpolarization. Tributyltin, a Cl/OH, ionophore enhanced EA at 0.25 microM with 120 mM Cl-o, but reduced EA with 10 mM Cl- as would be predicted with either cellular acidification or alkalinization, respectively. Amiloride at 100 microM elicited constant spike activity perhaps due to inhibition of Na/H exchange. Reduction of Na+o from 142.8 to 40.8 mM had a similar effect and enhanced the influence of amiloride. It appears therefore that interference with putative pH regulatory mechanisms in the B-cell are consistent with the hypothesis that cell pH is involved in regulation of EA.

No 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.