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.

The role of chemiosmotic lysis in the exocytotic release of insulin has been studied using perifused rat pancreatic islets of Langerhans. Established criteria for osmotic lysis of secretory granules requires proton translocation across the secretory granule membrane and the influx of a permeant anion. The consequent increase in granule osmolarity induces water entry and granule lysis. A proton gradient has been previously established to exist across the insulin secretory granule membrane. We have examined the sensitivity of insulin release to 1) hyperosmolar solutions, 2) replacement of medium Cl-, 3) replacement of medium Na+, and 4) anion transport inhibitors. The addition of 200-600 mM sucrose resulted in a 32-69% inhibition of insulin release due to 16.7 mM glucose. Replacement of Cl- by isethionate or SO4--reversibly inhibited glucose-induced insulin release by 47% and 78%, respectively. Na+ replacement by choline did not influence the secretory response. 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid (500 microM) and probenecid (10 mM) inhibited insulin release by 73% and 79%, respectively. These drugs are known to inhibit anion exchange in erythrocytes and may be influencing Cl- entry into the secretory granule fused to the plasma membrane by a similar mechanism. Furosemide inhibits NaKCl2 cotransport in erythrocytes, but had no influence on glucose-induced insulin release, suggesting that Cl- does not enter the secretory granule by this pathway. The primary criteria for the participation of a chemiosmotic mechanism subserving lysis of the insulin secretory granule are fulfilled by these results.

Protons (H+) generated by glucose metabolism have been proposed to serve as a coupling factor between cationic and secretory events in the B-cell. We have examined the influence of alteration of extracellular or intracellular pH (pHo or pHi) on dynamic secretory responses of perfused rat islets to 4.2, 8.4, or 16.7 mM glucose. Reduction of pHo from 7.4 to 7.0 inhibited the secretory response to 16.7, but not 8.4 mM glucose, by 47% during the 30-min period following medium change. Increase of pHo from 7.4 to 7.8 had no influence on the secretory response to glucose. Alteration of pHo had no influence on basal insulin release in the presence of 4.2 mM glucose. Sulfamerazine (5 mM), a permeable weak acid, augmented the secretory response to 8.4 mM glucose by 60% but had no influence on the response to 16.7 mM glucose. In contrast, imidazole (10 mM), a permeable weak base, inhibited the secretory response to both 8.4 (62%) and 16.7 mM (72%) glucose. Another weak base, NH4Cl (20 mM), also inhibited the secretory response to 8.4 (61%) and 16.7 mM (68%) glucose. Alteration of pHi by sulfamerazine and imidazole did not alter basal insulin release in the presence of 4.2 mM glucose. A comparison of the present findings to those obtained for the influence of pH on glucose-induced electrical activity indicates that alteration of pHi, and not pHo, induces parallel effects on glucose-induced electrical and secretory events.

No abstract

No abstract