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The amino acids L-leucine, L-isoleucine, and L-arginine require a subthreshold concentration of glucose to elicit insulin release and electrical activity from B-cells. There is evidence suggesting that protons couple the metabolism of glucose to the functional response of B-cells. In view of this, a permeable weak acid, sulfamerazine, was used to determine if the generation of intracellular protons could account for the permissive action of glucose. Addition of 10 mM sulfamerazine elicited constant spike activity only with 20 mM leucine. With 20 mM arginine or isoleucine, sulfamerazine induced silent depolarization no different from that caused by sulfamerazine alone. The pattern of the electrical activity of each amino acid plus 5.6 mM glucose or alpha-ketoisocaproic acid alone was qualitatively different; addition of sulfamerazine enhanced the electrical response. The permeable weak base NH4Cl at 20 mM immediately inhibited the electrical response to each amino acid plus glucose or alpha-ketoisocaproic acid alone. The effects of the permeable weak acid and base indicate that intracellular pH is important in maintaining amino acid-induced electrical activity. The permissive role of glucose may be due to provision of protons only with leucine.

The influence of exogenous phospholipase C and melittin on electrical activity in islet B cells was determined to assess the extent to which polyphosphatidylinositol turnover serves to generate components that influence the electrical events in the plasma membrane. Phospholipase C hydrolyzes polyphosphatidylinositol to diacylglycerol and polyphosphoinositol, whereas melittin increases the susceptibility of phospholipids to phospholipases and increases the permeability of the membrane to ions. Application of both 20 mU/ml phospholipase C or 0.5 mg/ml melittin to 11.1 mM glucose elicited a time-dependent enhancement of glucose-induced electrical activity that stabilized after 10 min. Phospholipase C increased both the active phase fraction and the burst frequency, whereas melittin only increased the burst frequency. These results indicate that both compounds, which disrupt the phospholipid environment of the plasma membrane, play a role in modulating the oscillatory pattern of electrical activity in the B cell, although melittin is obviously not influencing the factors controlling the ionic events in the same manner as phospholipase C.

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.

The electrical responses of rat islet cells maintained in culture with varying levels of glucose were determined. After culture with 2.8 or 4.2 mM glucose, the beta-cells did not respond electrically to 27.8 mM glucose. However, after culture in 5.6 mM glucose, the cells responded to 27.8 mM glucose in short term experiments with depolarization and an increase in the incidence of spike activity. Islet cells maintained in 8.4 or 14.0 mM glucose had increasingly greater (negative) membrane potentials. The incidence of glucose-induced spike activity was the greatest for cells which had been cultured in 14.0 mM glucose. Raising glucose in the culture medium from 2.8 to 14.0 mM for the last 2 days of culture restored the ability of the islets to respond electrically to glucose in short term experiments. These observations show that the electrical phenomena of the beta cell membrane are affected by alterations in the level of glucose in the culture medium and serve as reliable indicators of functional glucose sensitivity of the islet cells.