KETOISOCAPROIC ACID AND LEUCINE INCREASE CYTOPLASMIC PH IN MOUSE PANCREATIC B-CELLS - ROLE OF CYTOPLASMIC CA2-REGULATING EXCHANGERS( AND PH)

The effects of nonglucose nutrient insulin secretagogues on cytoplasmi c pH (pH(i)) in pancreatic B cells are unclear. These were studied wit h intact mouse islets loaded with BCECF and stimulated with ketoisocap roic acid (KIC) or leucine, which, unlike glucose, are exclusively met abolized in mitochondria. The changes in pH(i) were compared to those in cytoplasmic Ca2+ ([Ca2+](i); islets loaded with fura-2), metabolism [NAD(P)H fluorescence], and insulin release. In HCO3- buffer containi ng 3 mM glucose, 10 mM KIC produced a rapid and sustained increase in metabolism, [Ca2+](i), pH(i), and insulin release. In HEPES buffer, th e increases in metabolism, [Ca2+](i), and release were also rapid but not as sustained, whereas the alkalinization was delayed. The changes in release thus follow a time course more similar to that of [Ca2+](i) and metabolism than to that of pH(i). The role of [Ca2+](i) in pH(i) changes was next examined. A similar rapid rise in pH(i) was produced by KIC in both HCO3- and HEPES buffers when its effects on [Ca2+](i) w ere prevented, whether [Ca2+](i) was kept low (4.8 mM KCl plus diazoxi de) or high (30 mM KCl plus diazoxide). When the Na+:H+ exchanger was blocked by dimethylamiloride, the alkalinizing effect of KIC was unaff ected in HCO3- buffer, indicating that it does not result from an acti vation of this exchanger. In HEPES buffer, however, KIC strongly decre ased pH(i) unless the rise in [Ca2+](i) was prevented, in which case K IC increased pH(i). When the HCO3-/Cl- exchanger was blocked by 4,4'-d iisothiocyanostilbene-2,2'-disulfonic acid (DIDS), the effect of KIC i n HCO3- buffer became similar to that in HEPES buffer without or with DIDS. The effects of leucine on pH(i) were similar to those of KIC. In conclusion, the effect of KIC and leucine on islet cell pH(i), like t hat of glucose, is the complex result of an alkalinizing action of the ir metabolism and an acidifying action of the [Ca2+](i) rise that they also produce. Compensation of this acidification is achieved by opera tion of the pH(i)-regulating exchangers, of which the HCO3-/Cl- exchan ger plays a predominant role.