MULTIPHASIC ACTION OF GLUCOSE AND ALPHA-KETOISOCAPROIC ACID ON THE CYTOSOLIC PH OF PANCREATIC BETA-CELLS - EVIDENCE FOR AN ACIDIFICATION PATHWAY LINKED TO THE STIMULATION OF CA2+ INFLUX

Glucose stimulation raises the pH(i) of pancreatic beta-cells, but the underlying mechanisms are not web understood, We have now investigate d the acute effects of metabolizable (glucose and the mitochondrial su bstrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high Kand the K-ATP channel blocker tolbutamide) insulin secretagogues on th e pH(i) of pancreatic beta-cells isolated from normal mice, as assesse d by BCECF fluorescence from single cells or islets in the presence of external bicarbonate, The typical acute effect of glucose (22-30 mM) 0.11 the pH(i) was a fast alkalinization of approximately 0.11 unit, f ollowed by a slower acidification, The relative expression of the alka linizing and acidifying components was variable, with some cells and i slets displaying a predominant alkalinization, others a predominant ac idification, and others yet a mixed combination of the two, The initia l alkalinization preceded the [Ca2+](i) rise associated with the activ ation of voltage-sensitive Ca2+ channels, There was a significant over lap between the glucose-evoked [Ca2+](i) rise and the development of t he secondary acidification, Depolarization with 30 mM K+ and tolbutami de evoked pronounced [Ca2+](i) rises and concomitant cytosolic acidifi cations, Blocking glucose-induced Ca2+ influx (with 0 Ca2+, nifedipine , or the K-ATP channel agonist diazoxide) suppressed the secondary aci dification while having variable effects (potentiation or slight atten uation) on the initial alkalinization, KIC exerted glucose-like effect s on the pH(i) and [Ca2+](i), but the amplitude of the initial alkalin ization was about twice as large for KIC relative to glucose, It is co ncluded that the acute effect of glucose on the pH(i) of pancreatic be ta-cells is biphasic, While the initial cytosolic alkalinization is an immediate consequence of the activation of H+-consuming metabolic ste ps in the mitochondria, the secondary acidification appears to origina te from enhanced Ca2+ turnover in the cytoplasm, The degree of couplin g between glucose metabolism and Ca2+ influx as well as the relative e fficacies of these processes determines whether the acute pH(i), respo nse of a beta-cell (or of a tightly coupled multicellular system such as an islet of Langerhans) is predominantly an alkalinization, an acid ification, or a mixed proportion of the two.