OSCILLATIONS IN OXYGEN-CONSUMPTION BY PERMEABILIZED CLONAL PANCREATICBETA-CELLS (HIT) INCUBATED IN AN OSCILLATORY GLYCOLYZING MUSCLE EXTRACT - ROLES OF FREE CA2+, SUBSTRATES, AND THE ATP ADP RATIO/

To determine whether oscillations in glycolysis could underlie the osc illations in O-2 consumption observed in intact islets, we evaluated t he capacity of an islet extract to exhibit spontaneous oscillations in glycolysis, When a cell-free extract obtained from similar to 1,000 i slets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spont aneous oscillations in islet extracts, we bathed permeabilized clonal beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-glycerophosphate and pyr uvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. Thi s preparation was used to investigate whether changes in Ca2+ and poss ibly alpha-glycerophosphate or pyruvate supply could underlie observed oscillations in O-2 consumption and explain coordination between cyto solic and mitochondrial metabolism. We found that oscillations of O-2 consumption and Ca2+ of a similar period were induced. Removal of medi um Ca2+ with EGTA did not prevent the oscillations in O-2 consumption nor were they greatly affected by the substantial rise in medium Ca on treatment with thapsigargin to inhibit sequestration into the endopla smic reticulum. The O-2 oscillations were also not eliminated by the a ddition of relatively high concentrations of pyruvate or alpha-glycero phosphate. However, they were lost on addition of fructose-2,6-P-2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased O-2 consumpt ion and also prevented O-2 oscillations. These results suggest that th e changes in respiration reflected in the O-2 oscillations occur in re sponse to the oscillations in the ATP/ADP ratio or ADP concentration a nd that this parameter is a primary regulator of O-2 consumption in th e pancreatic beta-cell.