STABLE AND DIFFUSIBLE POOLS OF NUCLEOTIDES IN PANCREATIC-ISLET CELLS

Adenine nucleotides are thought to serve as second messengers in the c ontrol of beta-cell function by glucose, e.g. by regulating the activi ty of ATP-dependent K+ channels. However, their localization in differ ent intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The pla sma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplas mic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min a t 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin s tores when these were lowered by stimulating secretion to different de grees during culture before permeabilization. From these results, it c an be calculated that nondiffusible nucleotides are mainly contained i n insulin secretory granules, with a small proportion in another, prob ably mitochondrial, compartment. Approximately 80% GTP and 30% GDP wer e present in the diffusible pool, and their relative proportions in th e granular pool were only about 20% that of adenine nucleotides. Incub ation of the cells in 20 instead of 2 mM glucose before permeabilizati on did not affect the nondiffusible pool, which indicates that the inc rease in the ATP/ADP ratio measured in intact cells occurred in the di ffusible pool. Cytoplasmic nucleotide levels could be evaluated by sub tracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fal l in ADP.