digitonin and beta-glucono-1-5-lactone

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine Triphosphate; Adrenal Medulla; Animals; Antimycin A; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cattle; Cell Membrane Permeability; Cells, Cultured; Chromaffin Granules; Chromaffin System; Cytosol; Deoxyglucose; Digitonin; Gluconates; Half-Life; Kinetics; Lactones; Phosphates; Sodium Cyanide