Metabolic Pools of ATP in Cultured Bovine Adrenal Medullary Chromaffin Cells

Abstract

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 β‐glucono‐l,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 μM adenosine to the media resulted in a similar loss of [3HJATP 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. Permeabilization of the plasma membrane by treatment with digitonin resulted in a loss of 90–95% of the labeled ATP but only 20% of the endogenous ATP in cells examined immediately after labeling and in a 79% loss of [3H]ATP, a 32% loss of [32P]ATP, and a 23% loss of endogenous ATP in cells examined 48 h after labeling. These studies show that ATP contained within chromaffin vesicles is metabolically inert and does not exchange readily with cytosolic ATP, but they do not differentiate extravesicular pools of labeled ATP. Calculation of ATP disappearance after abrupt cessation of ATP production indicates that cultured adrenal chromaffin cells utilize ATP at a rate of 0.24 nmol/min/106 cells, corresponding to a turnover time of 12.5 min for the extravesicular pool.

The intravesicular cocktail and its role in the regulation of exocytosis

“…; Corcoran et al . ). However, the turnover of vesicular ATP seems to be very slow in chromaffin granules when measured using radioactive nucleotides (Corcoran et al .…”

“…This resistance to change suggests that this molecule can be considered as metabolically inert, scarcely entering into dynamic exchange with the cytoplasmic ATP (Corcoran et al . ).…”

“…Despite the data obtained in vitro regarding the formation of complexes between ATP and amines, little information exists about the role of intravesicular ATP in the storage of other neurotransmitters and in their exocytosis. Chromaffin granules behave as 'ATP sinks' and vesicular ATP accounts for 75% of the entire cellular ATP content (Bevington et al 1984;Corcoran et al 1986a). However, the turnover of vesicular ATP seems to be very slow in chromaffin granules when measured using radioactive nucleotides (Corcoran et al 1986b).…”

“…Pharmacological inhibition of ATP production through the application of cyanide and deoxyglucose successfully reduces the cytosolic ATP, yet these agents prove to have virtually no effect on the vesicular ATP content, even when treatments were prolonged over several days. This resistance to change suggests that this molecule can be considered as metabolically inert, scarcely entering into dynamic exchange with the cytoplasmic ATP (Corcoran et al 1986a).…”