Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and 3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that 3H]ATP and 32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the 32P]ATP was present in chromaffin vesicles while only 35% of the 3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of 32P]ATP and 3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with 3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.