Effects of calcium and phosphate on catecholamines, ATP and dopamine ?-hydroxylase of chromaffin medullary granules

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Abstract
Isolated bovine chromaffin granules lost their catecholamines to a significantly higher degree when incubated in isotonic sucrose-buffer of pH 7.4 containing 10 and 25 mM sodium phosphate, respectively, than during incubation in sucrose with 4 mM sodium phosphate. In 4 mM sodium phosphate-sucrose, CaCl2 in a final concentration of 1 and 2 mM, respectively, produced only traces of an amorphous precipitate of calcium phosphate which increased the efflux of catecholamines only to a small degree. The same concentrations of CaCl2 in 10 and 25 mM sodium phosphate containing sucrose solutions caused larger amounts of calcium phosphate precipitate and simultaneously a very high efflux of catecholamines. Small amounts of exogenous ATP (1 mM) and MgCl2 (0.3 mM) effectively antagonized the efflux of catecholamines from the granules evoked by calcium phosphate. The influx of catecholamines into the granules from the different incubation media was always inhibited by CaCl2. This inhibition of catecholamine influx was almost abolished by ATP-Mg2+. Incubation of granules in 25 mM sodium phosphate-sucrose in the presence of 1 and 2 mM CaCl2, respectively, caused a release of catecholamines and of ATP of about 90 and 98%, respectively. This nearly compete release of catecholamines and ATP was not accompanied by a simultaneous release of soluble proteins indicating that calcium phosphate did not produce a lysis of the granules. Determinations of the dopamine β-hydroxylase (DBH) in the granules as well as in the incubation medium after 30 min incubation in 25 mM phosphatesucrose revealed an inactivation of the DBH-activity in both extracts of approximately 40%. 2 mM CaCl2 reduced the DBH-activity in the granules by additional 27%, whereas ATP-Mg2+ diminished the degree of inactivation after incubation with and without CaCl2. The DBH-activity of the incubation medium represents only 1% of that in the granules and was not increased by incubation with calcium phosphate. Incubation in phosphate-sucrose diminished by about 40% the activity of the soluble as well as of the membrane-bound DBH of the granules. 2 mM CaCl2 did not change the activity of the membranebound DBH, but reduced that of the soluble form. This additional inactivation evoked by CaCl2 could be antagonized by ATP-Mg2+ as could that produced by incubation. The relevance of the present results for the release and uptake mechanisms of catecholamines is discussed.