Evidence for an inherent, ATP-stimulated uptake of calcium into chromaffin granules

Summary

Partially purified chromaffin granules (granular fraction), crude mitochondria (mitochondrial fraction) and a microsomal fraction were prepared from bovine adrenals by differential centrifugation and characterized by their catecholamine content, succinate dehydrogenase and glucose-6-phosphatase activity. During isotonic incubation with 0.1 mM ⁴⁵Ca²⁺ all fractions showed an uptake of ⁴⁵Ca²⁺, which was stimulated by ATP. In addition, after incubation the granular fraction was further fractionated by sucrose density gradient centrifugation (2.0–1.3 M sucrose; 170,000g·60 min). In some of these experiments the granular fraction was incubated simultaneously with ⁴⁵Ca²⁺ and [³H](-)noradrenaline to compare the uptake of both. The rate of uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction (characterized by the highest catecholamine content and the lowest succinate dehydrogenase activity) was doubled by ATP. The ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules was inhibited by N-ethylmaleimide (NEM) (0.1 mM), 2,4-dinitrophenol (DNP) (0.1 mM), azide (1 mM), carbonyl cyanide mchlorophenylhydrazone (CCCP) (20μM), atractyloside (50 μM), ruthenium red (40 μM) and amobarbital (1 mM). This inhibition pattern was different from that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the microsomal fraction, but it was similar to that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction. However, the following differences are incompatible with the view that a mitochondrial contamination, with a highly active uptake, is responsible for the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules: a) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was insensitive to 1 mM amobarbital, whereas this agent inhibited the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules. b) Replacement of ATP by succinate stimulated the uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction only. c) The dependence of the ATP-stimulated uptake of ⁴⁵Ca²⁺ on the concentration of ATP (0.1–5 mM) was determined: while uptake into the 2.0 M sucrose fraction of chromaffin granules exhibited saturation kinetics, that into the mitochondrial fraction was linearly related to the concentration of ATP. Interestingly, uptake of ⁴⁵Ca²⁺ into those fractions of chromaffin granules that are known to be contaminated with mitochondria (1.6 M sucrose fraction) exhibited bot a saturable and a nonsaturable component. d) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was more sensitive to 0.1 mM DNP than the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules.

The comparison of the ATP-stimulated uptake of ⁴⁵Ca²⁺ with that of [³H](-)noradrenaline into the 2.0 M sucrose fraction of chromaffin granules revealed that amobarbital, N,N′-dicyclohexylcarbodiimide (DCCD), DNP and increasing concentrations of ATP had the same influence on both uptake processes. From our results we conclude that an inherent, ATP-stimulated uptake of ⁴⁵Ca²⁺ exists in chromaffin granules. The effects of agents on the uptake of ⁴⁵Ca²⁺ and/or [³H](-)noradrenaline into chromaffin granules are discussed with regard to the granular uptake mechanisms.