UPTAKE OF CALCIUM BY CHROMAFFIN GRANULES IN VITRO

Abstract— Partly purified chromaffin granules were incubated in vitro with Ca2+ (with trace amounts of 45Ca2+) in concentrations ranging from 4 μm to 1 mm. After incubation the granules were washed with media containing EDTA and then subjected to density gradient centrifugation (1.3 to 2.0 m‐sucrose solutions) in order to characterize the particles which had taken up 45Ca2+. By using marker enzymes and various inhibitors of Ca2+ uptake into such cell particles as mitochondria it was established that under the conditions of the experiments chromaffin granules took up Ca2+ from the incubation medium. To characterize this uptake a simplified density gradient procedure was tested and found to be suitable. The uptake of Ca2+ into chromaffin granules was strongly dependent on temperature. It was not activated by ATP. The uptake was linear up to 10 min. At high calcium concentrations (above 200 μm) the rate of uptake levelled off. The uptake at 37°C was 1 nmol Ca2+/mg protein/min at a Ca2+ concentration of 500 μm. Mg2+ had no influence on Ca2+ uptake, whereas Sr2+ (1 mm) inhibited it. The methods established in this study should prove useful for a further characterization of this Ca2+ uptake into chromaffin granules which is likely to represent a useful model for the Ca2+ uptake occurring in the intact gland.