Bovine adrenal medulla: subcellular distribution of newly synthesised catecholamines, nucleotides and chromogranins.

Abstract

1. The subcellular distribution of newly synthesised catecholamines, nucleotides and proteins was investigated in bovine adrenal medulla. 3H-tyrosine, 3H-leucine and 32P-phosphate were used as radioactive precursors (“pulse label”). 2. Already 3 min after infusion of 3H-tyrosine the bulk of the labelled catecholamines was present in the “large granules” (mitochondria, lysosomes and chromaffin granules). In the fractions from the density gradient the distribution of the labelled and the total catecholamines was the same. Analogous results were obtained at longer time intervals. 3. 3 min after infusion of 32P-phosphate the labelled nucleotides present in the “large granules” were concentrated in the mitochondrial fraction. At longer time intervals after infusion of 32P-phosphate (45 min and 4h) chromaffin granules had accumulated a larger portion of the labelled nucleotides, mitochondria contained less. 4. After infusion of 45Ca2+, the isotope present in the large granules was found to be concentrated in the mitochondria and in chromaffin granules. 5. After infusion of 3H-leucine the soluble proteins of the adrenal medulla rapidly became labelled. 4 h after 3H-leucine newly synthesised proteins could be demonstrated in a particle which was present in the large granule fraction and which equilibrated in density gradients in a position corresponding to 1.6 M sucrose. This particle can be differentiated from mitochondria, microsomes, lysosomes and the bulk of the chromaffin granules. The labelled soluble proteins of this particle were identified as chromogranins. It seems likely that this particle represents a newly formed chromaffin granule which differs in its properties from the bulk of mature granules. The membrane proteins of this particle were not significantly labelled. 6. After infusion of 32P-phosphate the phospholipids of the adrenal medulla became labelled. The subcellular distribution of these phospholipids was similar to that of a microsomal marker enzyme, glucose-6-phosphatase. Lysoclecithin was not significantly labelled. 7. In the light of these results the subcellular events leading to the formation of complete chromaffin granules are discussed.