Synthesis of chromogranin A, dopamine beta-hydroxylase, and chromaffin vesicles

Primary cultures of bovine adrenal medullary cells synthesize chromogranin A (CgA) and dopamine beta-hydroxylase (DBH) and incorporate them into chromaffin vesicles. The incorporation of L-[35S]methionine into CgA, DBH, and total protein was approximately linear for 8 h at methionine concentrations of 12.5, 25, and 50 microM. Newly synthesized CgA and DBH were initially incorporated into vesicles of low buoyant density that matured over 24 h into vesicles having the greater buoyant density of chromaffin vesicles. Approximately 10% of the newly synthesized CgA is released constitutively within 4 h of formation, approximately 30-40% appears to be degraded, and the remainder is incorporated into chromaffin vesicles, which can secrete CgA in response to nicotinic stimulation. Newly synthesized DBH follows a similar course. Once incorporated into chromaffin vesicles, the newly synthesized CgA and DBH appear to be stable for 2-3 days and then decline with a half-time of 3-4 days. Primary cultures of bovine adrenal medullary cells are a good model system for studying factors regulating CgA and DBH synthesis and the formation of chromaffin vesicles.

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry.

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.