Chromogranin A: Secretion of processed products from the stimulated retrogradely perfused bovine adrenal gland

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N-terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N-terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS-PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA_1-40 and CGA_44-76 detected a 7 kD protein whose release was increased during stimulation. This component co-migrated with synthetic CGA_1-76, was not immunoreactive to antisera to CGA_79-113 or CGA_124-143) and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an ~ 18 kD component, which stained with antisera to CGA_1-40, CGA_44-76 and CGA_79-113, but not to chromostatin (CGA_124-143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA_1-40, CGA_44-76 and CGA_79-113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells. The major fragments secreted appear to be the N-terminal fragments of CGA, CGA_1-76 and CGA_1-133 which would arise as a result of processing of CGA at the first and second pairs of basic amino acids. A number of larger CGAFs, possibly containing the chromostatin sequence CGA_124-143 at their N-terminal, and components similar in size to intact CGA and to proteoglycan forms of CGA, are also secreted from the perfused bovine adrenal gland during stimulation.