Chromogranin A (CGA) is a ubiquitous 48-kDa secretory protein present in adrenal medulla, anterior pituitary, central and peripheral nervous system, endocrine gut, thyroid, parathyroid, and endocrine pancreas. Recently, we have demonstrated that the protein could be a precursor of bioactive peptides capable of modulating catecholamine secretion from cultured adrenal medullary chromaffin cells. Here we cleaved CGA purified from bovine chromaffmn granules with endoproteinase Lys-C, and we isolated and partially sequenced the peptide inhibiting catecholamine secretion from cultured chromaffim cells. A corresponding synthetic peptide composed of the first 20 N-terminal amino acids produced a dose-dependent inhibition in the 10-9 to 10-6 M range (with an ID.5 of 5 nM) of the catecholamine secretion evoked by carbamoylcholine or by potassium at a depolarizing concentration. This peptide affected secretagogue-induced calcium fluxes but did not alter sodium fluxes. It was found to increase desensitization of cell responses and to modify the kinetics of catecholamine release. Our results indicate that the peptide is extracellularly generated from CGA by a calcium-dependent proteolytic mechanism. We suggest that this peptide, named chromostatin, may be an endocrine modulator of catecholamine-associated responses.

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety ofendocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in 13 cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (a) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffm cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas ofnormal and pathological conditions may indicate that CST is involved in 13-cell function.Chromogranin A (CGA) is an acidic glycoprotein originally detected in the adrenal medulla but more recently found also in a variety of peptide hormone-producing cells (see refs. 1 and 2 for review). Despite its widespread distribution, the physiological function of CGA is still largely unknown.The primary amino acid sequence of CGA, elucidated by cDNA analysis, includes several pairs of basic residues, which by analogy with other peptide hormone precursors constitute potential sites of proteolytic cleavage (3-5). Thus, it was speculated that CGA may be a putative precursor of biologically active peptides (2, 6, 7). Pancreastatin (PST), a peptide fully contained in the porcine CGA sequence (3, 7), was isolated from the porcine pancreas (8). This peptide inhibits insulin secretion (8). Moreover, tryptic digestion of CGA yielded various peptides that were able to modulate catecholamine secretion (7). Recently, chromostatin (CST) has been identified as a CGA derivative, which is localized between amino acids 124 and 143 in the bovine CGA sequence (9). In contrast to PST, CST had potent inhibitory action on chromaffin cell secretion (9).Previous studies demonstrated that pancreatic islet cells and islet cell tumors contain various CGA-derived peptides (10)(11)(12)(13)(14)(15)(16)(17). In the human pancreas, CGA has been localized mainly to glucagon (a) cells (18). The cellular distribution of PST, however, is still a matter of controversy (see ref. 19 for review), which in part may be due to species specificities (20). Data on tissue distribution of CST are completely lacking.Using specific antisera against CST, PST, and CGA, we inves...

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 CGA1-40 and CGA44-76 detected a 7 kD protein whose release was increased during stimulation. This component co-migrated with synthetic CGA1-76, was not immunoreactive to antisera to CGA79-113 or CGA124-143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an approximately 18 kD component, which stained with antisera to CGA1-40, CGA44-76 and CGA79-113, but not to chromostatin (CGA124-143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1-40, CGA44-76 and CGA79-113. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

in the present study we investigated the regulation of chromogranin A (CGA) and chromogranin B (CGB) biosynthesis in bovine shromaffin cells maintained in primary culture. Cellular proteins were labelled with [35S]methionine and the incorporated radioactivity was used as an index of the s y n t h e s i s rate. The radioactivity incorporated into CGA was determined by immunoprecipitation, m d that into CGB was quantified by a dot immunobinding assay using specific antibodies. Incubation of cells with carbamylcholine, .iigh K' or histamine, three potent stimulators of catecholamine secretion in chromaffin cells, increased the rate of CGA and CGB synthesis. On the other hand bradykinin, angiotensin II and prostaglandin E, , which cause little secretion, also produced an ,ncrease in both CGA and CGB synthesis. These results suggest that in chromaffin cells, the biosynthesis of chromogranins is not closely linked to the secretory activity. Inhibition of protein kinase C by sphingosine or by long-term treatment with phorbol esters, completely abolished the synthesis of CGA and CGB induced by carbamylcholine, bradykinin and prostaglandin E, but decreased only partially the stimulating effect of histamine. Thus, protein kinase C may not be the sole effector involved in t h e secretagogueinduced modulation of chromogranin synthesis. Forskolin, an activator of adenylate cyclase had no effect on CGA synthesis, but significantly enhanced the incorporation of radioactivity into CGB. The effect of forskolin was not modified by protein kinase C inhibitors and was additive to that induced by phorbol esters indicating that cyclic AMP did not stimulate CGB synthesis through a protein kinase C-dependent pathway. These observations suggest that the biosynthesis of CGA and CGB in chromaffin granules IS independently regulated.The secretory granules of adrenal medullary chromaffin cells contain a high concentration of soluble peptides and proteins, [hat are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation. The soluble acidic proteins of chromaffin granules have been collectively called chromogranins. Chromogranin A (CGA) and chromogranin B (CGB) also known as secretogranin I, together constitute about 50% of the total soluble proteins. A third, highly acidic, though less abundant protein is found in chromafin granules: chromogranin C also named secretogranin 11. Chromogranins are widely distributed throughout tissues of the neuroendocrine system including thyroid and parathyroid, pituitary, pancreatic islets and various cells of the endocrine gut in addition to adrenal medulla and adrenergic neurons (for review see I). Chromogranins are therefore increasingly used as markers of normal and neoplastic neuroendocrine cells (2-4).The different members of the chromogranin family share certain structural features: they are extremely heat stable, highly acidic proteins, capable of binding C a Z f with low affinity but high capacity ( 5 , 6), and are post-translationally modified by ph...