Hongjiang Wu

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist.

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased approximately 82 kD and decreased approximately 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of approximately 71 and approximately 27 kD NH2-terminal chromogranin A fragments. Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.

Formation of the Catecholamine Release-inhibitory Peptide Catestatin from Chromogranin A DETERMINATION OF PROTEOLYTIC CLEAVAGE SITES IN HORMONE STORAGE GRANULES

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A332–364; identity of the peptide was confirmed by diagnostic Met346oxidation. Further analysis revealed two additional forms: bovine chromogranin A333–364 and A343–362. Synthetic longer (chromogranin A332–364) and shorter (chromogranin A344–364) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC50 ∼2.01 versus∼0.35 μm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A340–372, bounded by dibasic sites. We conclude that catestatin is cleaved extensivelyin vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

A NOVEL, CATECHOLAMINE RELEASE-INHIBITORY PEPTIDE FROM CHROMOGRANIN A : AUTOCRINE CONTROL OF NICOTINIC CHOLINERGIC-STIMULATED EXOCYTOSIS

Publisher Summary Proteolysis of chromogranin A takes place both within secretory granules and extracellularly, giving rise to several smaller, biologically active peptides, such as pancreastatin, which inhibits insulin and parathyroid hormone release, β-granin or vasostatin, which inhibits parathyroid hormone release and relaxes vascular smooth muscle, and parastatin which inhibits parathyroid hormone release. In search of the specific secretion-inhibitory domain within chromogranin A, 15 peptides (range, 19-25 residues) spanning 336 amino acids, or 78% of the length (431 amino acids) of the bovine chromogranin A mature protein were synthesized, and tested their efficacy on nicotine-induced norepinephrine secretion from rat pheochromocytoma (PC12) cells, screening each peptide at a 10-μM concentration. This chapter highlights that exposure of PC12 cells to a spectrum of nicotine doses (10-1000 μM), alone or with catestatin (10 μM), revealed that nicotine never overcame catestatins inhibition of norepinephrine release, even at the highest nicotine dosage. Thus, catestatin is likely to be a noncompetitive nicotinic cholinergic antagonist. To explore the minimal catestatin domain exerting catecholamine secretion-inhibitory effect, we synthesized several (N-terminal, C-terminal, and bidirectional) truncated catestatin peptides. Catecholamine-release inhibitory mapped toward the N-terminus of catestatin, in a region of 12 or fewer amino acids.

Regulation of Chromogranin a Transcription and Catecholamine Secretion by the Neuropeptide Pacap

We propose a model depicting putative signal transduction pathways underlying PACAP-induced CGA transcription and catecholamine secretion in PC12 cells (Fig 7). PACAP mediates both secretion and transcription through the PAC 1 receptor, but with quite different post-receptor signaling pathways. PACAP signals to CGA transcription through a Ca2+-independent pathway involving the CGA promoter CRE domain in cis and PKA and the transcription factor CREB in trans. PACAP-evoked secretion and transcription are subject to homologous desensitization in PC 12 cells; however, PACAP also provokes long-lasting secretion, even under dose and time circumstances where acute, DHP-sensitive secretion has been desensitized. While initial secretion is mediated by an L-type VOCC, extended secretion may involve a SOCC activated through a Gq/11/PLC-β/PI signaling pathway. Further characterization of PACAP signaling pathways will require definitive identification of the SOCC channel involved in the sustained catecholamine release.