Immunocytochemical localization of chromogranin A in the normal and stimulated hypothalamo-neurohypophysial system of the rat.

Chromogranin A (CGA) is a calcium-binding glycoprotein thought to be the precursor of several peptides with defined biological activity. Chromogranin A has been localized in most endocrine cells and many neurons in the CNS. Here we studied its expression in neurons of the hypothalamo-neurohypophysial system, which secrete the neurohormones oxytocin and vasopressin. Light and electron microscopic immunocytochemistry and immunoblot analysis with antibodies specific for CGA revealed high levels of chromogranin A immunoreactivity throughout the hypothalamo-neurohypophysial system. In the supraoptic and paraventricular nuclei, it was characterized by intracytoplasmic labelling of magnocellular somata and processes and of certain astrocytes. Extensive labelling of fibres and dilatations characterized the internal layer of the median eminence and the neurohypophysis, transit and terminal site of the neurosecretory axons, respectively. Tanycyte-like cells in the median eminence also displayed reaction. Simultaneous immunofluorescence showed that oxytocinergic and vaso-pressinergic neurons contain chromogranin A. Electron microscopy revealed that chromogranin A immunoreactivity (visualized by pre-embedding immunoperoxidase or silver-enhanced colloidal gold techniques) was associated with neuro-secretory granules in hypothalamo-neurohypophysial system neurons. In astrocytes and pituicytes, it was seen over the cytoplasm and glial filaments. In tissue from colchicine-treated or immobilization-stressed rats, it was clear that chromogranin A immunoreactivity in the hypothalamus was confined to the hypothalamo-neurohypophysial system. In rats in which neurohypophysial secretion was strongly stimulated by dehydration, immunocytochemistry showed that hypothalamo-neurohypophysial system immunoreactivity significantly increased in the magnocellular nuclei but decreased in the neurohypophysis. On the other hand, chromogranin A distribution was not markedly affected by stress or lactation. These observations demonstrate that chromogranin A is present in neurons and, to a lesser degree, glial cells of the hypothalamo-neurohypophysial system and that its expression is closely related to that of the neurohypophysial peptides.

Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614-626 with antibacterial activity.

Chromogranins constitute a family of acidic soluble proteins widely distributed in endocrine cells and neurons. Chromogranin A, the major soluble component in bovine adrenal medullary secretory granules in chromaffin cells, has been shown to be actively processed to peptide fragments [Metz-Boutigue, M. H., Garcia-Sablone, P., Hogue-Angeletti, R. & Aunis, D. (1993) Eur. J. Biochem. 217, 247-257]. In the present paper, the structural features of the proteolytic degradation mechanism of chromogranin B/secretogranin I have been characterized with regard to the possible function of this protein as a precursor of biologically active peptides. Chromogranin-B-derived fragments present in bovine chromaffin granules were identified by microsequencing after separation by two-dimensional gel electrophoresis or high-performance liquid chromatography. A similar approach was performed to characterize chromogranin-B-derived fragments released into the extracellular space from depolarized bovine cultured chromaffin cells. In chromogranin B, 18 cleavage sites were identified along the protein chain and chromogranin B/secretogranin I fragments were generated by proteolytic attack at both the N-terminus and C-terminus. A major fragment corresponding to residues 614-626 of the C-terminal sequence, was identified in the extracellular space; this peptide was found to share sequence and structural similarities with the lytic domain of cecropins and, as expected from this similarity, to display potent antibacterial properties. Endogenous and synthetic peptides were active on Micrococus luteus, killing bacteria in the micromolar concentration range. The synthetic peptide slows the growth of Bacillus megaterium and was inactive towards Escherichia coli. In addition, the synthetic peptide was unable to induce hemolytic activity. This antibacterial function might be of biological significance in the neuroendocrine system of living organisms. We propose to name this peptide secretolytin.

Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites.

Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a pI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and C-terminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65-76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.