Antonia Zanini

Preparation and characterization of anti-human chromogranin A and chromogranin B (secretogranin I) monoclonal antibodies

Chromogranin A, chromogranin B/secretogranin I and chromogranin C/secretogranin II are acidic sulphated and phosphorylated secretory proteins present in a large number of endocrine and neuronal tissues. It has been suggested that these proteins may be useful immunohistochemical markers for human tumours of endocrine origin and their measurement in plasma has been proposed as a diagnostic tool in patients with these tumours. In order to obtain anti-human chromogranins/secretogranins antibodies for clinical applications, we immunized mice with whole chromaffin granules isolated from human pheochromocytoma. The immune sera analysed by two-dimensional immunoblotting were found to recognize chromogranins/secretogranins and other unidentified proteins and to react in immunocytochemistry with pheochromocytoma as well as with a number of endocrine cells of different types. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analysed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach we were able to identify several monoclonal antibodies against human chromaffin granule components. In particular, we have characterized one anti-human chromogranin A and one anti-human chromogranin B/secretogranin I monoclonal antibody which showed a very specific pattern both in immunocytochemistry and in two-dimensional immunoblotting.

Presence of sulfated proteoglycans in prolactin secretory granules isolated from the rat pituitary gland

The composition of the segregated content of rat prolactin granules was investigated taking advantage of the fact that these organelles, isolated as a pure fraction, retain their structural organization after solubilization of their limiting membrane by mild detergent treatment. We found that these membraneless granules contain not only the hormone, but also a number of minor macromolecular components including sulfated glycosaminoglycans, which are labeled when pituitary slices are incubated in vitro with [35S] sulfate. In order to characterize the latter components, the isolated radioactive granules were solubilized (by treatment with either a high ionic strength solution orNaOH) and 35S-labeled acidic glycosaminoglycans precipitated by complexing with cetylpirydinium chloride. A high degree of heterogeneity was observed when the ensuing precipitates were analyzed by cellulose acetate electrophoresis: different components were found to co-migrate with authentic heparin and chondroitin sulfate A and C standards. Another component, which accounts for approx. 50% of the glycosaminoglycan-bound radioactivity, might be heparin sulfate. These acidic glycosaminoglycans are linked to peptide moieties to form proteoglycans.

Growth hormone-releasing hormone and clonidine stimulate biosynthesis of growth hormone in neonatal pituitaries

Comparative studies were performed to verify the effect of growth hormone releasing hormone (GHRH) or clonidine (CLON), a compound thought to act via release of endogenous GHRH, in stimulating GH biosynthesis in the pituitary from neonatal and adult rats. In vitro preincubation for 1 h with GHRH (hpGRF-40, 10(-8) M) increased the incorporation of L-[3H]leucine in the electrophoretic band of GH in the pituitary from 10-day-old rats, but not in the gland from adult rats. Ex-vivo treatment with GHRH or CLON for 5 days was effective in stimulating GH biosynthesis only in the pituitary from neonatal rats. These data demonstrate that neonatal somatotropes are particularly sensitive to the GH-synthesizing activity of GHRH or a GHRH-releasing stimulus.

Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies

Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of specific neuroendocrine tumours. In order to obtain anti-secretogranin II monoclonal antibodies for cell biology studies and, in particular, for clinical applications, we immunized mice with a secretogranin II-enriched fraction prepared from homogenates of bovine anterior pituitaries. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analyzed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach, we were able to identify two monoclonal antibodies (8G1 and 5A7) which recognize bovine secretogranin II. Both immunocytochemistry and immunoblotting revealed that one of them, the 5A7 antibody, cross-reacts with the human antigen. The distribution patterns of the immunoreactivity, obtained by immunocytochemistry with the 5A7 antibody in animal and human tissues, partially overlap those, obtained by using a polyclonal antiserum elicited against bovine secretogranin II, previously described. Moreover, the 5A7, but not the polyclonal antibody, reacts with some duodeno-jejunal cells. In conclusion, both the 5A7 and 8G1 antibodies can be useful for cell biology studies. The 5A7 antibody can be used for the detection of secretogranin II in human tissues and should be of help in clinical and pathological practices.

The gonadotrope polypeptide (GP 87) released from pituitary cells under luteinizing hormone-releasing hormone stimulation is a secretogranin II form.

Cultured gonadotrope cells from 14 day old female rat pituitaries have been shown to release a highly acidic protein when incubated with LHRH: the gonadotrope polypeptide (GP 87). Moreover, a new tyrosine-sulfated acidic protein, secretogranin II (Sg II), clearly distinct from the chromogranin species, was described in the secretory granule matrix of endocrine cells secreting peptide hormones by the regulated pathway. Recently, the release of Sg II from female rat pituitary stimulated by LHRH was demonstrated in vitro. Several physicochemical (Mr; pI) and biological (cellular localization in the pituitary; LHRH-stimulated release) properties are common to Sg II and GP 87. To verify if these 2 polypeptides are similar or distinct components, other physicochemical characteristics (heat-stability, sulfation, phosphorylation) were compared using isotope incorporation followed by either 1- or 2-dimensional polyacrylamide gel electrophoresis and autoradiography. Furthermore, the similarity of GP 87 to Sg II was studied by immunoblotting on nitrocellulose sheets following electrophoresis of intracellular and secreted proteins. Antisera raised against bovine Sg II (extracted from whole pituitaries) and against rat GP 87 (released into the medium of cultured pituitary cells stimulated by LHRH) were used. The overall data presented here suggest that GP 87 is the Sg II form contained in, and released by, gonadotrope cells.

Complex Carbohydrates of Secretory Organelles

Over the last few years, considerable attention has been devoted to the role played by complex carbohydrates in the structure and function of secretory granules and vesicles. These intracellular storage organelles, which are delimited by a single membrane, are endowed with the ability to discharge their content into the extracellular space by exocytosis. Since each secretory system is characterized by the specificity of its secretion products, the various granules and vesicles constitute an extremely heterogenous family of organelles. In some of them, complex carbohydrates represent secretion products of known physiological significance. This is the case, for instance, with the gonadotropin and thyrotropin granules of the anterior pituitary, the B granules of follicular thyroid cells, and the immunoglobulin vesicles of plasmocytes. Other secretory granules (e.g., those of the exocrine pancreas, parotid gland, and liver) contain a mixture of many physiologically important secretion products, some of which are complex carbohydrates. Finally, in a variety of other systems, the occurrence of complex carbohydrates (often in small amounts) in the segregated content of secretory organelles has also been reported, even if no clear information is yet available on whether these components have any function after their discharge. However, complex carbohydrates do not reside only in the content of secretory organelles, since in all systems so far investigated they have also been found in the limiting membrane, where at least some of them are oriented according to a specific geometry.

Intracellular Events in Prolactin Secretion

Considerable experimental evidence, obtained during the last several years, indicates that secretion of prolactin (PRL) is carried out in pituitary mammotrophs by means of a series of complex interconnected events(analogous to those occurring in other protein-secreting cells) that ultimately result in hormone discharge by exocytosis (Fig. 1). This chapter will focus primarily on one such event, namely, the packaging of PRL within secretion granules. The others, namely, the biosynthesis of the hormone and its transport to the Golgi periphery, exocytosis, and crino-phagy, as well as the recycling of membranes from the cell surface, will be dealt with only briefly, because they have been covered in detailed reviews that have been published recently or will appear soon (Farquhar et al., 1975; Palade, 1975; Farquhar, 1977; Meldolesi et al., 1978; De Camilli et al., 1979; Herzog and Miller, 1979). In addition, specific problems related to these events are discussed in other chapters of this volume.

Small GTP-binding proteins in human neuroblastoma cell lines

The presence of small G proteins was investigated by [gamma-35S]GTP-binding in 3 human neuroblastoma cell lines. IMR-32, SK-N-BE and SH-SY5Y, before and after treatment with differentiating agents (dibutyryl-cAMP, 5-bromodeoxyuridine or retinoic acid) which induce the appearance of secretory organelles. One major component of about 24 kDa and 3 minor components of smaller Mr were found to bind specifically [gamma-35S]GTP in all 3 cell lines already before differentiation. Differentiation did not affect the expression of small G proteins in IMR-32 cells and only modestly affected it in the other two cell lines. The possibility that the expression of small G proteins in neuroblastoma cells is not coupled with the assembly of secretory organelles is discussed.