Drukker, Jan

Ehrhart, M.

Gratzl, Manfred

English

Open Access LMU

Acta histochemica Zeitschrift für histologische Topochemie Supplementband XXXVIII Herausgegeben von Joachim-Hermann Scharf, Halle, Gerhard E.Voigt, Lund, Werner Linß, Jena Verhandlungen der Gesellschaft für Histochemie auf dem XXX. Symposion in Gargellen, Montafon (Österreich) vom 21. bis 24. September 1988 Cytochemical Markers of Neural and Endocrine Cells Herausgegeben von Jan Drukker, Maastricht Mit 129 Abbildungen und 34 Tabellen fSEMPER BONIS Gustav Fischer Verlag Jena -1990 Table of contents Robert Feulgen Lecture Larsson, L.I . : Tracing of neuroendocrine peptides and their corresponding mRNA's: unra-velling the neuroendocrine network 9 Symposium: Cytochemical markers of neural and endocrine cells—cell biology and pa-thology Pilgrim, Ch.: Introductory remarks 15 Bock, E.f and Linnemann, D.: Cell adhesion molecules in brain 19 O'Connor, D. T., Takiyyuddin, M.A., Cervenka, J. H., Parmer, R.J., Barbosa, J. A., Chang, Y. M., and Hsiao, R. J. : Circulating chromogranin A as a diagnostic tool in clinical chem-istry 27 Heitz, Ph. U.: Neuroendocrine markers in pathology 35 Workshop I: Cell surface markers Organizer and moderator: 0. K. Langley, Strasbourg Aletsee-Ufrecht, M. C, Langley, 0. K., and Gratzl, M.: NCAM expression in endocrine cells 45 Rougon, G., Nedelec, J., Malapert, P., Goridis, C, and Chesselet, M.F.: Post-translation modifications of neural cell surface molecules 51 Rathjen, F. G., Wolff, J. M., Chang, S., and Raper, J. : Membrane glycoproteins involved in neurite-neurite interactions 59 Gennarini, G., Rougon, G., and Goridis, C : F3: a new developmental^ regulated member of the HNK-1 family 65 Workshop II: Intracellular markers Organizer and moderator: M. Gratzl, Ulm Grant, N.J., Aunis, D., and Langley, 0. K.: Neurofilament expression in bovine chromaffin cells 71 Schilling, K., and Pilgrim, Ch.: Expression of neurofilament proteins in developing neurons 77 Grube, D., Bargsten, G., Cetin, Y., Jörns, A., und Yoshie, S.: Chromogranine in den endo-krinen Zellen des Verdauungsapparats 81 Ehrhart, M., and Gratzl, M.: Chromogranin A in the endocrine pancreas: Extracellular or in-tracellular function? 87 Workshop III: Neural and endocrine markers as diagnostic tools Organizer and moderator: H. Höfler, Graz Klöppel, G., and In't Veld, P.: Neural and endocrine markers as diagnostic tools in pancrea-tic and gastrointestinal endocrine tumors 93 de Brume, A. P., and Bosman, F. T.: Neuroendocrine tumours in the respiratory tract . . 99 Becker, H., Wirnsberger, G., Ziervogel, K., and Höfler, H.: Immunohistochemical markers in (ganglio)neuroblastomas 107 Table of contents 7 Larsson, L.T., and Sundler, F.: Neuronal markers in Hirschsprung's disease with special reference to neuropeptides ^ 5 Falkmer, S., Askensten, U., Grimelius, L, and Abrahamsson, P.-A.: Cytochemical markers and DNA content of neuroendocrine cells in carcinoma of the prostate gland during tu-mour progression ^27 Schmid, K. W., und Weiler, R.: Unterschiedliches Vorkommen von Chromogranin A und B in normalen menschlichen endokrinen und nervalen Geweben und Tumoren neuroendo-krinen Ursprungs 133 Contributed papers Viebahn, Ch., Lane, E. B., and Ramaekers, F. C. S.: Vimentin and keratin are expressed in the neurogenic tissue of the rabbit embryo during primary neurulation 139 Layer, P. G.: Cholinesterases reveal early patterns of neurogenesis in the chick . . . . 145 Becker, I., Paulus, W., Roggendorf, W., and Peiffer, J. : Immunohistologic analysis of neu-ron-specific enolase and neuropeptides in stromal cells of cerebellar hemangioblasto-mas Graeber, M. B., Streit, W. J., and Kreutzberg, G. W.: The third glial cell type, the microglia: cellular markers of activation in situ 157 Brückner, G., Delpech, B., Delpech, A., and Girard, N.: Concentration of hyaluronectin and anionic glycoconjugates in perineuronal glial cell processes at GABAergic synapses of rat cerebellum 161 Nitsch, C , and Riesenberg, R.: Ultrastructure of the dynorphin-immunoreactivity in rat brain hippocampal mossy fiber system 167 Christmann, M. Ph., Nokihara, K., Feller, S., and Forssmann, W. G.: Demonstration of the storage form of cardiac hormones by the use of segment-specific antibodies against hu-man Cardiodilatin/human Atrial Natriuretic Peptide (CDD/hANP) 173 Vyberg, M., Horn, T., Francis, D., and Askaa, J. : Immunohistochemical identification of neuron-specific enolase, synaptophysin, chromogranin and endocrine granule constitu-ent in neuroendocrine tumours 179 Schwenk, J. , and Makovitzky, J. : The occurrence of carbohydrate antigens (CA 19-9, CA-50) in chronic pancreatitis and pancreatic carcinoma 183 Makovitzky, J. : The sterical order of the sialic acid in the microvillous layer of the human duodenal mucosa 189 Budde, R., Gerok, K., von Deimling, 0., and Schaefer, H. E.: The occurrence of ß-glucuronid-ase in erythrocyte inclusions 195 Haider, S. G., Passia, D., and Rommert, F. F. G.: Histochemical demonstration of 11 ß-hy-droxysteroid dehydrogenase as a marker for Leydig cell maturation in rat 203 Aumüller, G., Hüntemann, S., Larsch, K. P., and Seitz, J. : Transglutaminase immunoreacti-vity in the male genital tract of the rat 209 Staneva, L.: Histochemical and ultracytochemical studies on complex carbohydrates in the cyclic rat endometrium 213 Graf, R., Goßrau, R., and Frank, H.G.: Enhancement of immunoreactivity of von Wille-brand factor in vascular endothelial cells of rat organs after glucocorticoid administra-tion 219 Ribitsch, D., Dohr, G., Hartmann, M., Pilz, G., Salmhofer, H., and Desoye, G.: GZ 100, 101, 106, 107, 111, 112, 116, 121: A series of monoclonal antibodies against human tropho-blast antigens 227 Manfredi Romanini, M.G., Fraschini, A., Fuhrmann Conti, A .M. , Gasperi, G., Giuliani, A., and Pellicciari, C : Changes of gene expression during long term adaptation of human EUE cells to a hypertonic medium: electrophoretic protein patterns and DNase I dige-stion in situ 233 Fritz, P., Hoenes, J., Multhaupt, H., Schenk, J., Mischlinski, A., Klein, C , Tuczek, H. V., and Wolf, M.: Application of the chromogenic reaction to conventional silver staining, the Ag-NOR staining and the silver-intensified immunogold technique 239 8 Table of contents Papers read by title Nöhammer, G., Bajardi, F., Benedetto, C, Kresbach, H., Rojanapo, W., Schauenstein, E., and Slater, T. F.: Histophotometry of protein thiols and disulphides in tissue samples from the human uterine cervix and the skin reveals a "field effect" as well as an "extend-ed field effect" of malignant tumours 247 Schulte, E.: The influence of embedding on the stoichiometry of the pararosaniline-Feul-gen stain in histological material 255 Lecture by the Robert-Feulgen-Laureate Thanos, S.: Bidirectional fluorescent labelling techniques for the developing and regenerat-ing visual system 259 Acta histochemica, Suppl.-Band XXXVIII, S. 87-91 (1990) Chromogranin A in the endocrine pancreas: Extracellular or intracellular function? By M. Ehrhart and M. Gratzl (Abteilung Anatomie und Zellbiologie der Universität Ulm, FRG) With 3 figures Chromogranin A belongs to a family of highly acidic proteins, the chromogranins/se-cretogranins. These proteins are widely distributed in endocrine cells storing hor-mones or amines in dense cored vesicles. Chromogranin A has first been detected in the adrenal medulla, were it is costored and coreleased together with the catechol-amines (Banks and Helle, 1965; Blaschko et al., 1967). Both intracellular and extracellu-lar functions of chromogranin A are currently discussed: Within the chromaffin gra-nules of the adrenal medulla chromogranin A is probably involved in the regulation of the osmotic pressure (Helle et al., 1985). In addition, chromogranin A is part of the Ca 2 + storage complex in the matrix of the chromaffin vesicles (Reiffen and Gratzl, 1985a, b; Bulenda and Gratzl, 1985; Gratzl, 1987, 1988). Chromogranin A derived pep-tides have been shown to inhibit nicotine induced catecholamine secretion from adrenal medullary chromaffin cells (Simon et al., 1988). Thus, in the adrenal medulla beside the established intracellular functions also an extracellular function is likely. No functional role has yet been described for chromogranin A in other endocrine cells. However, it was shown recently that chromogranin A contains the amino acid sequence of the putative hormone pancreastatin (Eiden, 1987; Huttner and Benedum, 1987; lacangelo et al., 1988). Porcine pancreastatin inhibits the first phase of glucose induced insulin secretion of rat pancreas (Tatemoto et al., 1986; Efendic et al., 1987). Thus, pancreatic chromogranin A has a potential function as a prohormone. There-fore, the cellular and subcellular distribution in the endocrine pancreas, the molecular form of pancreatic chromogranin A as well as its amount in relation to the hormones are of special interest. Methods Immunocytochemistry: Small pieces of bovine pancreas were fixed with Acrolein (5% Acrolein, 0.1 M phosphate buffer, 2% sucrose) for 1 h and embedded in epoxy resin. For lightmicroscopical investigations semithin sections were immunostained for chromogranin A using the peroxidase-anti-peroxidase method according to Sternberger (1986). For immunoelectron microscopy, ul-trathin sections were immunostained using the protein A gold technique (Ehrhart et al., 1986). For lightmicroscopical investigations, the antiserum was diluted 1:5,000, for electronmicroscopy it was diluted 1:10,000. Immunochemical analyses: The extraction of chromogranin A of calf pancreatic tissues was carried out in 1 N HCl to prevent proteolysis. Soluble proteins of chromaffin granules from the adrenal medulla were isolated as described previously (Reiffen and Gratzl, 1986a). The extracts were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and im-munostained for chromogranin A using 1 2 5 l-protein A (Burnette, 1981). The immunocytochemical and the immunochemical studies were carried out using a poly-clonal antiserum raised against bovine adrenal medullary chromogranin A kindly provided by M.F. Bader and D.Aunis (Centre de Neurochimie, Strasbourg, France). 88 M. Ehrhart and M. Gratzl Results and discussion Fig. 2. Photomicrograph of an ultrathin section of B-cell vesicles. The section is immunostained for chromogranin A. Bar = 0.5 pm. Chromogranin A in the endocrine pancreas 89 Within the bovine pancreas, the islets of Langerhans were heavily stained for chromo-granin A (Fig. 1). The comparison of chromogranin A-immunoreactivity with that of the hormones showed that the insulin-, glucagon-, and somatostatin-containing cells reacted intensively with the chromogranin A-antiserum (Ehrhart et al., 1986). The chromogranin A-immunoreactivity is confined exclusively to the hormone containing vesicles (Fig.2). The immunocytochemical studies showed that chromogranin A in the endocrine pancreas is stored together with the hormones in the same vesicles. Consequently, chromogranin A can be secreted together with the hormones of the endocrine pan-creas. To elucidate the function of pancreatic chromogranin A it is essential to know, whether or not it is processed within the cells of the endocrine pancreas. Using immu-noblots, the molecular weight of pancreatic chromogranin A was compared with that of chromogranin A of the adrenal medulla. When proteolysis of chromogranin A was inhibited during extraction either by heating (Yoshie et al., 1987) or acidification of the extracts mainly the unprocessed form of pancreatic chromogranin A was detected (Fig.3). It has the same apparent molecular weight of 74 kD as the main component of adrenal medullary chromogranin A (Fig. 3). Using immunoblotting as a quantitative method, it turned out that the endocrine pancreas contains on a molar basis 2,000 times more insulin than chromogranin A (460 pmol/mol insulin; Ehrhart et al., 1988). The wide distribution of chromogranin A in endocrine cells indicates an universal function of this protein. For the adrenal medulla, intravesicular functions as well as ex-205-116-97.4-45-66-29-20-A B Fig. 3. Immunoblotting of soluble proteins of bovine chromaffin vesicles and of an extract of bovine pancreas. Samples were subjected to Polyacrylamide gel electrophoresis (11%) fol-lowed by blotting and immunostaining for chromogranin A. (A) chromaffin vesicle con-tent; (B) pancreatic extract. 90 M. Ehrhart and M. Gratzl tracellular functions of chromogranin A are known (see above). Within the endocrine pancreas, the estimated amount of chromogranin A is more than 100 times less than the amount of chromogranin A that would be necessary to bind the intravesicular cal-cium (Ehrhart et al., 1988). Thus, a functional role of chromogranin A in the storage of C a 2 + within the secretory vesicles of the endocrine pancreas is not likely. No regulatory effect on hormone secretion has so long been shown for chromo-granin A itself. However, the aminoacid sequence of porcine chromogranin A includes the sequence of the putative hormone pancreastatin (lacangelo et al., 1988). Recently, it has been described that pancreastatin is released together with insulin from the por-cine pancreas (Östenson et al., 1988). A sequence homologous to pancreastatin is also contained in bovine, human and rat chromogranin A (cf. lacangelo et al., 1988). How-ever, within the bovine endocrine pancreas, chromogranin A is stored in its unpro-cessed form. Thus chromogranin A has to be degraded shortly before or after secre-tion to yield pancreastatin which subsequently could inhibit the secretion of insulin. Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft (Gr 681), by Forschungsschwerpunkt n.24 of the State of Baden-Württemberg and by a twinning exchange Procope fellowship. References Banks, P., Helle, K. B., and Mayor, D.: Evidence for the presence of a chromogranin-like protein in bovine splenic nerve granules. Molec. Pharmac. 5, 210-212 (1968). Blaschko, H., Comline, R.S., Schneider, F.H., Silver, M., and Smith, A.D.: Secretion of a chrom-affin granule protein, chromogranin, form the adrenal gland after splanchnic stimulation. Na-ture 215, 58-59 (1967). Bulenda, D., and Gratzl, M.: Matrix free Ca 2 + in isolated chromaffin vesicles. Biochemistry 24, 7760-7765 (1985). Burnette, W. N.: "Western Blotting": elektrophoretic transfer of proteins from sodium dodecylsul-fate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203 (1981). Efendic, S., Tatemoto, K., Mutt, V., Quan, C, Chang, D., and Östenson, C.G.: Pancreastatin and islet hormon release. Proc. Natl. Acad. Sei. USA 84, 7257-7260 (1987). Ehrhart, M., Grube, D., Bader, M.F., Aunis, D., and Gratzl, M.: Chromogranin A in the pancreatic islet: Cellular and subcellular distribution. J. Histochem. Cytochem. 34, 1673-1682 (1986). Ehrhart, M., Jörns, A., Grube, D., and Gratzl, M.: Cellular distribution and amount of chromo-granin A in the bovine endocrine pancreas. J. Histochem. Cytochem. 36, 467-472 (1988). Eiden, L. E.: Is chromogranin a prohormone? Nature 325, 301 (1987). Gratzl, M.: Uptake and release of Ca 2 + by chromaffin vesicles. In: Rosenheck, K., and Lelkes, P.I. (eds.): Stimulus-secretion coupling in chromaffin cells. CRC Press Inc., Boca Raton, 111-123 (1987) . Gratzl, M.: Metabolism and function of Ca 2 + in secretory cells. In: Thorn, N. A., Treiman, M., Pe-terson, O.H., and Thaysen, J.H. (eds.): Molecular mechanisms in secretion. Munksgaard, Co-penhagen, 364-376 (1988). Helle, K. B., Reed, R. K., Pihl, K. E., and Serck-Hanssen, G.: Osmotic properties of the chromogra-nins and relation to osmotic pressure in catecholamine storage granules. Acta Physiol. Scand. 123, 21-33 (1985). Huttner, W.B., and Benedum, U.M.: Chromogranin A and pancreastatin. Nature 325, 305 (1987). lacangelo, A.L., Fischer-Colbrie, R., Koller, K.J., Brownstein, M.J., and Eiden, L. E.: The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin. Endocrinology 122, 2339-2341 (1988) . Östenson, C G . , Holst, J .J . , and Efendic, S.: Pancreastatin and insulin are released in parallel from the perfused porcine pancreas. Diabetologia, in press (1988). Chromogranin A in the endocrine pancreas 91 Reiffen, F. U., and Gratzl, M.: Chromogranins, widespread in endocrine and nervous tissue, bind Ca 2 + . FEBS Lett. 195, 327-330 (1986a). Reiffen, F. U., and Gratzl, M.: Ca 2 + binding to chromaffin vesicles matrix proteins: effect of pH, M g 2 + , and ionic strength. Biochemistry 25, 4402-4406 (1986b). Simon, J. P., Bader, M. F., and Aunis, D.: Secretion from chromaffin cells is controlled by chromo-granin A-derived peptides. Proc. Natl. Acad. Sei. USA 85, 1712-1716 (1988). Sternberger, L. A.: Immunocytochemistry. 3rd ed. J.Wiley, New York 1986. Tatemoto, K., Efendic S., Mutt, V., Makk, G., Feistner, G.J., and Barchas, J.D.: Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 324, 476-478 (1986). Yoshie, S., Hagn, C, Ehrhart, M., Fischer-Colbrie, R., Grube, D., Winkler, H., and Gratzl, M.: Im-munological characterization of chromogranins A and B and secretogranin II in the bovine pan-creatic islet. Histochemistry 87, 99-106 (1987). First author's address: Dr. Monika Ehrhart, Abteilung Anatomie und Zellbiologie der Universität Ulm, Postfach 4066, D-7900 Ulm, FRG. 

Ca + binding to chromaffin vesicles matrix proteins: effect of pH, Mg 2+, and ionic strength.

Cellular distribution and amount of chromogranin A in the bovine endocrine pancreas.

Chromogranin A and pancreastatin.

Chromogranin A in the pancreatic islet: Cellular and subcellular distribution.

E.: Is chromogranin a prohormone?

Evidence for the presence of a chromogranin-like protein in bovine splenic nerve granules.

Immunological characterization of chromogranins A and B and secretogranin II in the bovine pancreatic islet.

Matrix free Ca + in isolated chromaffin vesicles.

Metabolism and function of Ca + in secretory cells.

Osmotic properties of the chromogranins and relation to osmotic pressure in catecholamine storage granules.

Pancreastatin and insulin are released in parallel from the perfused porcine pancreas. Diabetologia, in press

Pancreastatin and islet hormon release.

Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion.

Secretion from chromaffin cells is controlled by chromogranin A-derived peptides.

Secretion of a chromaffin granule protein, chromogranin, form the adrenal gland after splanchnic stimulation.

The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin.

Uptake and release of Ca + by chromaffin vesicles.

Western Blotting&quot;: elektrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.

Chromagranin A in the endorine Pancreas: Intracellular or extracellular function?

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Chromagranin A in the endorine Pancreas: Intracellular or extracellular function?

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