Journal of Histochemistry and Cytochemistry, Vol. 45, 815-822, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Complex Co-localization of Chromogranins and Neurohormones in the Human Gastrointestinal Tract

Guida Maria Portela-Gomesa,d, Mats Stridsbergb, Henry Johanssonc, and Lars Grimeliusa
a Department of Pathology, University Hospital, Uppsala, Sweden
b Department of Clinical Chemistry, University Hospital, Uppsala, Sweden
c Department of Surgery, University Hospital, Uppsala, Sweden
d Department of Medicine II, University Hospital of Santa Maria, Lisbon, Portugal

Correspondence to: Lars Grimelius, Pathology Dept., University Hospital, S-75185 Uppsala, Sweden.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Co-localization of chromogranin (Cg) A, B, and C has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum, and colon) using single-, double-, and triple-immunofluorescence stainings. Virtually all enterochromaffin (EC) cells contained CgA, and those in the luminal two thirds of the antral mucosa and villi of small intestine often also contained CgB. A few EC cells in the duodenal crypts contained CgC. Most gastrin cells harbored both CgB and CgA, although rather more CgB than CgA, but some gastrin cells contained all three types, i.e., also CgC. Some CCK cells also contained all three chromogranins. Enteroglucagon cells in the duodenal villi contained CgA and some CgB. CgA (but not B or C) was found in some secretin, GIP, enteroglucagon/peptide YY, and neurotensin cells. A few somatostatin cells contained CgA but neither CgB nor CgC. CgA and C were found mainly in the basal cell region, whereas CgB occurred more diffusely throughout the cytoplasm. This varying distribution suggests that not all secretory granules contain CgA, or that CgB may occur in a nongranular form. The varying composition of the different chromogranins may reflect their complex functional roles in the widespread neuroendocrine system. (J Histochem Cytochem 45:815-822, 1997)

Key Words: chromogranin A, chromogranin B, chromogranin C, peptides, serotonin, human, gastrointestinal tract


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The CHROMOGRANIN FAMILY contains several members of glycoproteins: chromogranin (Cg) A, B, and C (secretogranin II), and proteins derived from these. CgA and CgB were first isolated from chromaffin cells in the adrenal medulla and CgC from the anterior pituitary gland. Most of the soluble proteins in the secretory granules of the adrenal chromaffin cells consist of CgA and B, but only to a minor extent of CgC. Chromogranins, especially CgA, also occur in the secretory granules of most neuroendocrine cell types. The main source of circulating CgA appears to be the adrenal medulla, although other neuroendocrine cell systems may also contribute (Winkler 1976 ; O’Connor et al. 1983 ; Rindi et al. 1986 ; Fischer-Colbrie et al. 1987 ). CgA has long served as an important broad-spectrum marker for the identification of neuroendocrine cells and tumors in tissue sections. CgA antibodies have been commercially available for the past 10 years and are widely used in routine pathology. CgB and C antibodies, on the other hand, have been less readily available, and knowledge of their usefulness in both routine pathology and research has therefore been more limited (Lloyd and Wilson 1983 ; Facer et al. 1985 ; Wiedenmann and Huttner 1989 ; Schmid et al. 1994 ; Fahrenkamp et al. 1995 ). In these previous location studies on the chromogranins in different endocrine cell types, consecutive sections and immunoelectronmicroscopic techniques were used.

The purpose of the present study was, by applying double- or triple-immunofluorescence staining of the same section, to ascertain the extent to which the three main chromogranins (A, B, and C) can occur in different endocrine cell types and to determine their intracellular distribution in relationship to the various neuroendocrine hormones in different parts of the human gastrointestinal tract.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Tissue specimens from adult human gastric corpus and antrum, proximal duodenum, distal ileum, and sigmoid colon were obtained from surgical samples removed at operations for adenocarcinoma. The specimens examined were taken at least 3 to 5 cm from the neoplasm in macroscopically normal mucosa. All were histologically normal.

Fixation of the specimens was either in neutral picric acid-formaldehyde (Stefanini et al. 1967 ) or in 10% buffered neutral formalin, for 18-20 hr at room temperature (RT), followed by embedding in paraffin. Sections 5 µm thick were cut and attached to poly-L-lysine-coated glass slides.

The sections were stained with hematoxylin-eosin or were immunostained by different methods to demonstrate various secretory granule products. The sensitive streptavidin-biotin complex (ABC) technique (Hsu et al. 1981 ), with diaminobenzidine as chromogen, was applied as a single immunostain mainly to reveal the distribution of endocrine cell types in the respective gastrointestinal regions, as well as to perform the control stainings specified below.

In the co-localization studies, immunofluorescence methods were used in single, double, or triple staining. In triple-immunofluorescence staining, the procedure was as follows. The sections were incubated with a cocktail of antibodies [one monoclonal + two polyclonal (anti-rabbit and anti-guinea pig)], overnight incubation at RT &Aelig; biotinylated goat anti-guinea-pig IgG, 30 min at RT &Aelig; a mixture of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG + Texas Red (TXRD)-conjugated goat anti-mouse IgG + aminomethyl coumarin acetic acid (AMCA)-conjugated streptavidin, 30 min at RT. Double immunostaining was performed in the same way except for exclusion of one of the primary antibodies and corresponding secondary antibodies. Before applying the respective primary antibodies, the sections were incubated with nonimmune sera from the animal species producing the secondary antibodies, at a dilution of 1~10.

If two primary polyclonal antisera raised in the same species (rabbit) had to be used, the staining technique was modified as follows: first, primary anti-rabbit antiserum was applied overnight at RT &Aelig; monovalent FITC-conjugated goat Fab anti-rabbit IgG (0.2 mg/ml), overnight at RT &Aelig; second primary anti-rabbit antiserum, overnight at RT &Aelig; biotinylated swine anti-rabbit IgG, 30 min at RT &Aelig; TXRD-labeled streptavidin, 30 min at RT (adapted from Negoescu et al. 1994 ).

The Fab concentration of 0.2 mg/ml overnight was found to saturate the epitopes of the first-step anti-rabbit antibody.

The primary antibodies are characterized in Table 1.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Antisera used in this study

Polyclonal antibodies to CgA were raised against a fragment covering amino acids 116-439 of human CgA (Stridsberg et al. 1993 ). The CgA fragment was purified from urine from a patient with a carcinoid tumor. CgB and CgC were raised in rabbits against synthetic peptides (Stridsberg et al. 1995 ). The sequences were selected to be specific for the respective proteins. The selected amino acid sequence for CgB was amino acids 312-331 (Benedum et al. 1987 ) and for CgC, 223-249 (Gerdes et al. 1989 ), with an additional tyrosine residue at the N-terminal. The CgB and CgC peptides were both amidated at the C-terminal. The peptides were coupled to bovine serum albumin (BSA) with glutaraldehyde and injected into rabbits to produce polyclonal antibodies.

Gastrin antibodies raised against the C-terminal portion were used in antrum and N-terminal gastrin antibodies in the duodenum. The latter antibodies did not crossreact with CCK. The CCK antibodies used did not crossreact with gastrin.

The labeled secondary antisera were biotinylated swine anti-rabbit IgG (Dakopatts; Glostrup, Denmark), biotinylated goat anti-guinea pig IgG, TXRD- and AMCA-labeled streptavidin (Vector Laboratories; Burlingame, CA), TXRD-conjugated goat anti-mouse IgG (Southern Biotechnology Associates; Birmingham, AL), FITC-conjugated goat anti-rabbit IgG, both the monovalent Fab fragment and the whole molecule (Sigma Chemical; St Louis, MO).

The control stainings included (a) omission of the primary antisera, (b) replacement of the first layer of antibody by nonimmune serum diluted 1:10 and by the diluent alone, (c) preincubation (24 hr) of primary antiserum with the relevant antigen (0.5, 2.5, and 7 mmol per ml diluted antibody solution, respectively) before application to the sections. These control tests were performed with both immunofluorescence and ABC techniques.

The sections were examined in a Vanox AHBS3 fluorescence microscope (Olympus; Tokyo, Japan) equipped with filters (Olympus) giving excitation at a wavelength of 475-555 nm for TXRD (filter no. 32821, dichroic mirror BH2-DMG), 453-488 nm for FITC (no. 32822, BH2-DMIB), and 340-375 nm for AMCA (no. 32817, BH2-DMU), and a double-band filter set (no. 39538, BH2-DFC5) for simultaneous visualization of TXRD- and FITC-labeled cells was also used (excitation at 550-570 nm and 480-495 nm, respectively). Photographs were taken with Fujicolor 400 film; the triple stainings were photographed by double-exposing the film first through the double band and then with the AMCA filter sets.

Co-localization of two or three of the fluorochromes was revealed by the resulting additive colors: the co-localization of green and red is perceived as yellow, blue (AMCA) and red as magenta, blue and green as cyan, and all three fluorochromes as white.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Distinctly immunoreactive (IR) cells were revealed by all tested antisera in the relevant regions of the gastrointestinal tract when ABC and immunofluorescence methods were used with both fixatives. In double- and triple-immunofluorescence stainings, omission of one or two of the primary antibodies gave a staining pattern corresponding to the remaining primary antibody or antibodies. The other staining controls were all negative.

Distribution of CgA, B, and C in the Gastrointestinal Tract
CgA-IR cells were observed in all parts of the gastrointestinal tract and were more numerous than CgB- and CgC-IR cells. They were seen at all levels of the mucosa, although mainly in the middle third portion. The staining intensity of the CgA-IR cells was strong and even in all regions except for the antrum, where it varied from strong to very weak; the weak IR cells predominated in the antrum.

CgB-IR cells were most numerous in the antrum, where they occurred at almost the same frequency as CgA-IR cells. In duodenum they were sparse, in the ileum they were sporadic, and in the corpus and colon they were virtually nonexistent. In the antral mucosa, CgB-IR cells were observed mainly in the midportion, in the regions of the small intestine in the various levels of the villi, but only occasionally in the crypts, where they were seen in the upper half. A few CgB-IR cells were also observed in Brunner's glands.

CgC-IR cells were few in number, being present mainly in the antrum, where they were seen in patches in the middle third of the mucosa. A few cells were present in the duodenum (mainly in the crypts, occasionally in the villi) but were completely absent in Brunner's glands and the other gastrointestinal regions.

Co-localization of CgA, B, and C in Endocrine Cells
The results are summarized in Table 2.


 
View this table:
[in this window]
[in a new window]
 
Table 2. Co-localization of chromogranins A, B, and C (A,B,C) in various endocrine cell types in different parts of the human gastrointestinal tractab

Corpus. Serotonin (enterochromaffin, EC) cells but not somatostatin cells displayed CgA immunoreactivity. Most of the CgA-IR cells were probably enterochromaffin-like (ECL) cells, a type predominating in this region but which could not be identified immunocytochemically because available histamine antibodies are unsuitable for use with formalin-fixed tissue.

Antrum. All EC cells were CgA-immunoreactive, and a subpopulation thereof also contained either CgB or CgC. Those cells harboring CgB and the few with CgC were located in the middle and upper parts of the mucosa. Only occasionally did the antral EC cells display immunoreactivity for all three chromogranins.

Most gastrin cells were immunoreactive for CgB and a fraction of them also for CgA, but a few contained all three chromogranins. CgA immunoreactivity in gastrin cells varied in intensity, being weak in most cells and sometimes virtually absent.

Immunoreactivity for CgB was stronger than for CgA or C (Figure 1A-D and Figure 2). A few gastrin cells were also serotonin-reactive and somatostatin cells were occasionally CgA immunoreactive (Figure 3).



View larger version (46K):
[in this window]
[in a new window]
 
Figure 1. Human antral mucosa triple stained for (A) gastrin (AMCA), (B) chromogranin A (FITC), and (C) chromogranin B (Texas red). (D) Double exposure through the double-band filter for FITC and Texas red and through the AMCA filter is shown. Most gastrin cells (blue) (A) show stronger immunoreactivity for chromogranin B (red) (C) than for chromogranin A (green) (B), evident as varying intensities of magenta color (D). A few gastrin cells are more strongly immunoreactive to chromogranin A, visualized as cyan color (D) (arrow). The white cells in D reflect the co-localization of the three fluorochromes together. Bar = 27 µm.

Figure 2. Human antral mucosa double stained for gastrin (FITC) and chromogranin C (Texas red). Most of the immunostained cells show co-localization of these two substances (yellow). This co-localization is mainly seen in the basal region of the gastrin cells, whereas only gastrin immunoreactivity (green) occurs in the entire cytoplasm. In a minority of the gastrin cell population, chromogranin C immunoreactivity (red) appears to occur alone in parts of the cytoplasm. Bar = 27 µm.

Figure 3. Human antral mucosa double stained for somatostatin (FITC) and chromogranin A (Texas red), showing distinct co-localization of these two substances in only one cell (yellow). Bar = 27 µm.

Figure 4. Tip of a villus of human duodenum triple stained for serotonin (Texas red), chromogranin A (FITC), and chromogranin B (AMCA). Serotonin (A) is evident throughout the cytoplasm of the three EC cells, whereas chromogranin A (B) is evident basally in these cells. (C) Co-localization of serotonin and chromogranin A, which is illustrated by the yellow color (double-band filter set). Chromogranin B immunoreactivity (D) is present throughout the cytoplasm, i.e., as for serotonin immunoreactivity. Bar = 27 µm.

Duodenum--Mucosa. The EC cells located in the crypts were CgA-IR, but only some were located in the villi. CgB immunoreactivity was observed in a subpopulation of EC cells in the villi, but not in EC cells in the crypts (Figure 4A and Figure 4B); occasional EC cells in the crypts contained CgC. Gastrin and CCK cells reacted to CgB and CgA antibodies, and some also to CgC. Some secretin and GIP cells and virtually all enteroglucagon cells were also immunoreactive to CgA, but somatostatin cells only occasionally so. A few enteroglucagon cells in this region were also CgB-immunoreactive.

Brunner's Glands. The identified EC, gastrin, and CCK cells (but not somatostatin or enteroglucagon cells) were CgA-IR. Gastrin and CCK cells also contained CgB.

Ileum. The EC cells were CgA-positive and some of those located in the villi also contained CgB. The somatostatin cells proved negative for all three chromogranins. Some enteroglucagon/PYY and neurotensin cells were CgA-IR, whereas CgB and C staining was absent.

Colon. The EC cells and a subpopulation of EG/PYY cells were found immunoreactive to CgA, but not to B or C. The somatostatin cells reacted negatively to all the Cg antibodies. The few CgB cells visualized could not be correlated to any of the endocrine cell types identified.

Intracellular Localization of Chromogranins and Corresponding Hormones
CgA and C were concentrated mostly in the basal (infranuclear) region of the cells, whereas CgB was more diffusely spread in the cytoplasm, as was serotonin immunoreactivity, involving the cell processes occasionally seen in some of these cells. CgA immunoreactivity in antral EC cells in the lower third of the mucosa was located mainly adjacent to the basal cell membrane. In gastrin cells, CgA was found mainly in the infranuclear region, although occasionally occupying the entire cytoplasm. CgA immunoreactivity was mainly basal in CCK, secretin, GIP, enteroglucagon, enteroglucagon/PYY, and neurotensin cells, whereas the entire cytoplasm was stained in the (few) CgA-IR somatostatin cells.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

For co-localization studies, the present multiple immunostaining technique using fluorochromes proved superior to single immunostaining in consecutive sections, because the substances could be determined more exactly in the individual cells. A similar technique--but with chromogens for light microscopy--is excellent if the substances occur in different cells, but difficulties may arise in determining the resulting color if they are located in the same cells. Immunoelectron microscopy can also be used in co-localization studies by using various sizes of gold particles, although the more complicated staining steps are a disadvantage compared with the present fluorescence methods. Furthermore, only a small number of cells can be studied at one time. With single-band filter sets, the different substances can be located individually. With the present technique, using a double-band filter, co-localization of two of these substances can be studied simultaneously, and with a further single-band filter and double exposure of the film it was possible to identify a third substance at the same time and even to determine its location in relationship to the other two. A triple-band filter for the three fluorochromes has also been tested, but it did not help us assess the stainings.

With double or triple immunostaining, crossreactivity of the antibodies must be avoided. In our investigation we used both monoclonal and polyclonal antibodies raised in different animal species. Only exceptionally did we use antibodies raised in the same animal species, and in these cases we blocked the epitopes of the first antibody by using monovalent Fab fragments of IgG. Appropriate control stainings, necessary to evaluate the staining specificity, were carried out. Furthermore, staining specificity was evaluated for single immunostaining, not only with fluorescence but also with the ABC technique.

The distribution of chromogranin immunoreactivity along the gastrointestinal tract is fairly similar to that demonstrated in previous reports (Varndell et al. 1985 ; Rindi et al. 1986 ; Bishop et al. 1989 ; Buffa et al. 1989 ; Schmid et al. 1989 ; Pelagi et al. 1992 ). CgA-IR cells predominated in all gastrointestinal regions studied, indicating the conventional wisdom of using CgA as a panendocrine marker. However, not all cells displayed CgA immunoreactivity. CgB-IR cells, were slightly fewer in the antrum compared with CgA-IR cells, and were sparse in duodenum and almost nonexistent in the corpus and colon. CgC was less common than the other members of the Cg family, and was present only in the antrum and duodenum.

As mentioned earlier, the ECL cells were indirectly identified, because the commercially available histamine antibodies tested were ineffective in our tissue. These antibodies need a special fixative containing carbodiimide. ECL cells predominate in the corpus and their frequency and distribution are as for the CgA cells, i.e., at least the majority of ECL cells must be CgA-positive, which would be consistent with other reports (Buffa et al. 1989 ), although they are both CgB- and C-negative.

EC cells displayed CgA immunoreactivity in all gastrointestinal regions examined, as in other reports (Varndell et al. 1989; Rindi et al. 1986 ; Buffa et al. 1985). CgA was found mainly in the infranuclear region, whereas CgB immunoreactivity, occurring in some EC cells in antrum, duodenum, and ileum, had a cytoplasmic distribution similar to that of serotonin, i.e., throughout the cytoplasm. This difference in the immunostainings suggests that either CgA is not stored in all secretory granules or else occurs in quantities too small to be detected by our immunostaining techniques, or that serotonin and CgB may occur in a nongranular form in the supranuclear cell region. CgB was demonstrated in a small subpopulation of EC cells in duodenum and ileum, located mainly in the villi. Earlier studies have produced various results regarding the occurrence of CgB in EC cells in the intestinal tract. Buffa et al. 1989 reported CgB-IR and non-CgB-IR EC cells, but they did not mention whether these subpopulations were located in the villi or in the crypts. Rindi et al. 1986 did not find CgB in EC cells, and Bishop et al. 1989 , using electron microscopy, reported both CgB and GAWK (a fragment of CgB) in this cell type. Two subpopulations of EC cells have previously been identified, on the basis of their content of motilin and substance P, respectively (Pearse et al. 1974 ; Heitz et al. 1976 ; Heitz et al. 1978 ). These subpopulations have different distributions in the gastrointestinal tract, the former in the proximal small intestine, the latter in the distal part. However, the localization of these two subpopulations cannot be related to the presence or absence of CgB-IR cells because the latter occur in both proximal and distal small intestine, and mostly in the villi. This difference in the content of CgB in EC cells may suggest different cellular functions in the villi and in the crypts. Our finding of CgC-IR EC cells in the antrum and in duodenal crypts has not been reported earlier, possibly due to the quality of the antibodies and/or the fact that these cells are so sparse.

The antral gastrin cells were immunoreactive for both CgA and B, and some also for CgC. The latter glycoprotein has not been reported earlier in this cell type in humans, but was found in guinea pig (Cetin and Grube 1991 ; Cetin et al. 1992 ). It is less probable that these CgC-IR gastrin cells correspond to the cell population expressing both gastrin and serotonin; the former were more numerous. In the duodenum, few gastrin-IR cells reacted to CgA antibodies.

With very few exceptions, somatostatin cells were the only cell type in which we were unable to find CgA. The reason for this is unclear, but one possible explanation may be that CgA is present at such a low concentration that it cannot be detected with the immunostaining used, although Wiedenmann et al. 1988 reported similar results using the more sensitive PAP stain. Other explanations could be that CgA may be masked by the granule-related proteins, or is proteolytically fragmented in such a way that it does not react with our antibodies, or that with few exceptions it does not exist in these cell types. We have not succeeded in demonstrating any CgB or C in this cell type.

Regarding the secretin cells and CgA, our results differ somewhat from those reported by Buffa et al. 1989 . Unlike these authors, we found CgA in only some of the secretin cells.

The co-localization of enteroglucagon and CgA varied in the different intestinal regions. In the duodenal mucosa, enteroglucagon cells reacted to CgA, which agrees with the results reported by Varndell et al. 1985 . Surprisingly, this cell type did not react to CgA in Brunner's glands. In ileum and colon, where enteroglucagon is to a large extent co-localized with PYY, CgA appeared in only some of these cells. Neurotensin cells also had a variable CgA content.

It has been demonstrated ultrastructurally that CgA and B are present in secretory granules in the gastrointestinal tract (Varndell et al. 1985 ; Bishop et al. 1989 ). CgC has been found in secretory granules in the pituitary gland (Rosa et al. 1992 ), and may also have the same location in antrum and duodenum. In antral gastrin cells the distribution of chromogranins suggests at least two subpopulations of secretory granules, one in the infranuclear region, which may contain all the three chromogranins, the other in the supranuclear region containing only CgB. It remains to be shown whether or not the different chromogranins are co-localized in the same secretory granules, or whether chromogranin B may also exist in a nongranular form.

Our knowledge of the physiological functions of chromogranins is limited. Most studies have concentrated on CgA and its proteolytic fragments, which may have several biological roles, among others an intracellular hormone-binding function, as precursors to various smaller fragments that may be biologically active, and/or an inhibitory effect on the secretion of different hormones belonging to the neuroendocrine system, e.g., parathyroid hormone, parathyroid hormone-related protein, insulin, calcitonin, and catecholamines (cf. Winkler and Fischer-Colbrie 1992 ; O’Connor et al. 1994 ).

The function of CgB and CgC is still virtually unknown. In addition to having a role as precursors of smaller peptides (Huttner et al. 1991 ), CgB has been reported to bind calcium (Gorr et al. 1989 ), a function also attributed to CgA, and to have a trophic action in neurons (Chen et al. 1992 ). It has been suggested that CgC is associated with secretory granule maturation (Tooze et al. 1994 ), but this would contradict the finding of this glycoprotein in only a limited number of endocrine cell types.

Immunoelectron microscopic studies have shown that serotonin (Fujimiya et al. 1995 ; Okumiya et al. 1996a ) and gastrin (Okumiya et al. 1996b ) may exist in a nongranular form in the supranuclear cytoplasm of EC and gastrin cells, respectively. Our findings of an association between the intracellular distribution of the immunoreactivity of serotonin and gastrin with CgB, particularly in the luminal parts of the cells, may suggest a functional role for CgB as a carrier protein, at least for serotonin and gastrin. A surprising finding was the variation in CgA, B, and C immunoreactivity within the different cell types. There is still no plausible explanation for the varying extent and composition of the different chromogranins, but this may be a reflection of their complex roles in the neuroendocrine system related to their biological activities as hormone-binding substances, carrier proteins, precursors to smaller fragments, and proteins contributing to secretory granule maturation and hormone release. A further interesting question about their interrelationship is whether or not the chromogranins may be present in the same secretory granules. Such a co-localization study at the ultrastuctural level could indicate if different granule populations exist, and thereby provide further knowledge concerning the functions of the different members of the chromogranin family.


  Acknowledgments

Supported by a grant from the Swedish Medical Research Council (project no. 102), by the Lion's Foundation, Uppsala, and by Hässle Läkemedel AB, Mölndal, Sweden.

We thank Ms Birgitta Vagnhammar for excellent technical assistance.

Parts of this work were presented at the 11th International Symposium on Regulatory Peptides, Copenhagen, Denmark, 3-7 September, 1996.

Received for publication October 25, 1996; accepted February 13, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Benedum UM, Lamouroux A, Konechi DS, Rosa P, Hille A, Baeuerle PA, Frank R, Lottspeich F, Mallet J, Huttner WB (1987) The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region. EMBO J 6:1203-1211[Abstract]

Bishop AE, Sekiya K, Salahuddin MJ, Carlei F, Rindi G, Fahey M, Steel JH, Hedges M, Domoto T, Fischer-Colbrie R, Winkler H, Krausz T, Ghatei MA, Bloom SR, Polak JM (1989) The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B. Histochemistry 90:475-483[Medline]

Buffa R, Gini A, Pelagi M, Siccardi AG, Bisisani C, Zanini A, Solcia E (1989) Immunoreactivity of hormonally-characterized human endocrine cells against three novel anti-human chromogranin B (B11 and B13) and chromogranin A (A11) monoclonal antibodies. Arch Histol Cytol 52(suppl):99-105[Medline]

Cetin Y, Bargsten G, Grube D (1992) Mutual relationships between chromogranins A and B and gastrin in individual gastrin cells. Proc Natl Acad Sci USA 89:2912-2916[Abstract]

Cetin Y, Grube D (1991) Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea-pig entero-endocrine system: cellular distributions and intercellular heterogeneities. Cell Tissue Res 264:231-241[Medline]

Chen M, Tempst P, Yankner BA (1992) Secretogranin I/chromogranin B is a heparin-binding adhesive protein. J Neurochem 58:1691-1698[Medline]

Facer P, Bishop AE, Lloyd RV, Wilson BS, Hennessy RJ, Polak JM (1985) Chromogranin: a newly recognized marker for endocrine cells of the human gastrointestinal tract. Gastroenterology 89:1366-1373[Medline]

Fahrenkamp AG, Wibbeke C, Winde G, Öfner D, Böcker W, Fischer-Colbrie R, Schmid KW (1995) Immunohistochemical distribution of chromogranins A and B and secretogranin II in neuroendocrine tumors of the gastrointestinal tract. Virchows Arch 426:361-367[Medline]

Fischer-Colbrie R, Hagn C, Schober M (1987) Chromogranins A, B and C: widespread constituents of secretory vesicles. Ann NY Acad Sci 493:120-134[Medline]

Fujimiya M, Okumiya K, Maeda T (1995) Immuno-electron microscopic demonstration of luminal release of serotonin from enterochromaffin cells of rat embryo. Acta Histochem Cytochem 28:555-563

Gerdes HH, Rosa P, Philips E, Baeuerle PA, Frank R, Argos P, Huttner WB (1989) The primary structure of human secretogranin II, a widespread tyrosine-sulphated secretory granule protein that exhibits low pH- and calcium-induced aggregation. J Biol Chem 264:12009-12015[Abstract/Free Full Text]

Gorr SU, Shioi J, Cohn DV (1989) Interaction of calcium with porcine adrenal chromogranin A (secretory protein-I) and chromogranin B (secretogranin I). Am J Physiol 257:E247-E254[Abstract/Free Full Text]

Heitz P, Kasper M, Krey G, Polak JM, Pearse AGE (1978) Immunoelectron cytochemical localization of motilin in human duodenal enterochromaffin cells. Gastroenterology 74:713-717[Medline]

Heitz P, Polak JM, Timson CM, Pearse AGE (1976) Enterochromaffin cells as the endocrine source of gastrointestinal substance P. Histochemistry 49:343-347[Medline]

Hsu SM, Raine T, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immmunoperoxidase techniques: a comparison between ABC and unlabeled (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]

Huttner WB, Gerdes H, Rosa P (1991) The granin (chromogranin/secretogranin) family. Trends Biochem Sci 16:27-30[Medline]

Lloyd RV, Wilson BS (1983) Specific endocrine tissue marker defined by a monoclonal antibody. Science 222:628-630[Medline]

Negoescu A, Labat-Moleur F, Lorimier P, Lamarcq L, Guillermet C, Chambaz E, Brambilla E (1994) F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 42:433-437[Abstract/Free Full Text]

O'Connor DT, Burton D, Deftos LJ (1983) Chromogranin A: immunohistology reveals its universal occurrence in normal polypeptide hormone producing endocrine glands. Life Sci 33:1657-1664[Medline]

O'Connor DT, Wu H, Gill BM, Rosansky DJ, Tang K, Mahata SK, Mahata M, Eskeland NL, Videen JS, Zhang X, Takiyyuddin MA, Parmer RJ (1994) Hormone storage vesicle proteins. Transcriptional basis of the widespread neuroendocrine expression of chromogranin A, and evidence of its diverse biological actions, intracellular and extracellular. Ann NY Acad Sci 733:36-45[Abstract]

Okumiya K, Kuwahara A, Fujimiya M (1996a) Immunoelectron microscopic demonstration of luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure. Regul Pept 64:146

Okumiya K, Matsubayashi K, Maeda T, Fujimiya M (1996b) Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol. Peptides, in press

Pearse AGE, Polak JM, Bloom SR, Adams C, Dryburgh JR, Brown JC (1974) Enterochromaffin cells of the mammalian small intestine as the source of motilin. Virchows Arch [Cell Pathol] 16:111-120

Pelagi M, Zanini A, Gasparrini A, Ermellino L, Giudici AM, Ferrero S, Siccardi AG, Buffa R (1992) Immunodetection of secretogranin II in animal human tissues by new monoclonal antibodies. Regul Pept 39:201-214[Medline]

Rindi G, Buffa R, Sessa F, Tortora O, Solcia E (1986) Chromogranin A, B and C immunoreactivities of mammalian endocrine cells. Distribution, distinction from costored hormones/prohormones and relationship with the argyrophil component of secretory granules. Histochemistry 85:19-28[Medline]

Rosa P, Bassetti M, Weiss U, Huttner WB (1992) Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas. J Histochem Cytochem 40:523-533[Abstract/Free Full Text]

Schmid KW, Brink M, Freytag G, Kirchmair R, Böcher W, Fischer-Colbrie R, Heitz P, Klöppel G (1994) Expression of chromogranin A and B and secretoneurin immunoreactivity in neoplastic and nonneoplastic pancreatic alpha cells. Virchows Arch 425:127-132[Medline]

Schmid KW, Weiler R, Xu RW, Hogue-Angeletti R, Fischer-Colbrie R, Winkler H (1989) An immunological study on chromogranin A and B in human endocrine and nervous tissues. Histochem J 21:365-373[Medline]

Stefanini M, De Martino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173-174[Medline]

Stridsberg M, Hellman U, Wilander E, Lundqvist G, Hellsing K, Öberg K (1993) Fragments of chromogranin A are present in the urine of patients with carcinoid tumours: development of a specific radioimmunoassay for chromogranin A and its fragments. J Endocrinol 139:329-337[Abstract]

Stridsberg M, Öberg K, Li Q, Engström U, Lundqvist G (1995) Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 144:49-59[Abstract]

Tooze SA, Hollinshead M, Dittié AS (1994) Antibodies to secretogranin II reveal potential processing sites. Biochemie 76:271-276[Medline]

Varndell IM, Lloyd RV, Wilson BS, Polak JM (1985) Ultrastructural localization of chromogranin: a potential marker for the electron microscopic recognition of endocrine cell secretory granules. Histochem J 17:981-992[Medline]

Wiedenmann N, Huttner WB (1989) Synaptophysin and chromogranins/secretogranins--widespead constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis. Virchows Arch [Cell Pathol] 58:92-121

Wiedenmann B, Waldherr R, Buhr H, Hille A, Rosa P, Huttner WB (1988) Identification of gastroenteropancreatic neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II. Gastroenterology 95:1364-1374[Medline]

Winkler H (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience 1:65-80[Medline]

Winkler H, Fischer Colbrie R (1992) The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497-528[Medline]