Guida M. Portela-Gomes

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

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.

Expression of the five different somatostatin receptor subtypes in endocrine cells of the pancreas.

Knowledge concerning tissue-specific expression of the five somatostatin receptor subtypes is of great importance in understanding their physiological function. We developed rabbit polyclonal antibodies specific for each human somatostatin receptor subtype and report our results concerning the expression in normal endocrine pancreatic cells. The antibodies were produced by immunizing rabbits with fragments specific for the five cloned somatostatin receptor subtypes. Colocalization of these somatostatin receptors with the four major islet hormones--insulin, glucagon, somatostatin, and pancreatic polypeptide--was studied in normal human endocrine pancreatic cells, using double-immunofluorescence staining. High expression of somatostatin receptor subtypes 1, 3, and 4 was found in all endocrine pancreatic cells. Somatostatin receptor subtype 2 was frequently expressed in alpha and beta cells, whereas expression was low in pancreatic polypeptide cells and intermediate in delta cells. Somatostatin receptor subtype 5 was expressed in most beta and delta cells but almost absent in alpha and pancreatic polypeptide cells. There is a variability in the normal expression of somatostatin receptor subtypes among the different human endocrine pancreatic cells. Knowledge of this expression and the physiological function mediated by these receptors will be valuable in the future when considering treatment of endocrine disorders.

Chromogranin A in human neuroendocrine tumors. An immunohistochemical study with region-specific antibodies.

Antibodies to six specific regions of the chromogranin A (CgA) molecule were used to study their immunoreactivity in human neuroendocrine (NE) tumors. Tissue specimens from endocrine pancreatic tumors (n = 14), duodenal carcinoids (n = 2), bronchial carcinoids (n = 5), ileal carcinoids (n = 5) appendix carcinoids (n = 2), medullary thyroid carcinomas (n = 6), parathyroid adenomas (n = 2), and pheochromocytomas (n = 8) were analyzed. The results showed that the NE tumor types expressed varying numbers of CgA fragments. A variation in frequency of the expression of immunoreactive cells was sometimes seen also within the same tumor type. The midportion fragment CgA 176–195 (chromacin) was the only fragment expressed in all tumors. Benign and malignant tumors expressed different patterns, being especially true of insulinomas and pheochromocytomas. These findings suggest that region-specific antibodies to CgA fragments can be used as a diagnostic tool for the characterization of NE tumors.

Chromogranin A in the Human Gastrointestinal Tract: An Immunocytochemical Study with Region-specific Antibodies

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411–424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 17–38 and 176–195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.

The distribution of endocrine cell types of the gastrointestinal mucosa in genetically diabetic (db/db) mice

BACKGROUND/AIMS Genetically diabetic (db/db) mice are a model for non-insulin-dependent diabetes in humans. The gastrointestinal tracts in 12-week-old db/db and nondiabetic control (db/+) mice were studied with particular emphasis on the endocrine cells. METHODS Immunocytochemical and quantification techniques were used to localize and determine the number of cells containing serotonin and various regulatory peptides. RESULTS In the antrum, the gastrin- and serotonin-immunoreactive cells were increased in number. In the large intestine, the enteroglucagon and the peptide tyrosine-immunoreactive cells were increased in number, whereas there were fewer serotonin-immunoreactive cells. There were also fewer somatostatin-immunoreactive cells in most gastrointestinal regions. In diabetic mice, the intestine was longer and its mucosa thicker than in control mice. CONCLUSIONS The results indicate that the genetic diabetic (db/db) condition exerts a significant influence on the gastrointestinal tract and on the endocrine cell systems studied. The observed alterations may reflect the effect of indirect factors rather than the diabetes per se.

Overexpression of von Hippel-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease

Background: A significant number of patients with chronic obstructive pulmonary disease (COPD) exhibit skeletal muscle wasting and decreased capillary area formation, which correlate with increased mortality. Aim: To determine the molecular mechanisms mediating decreased capillary formation in COPD. Methods: 24 patients with COPD and 12 matching controls were recruited. Patients with COPD were classified into mild, moderate and severe groups according to GOLD (global initiative for chronic obstructive lung disease) criteria. Biopsy specimens were obtained from the tibialis anterior muscle. Fibre typing and capillary formation, together with messenger RNA (mRNA) expression of hypoxia-inducible factors (HIF1α and HIF3α), vascular endothelial growth factors (VEGF-A, VEGF-B and VEGF-C isoforms) and von Hippel-Lindau (VHL) protein, were determined. VHL expression and localisation were further studied by immunohistochemistry. Results: Skeletal muscle capillary formation decreased significantly with increasing disease severity. Compared with controls, a tendency to mRNA overexpression of HIF1α, HIF3α and VEGF isoforms was observed in mild and moderate COPD, which decreased at the severe stage. In contrast, skeletal muscle biopsy samples from patients with COPD exhibited significant overexpression of VHL at both the mRNA and protein level by immunohistochemistry. VHL protein was further determined to be localised to satellite cells. Conclusions: Overexpression of VHL was identified in the skeletal muscle of patients with COPD. Increased VHL activity may have a negative effect on transduction of the hypoxic signal and may contribute to decreased capillarisation in skeletal muscles of patients with COPD.

Neuroendocrine cell markers for pancreatic islets and tumors.

The authors review the application of a variety of neuroendocrine cell markers to identify pancreatic islet cells and tumors. In the past, several empiric histochemical techniques had been used to demonstrate neuroendocrine cells, particularly the Grimelius argyrophilic stain. The development of immunohistochemistry made it possible to demonstrate specific cell products such as regulatory peptides, thus allowing the classification of pancreatic neuroendocrine tumors with a view to clinical symptoms. However, it is not always possible to visualize regulatory peptides in these tumors. It is therefore important to use broad-spectrum neuroendocrine cell markers to identify the neuroendocrine nature. These markers are proteins localized in the secretory granules (core- or membrane-related), in the cytosol, or in the cellular membrane. The markers most commonly used in routine histopathology are the secretory granule proteins chromogranin A and synaptophysin and the cytosolic enzyme neuron-specific enolase. Other new markers (eg, synaptic vesicle protein 2) are of general diagnostic value. Region-specific antibodies to chromogranin A can be valuable in differentiating between benign and malignant neuroendocrine tumors. Some markers may be related to the functioning characteristics of pancreatic neuroendocrine tumors, such as prohormone convertases. In addition, markers giving further complementary information have been identified, such as five somatostatin receptor subtypes, the expression of which varies markedly in pancreatic neuroendocrine tumors. Antibodies against all somatostatin receptor subtypes are now commercially available, and immunohistochemical investigation of its expression should be routinely applied when considering treatment with somatostatin analogs.

Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas An immunohistochemical study with region-specific antibodies

In a recent immunohistochemical study of pheochromocytomas, a difference was observed between benign and malignant pheochromocytomas in their expression in different parts of the chromogranin (Cg) A molecule. The purpose of the present study was to extend the investigations by including two other members of this Cg family, CgB and C. Twenty‐five patients operated on for clinicopathologically benign pheochromocytomas, and four for metastasizing pheochromocytomas, were studied. Expression of the different Cg regions was studied immunohistochemically by means of region‐specific antibodies: four raised against CgA epitopes, five against CgB, and two against CgC. Adrenal medulla parenchyma from three surgical adrenalectomy specimens was used as non‐neoplastic control. All cells of normal adrenal medulla were immunoreactive to all 11 region‐specific Cg antibodies. In the pheochromocytomas, variations in the expression pattern occurred, but no significant quantitative differences were noted between benign and malignant tumours. Nevertheless, in all four malignant pheochromocytomas, the antibodies raised against the C‐terminal regions of both CgB and CgC visualised a noticeable population of large spindle‐shaped tumour cells, characterised by elongated processes. This cell type occurred in all four malignant pheochromocytomas but only in one benign tumour. Their structure and immunoreactivity differed from those of the sustentacular cells in the pheochromocytoma parenchyma. The use of region‐specific antibodies raised against epitopes in the C‐terminal region of CgB and CgC can facilitate the diagnosis of malignant pheochromocytoma.

PACAP is expressed in secretory granules of insulin and glucagon cells in human and rodent pancreas. Evidence for generation of cAMP compartments uncoupled from hormone release in diabetic islets.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet neuropeptide with potent insulinotropic action. The current study investigates PACAP expression in normal human and rat pancreatic islets, and whether it is altered in diabetic state. To that end, PACAP immunoreactivity was studied by immunofluorescence methods enhanced by the catalyzed reporter deposition (CARD) technique. Insulin and cyclic adenosine monophosphate (cAMP) generation induced by PACAP were investigated in islets isolated from the spontaneously diabetic Goto-Kakizaki (GK) rat. PACAP immunoreactivity was observed in virtually all insulin and glucagon cells in both species, but not in somatostatin or pancreatic polypeptide (PP) cells; this co-localization pattern was unaltered in diabetic pancreata. In normal human pancreas, PACAP was further localized ultrastructurally to the secretory granules of insulin and glucagon cells. PACAP significantly potentiated glucose-stimulated insulin release in isolated islets of normal but not of GK rats. PACAP failed to enhance cAMP generation in normal islets, but induced approximately 5-folds exaggeration in the diabetic islets. In conclusion, using improved immunocytochemistry techniques and electron microscopy (EM), PACAP was shown to be expressed both in normal and diabetic islet cells and localized to secretory granules of insulin and glucagon cells. Furthermore, the insulinotropic action of PACAP was markedly impaired in diabetic islets in spite of exaggerated cAMP response.

Region-specific Antibodies to Chromogranin B Display Various Immunostaining Patterns in Human Endocrine Pancreas

Chromogranin (Cg) B is an acidic glycoprotein present in neuroendocrine tissue. The sequence shows several dibasic amino acid positions susceptible to proteolytic cleavage. The purpose of this study was to elucidate the expression of CgB epitopes in the human endocrine pancreas. Tissue sections of six human pancreata were immunostained with 16 different region-specific antibodies to the CgB molecule, using double immunofluorescence techniques. The CgB epitope pattern varied in the four major islet cell types. B (insulin)-cells expressed immunoreactivity to all region-specific antibodies. The antibodies to the N-terminal and mid-portions of CgB showed moderate immunoreactivity, the C-terminal antibodies weak. A (glucagon)-cells were reactive only to the N-terminal and mid-portion antibodies but, after microwave pretreatment, to all antibodies, whereas D (somatostatin)-cells expressed only the sequence CgB 244–255 and a subpopulation CgB 580–595. PP (pancreatic polypeptide) cells were immunostained with antibodies between CgB 1–417 and a few with CgB 580–593. The fragment CgB 244–255 was expressed in all four cell types. The cause of these differences may be cell-specific cleavage or masking of the molecule, but varying translation of CgB mRNA is also possible. The extent to which these epitopes reflect fragments having biological functions remains to be evaluated.