S. Van Noorden

Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas

SummaryIn this study we report the localisation of PYY immunoreactivity in intestinal mucosa endocrine (EG) cells containing glucagon-related peptides and also in foetal pancreatic A cells of rat and man. Radioimmunoassay of human foetal pancreatic extracts revealed the presence of PYY immunoreactivity, the concentration of which declined with age (from 65.42 pmol/g at week 20 to 17.0 pmol at week 40; correlation coefficient=−0.893), in contrast to the amount of glucagon which remained statistically constant throughout the same foctal period. The identity of this PYY immunoreactive material with the original 36 amino acid porcine peptide has been shown by high pressure liquid chromatography (HPLC).

Cytochemical and immunofluorescence investigations on insulin-like producing cells in the intestine of Mytilus edulis L. (Bivalvia)

SummaryInsulin-like immunoreactivity can be localized to cells of the intestine in the-area of the hepatopancreas of Mytilus edulis L. No cross-reactivity can be obtained with anti-glucagon, anti-gastrin, anti-pentagastrin or anticaerulin. The cells containing the substance immunoreactive to mammalian anti-insulin, can be restained with paraldehyde-fuchsin.

Localization of somatostatin-like immunoreactivity in the pancreatic islets of the hagfish, Myxine glutinosa and the lamprey Lampetra fluviatilis

SummarySomatostatin-like immunofluorescence has been found by immunostaining in cells of the bile duct mucosa and pancreatic islet parenchyma of the hagfish, Myxine glutinosa, and the islet lobules of the lamprey, Lampetra fluviatilis.

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats

SummaryWe have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.

Neuropeptides and 5-HT immunoreactivity in the gastric nerves of the dogfish (Scyliorhinus stellaris)

The gastric autonomic innervation of the dogfish was examined for regulatory peptides and serotonin by immunochemical techniques. Bouins-fixed, paraffin-embedded or benzoquinone-fixed frozen sections were used for light microscopical immunocytochemistry and glutaraldehyde-fixed resin-embedded sections for electron microscopical immunocytochemistry. Bombesin-, somatostatin-, gastrin/cholecystokinin-, substance P-, peptide histidine isoleucine-, vasoactive intestinal peptide- and serotonin-immunoreactive nerves were found in all layers of the stomach wall. Bombesin and vasoactive intestinal peptide-containing nerves were identified at ultrastructural level. Radioimmunoassay of acetic acid extracts of tissue confirmed the presence of immunoreactivity for bombesin, somatostatin, substance P, peptide histidine isoleucine and vasoactive intestinal peptide. Reverse phase high performance liquid chromatography indicated that the peptides identified were broadly similar to their mammalian counterparts.

Immunofluorescent localization of calcitonin in the ultimobranchial gland of Rana temporaria and Rana pipiens

SummaryCalcitonin-like immunoreactivity has been demonstrated by immunofluorescence in the cells of the ultimobranchial gland of two species of Rana, using an indirect (sandwich) technique with anti-pure porcine calcitonin serum. The possibility of cross-reactivity, between the amphibian hormone and anti-porcine calcitonin antibody, had been anticipated on account of the observation that injected porcine calcitonin lowered the plasma calcium levels in one of the species under investigation.

Co-localisation of substance P-, bombesin-and peptide histidine isoleucine (PHI)-like peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris

SummaryThe gastrointestinal tract of cartilaginous fishes, like that of higher vertebrates, is known to contain endocrine cells and nerves immunoreactive for a wide variety of peptides, some of which have been structurally characterised. Since we have found that substance P-, bombesin-and peptide histidine isoleucine-like immunoreactivities are similarly distributed in the endocrine cells of the dogfish pyloric stomach, we have tried to establish whether any of these peptides are co-localised. The cells were compared in thin serial sections with both ligh-and electron microscopical immunocytochemistry. Double immunolabelling was also used to show two immunoreactive peptides in the same tissue section. Further characterisation of the immunoreactivity was attempted by preabsorbing the antibodies with various peptides or synthetic fragments of peptide molecules. Immunoreactivity for all three peptides was frequently present in the same cells, whereas antibodies to other peptides such as gastrin and somatostatin marked different cells. Electron microscopy indicated that all the secretory granules in three morphologically different cell types reacted with antibodies to all three peptides. Dual localisation of unrelated peptides in endocrine cells or nerves is established in many cases, but triple localisation is as yet unusual. The immunoreaction for bombesin-like peptides is different in endocrine cells and nerves, indicating that dogfish bombesin may be present in two forms, in agreement with biochemical evidence.

Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalisation depends on 1) adequate preservation of the peptide antigen and the tissue structure in which it resides; 2) a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several ‘families’ of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.

Histochemical and ultrastructural studies on the enterochromaffin-like cell in the gastric mucosa of the opossum Didelphis albiventris (Marsupialia)

SummaryAn ultrastructural study of enterochromaffin-like (ECL) cells in the gastric mucosa of the white-belly opossum Didelphis albiventris (Marsupialia) was carried out. In parallel, histochemical methods were used at the light-microscopical level to demonstrate argentaffin cells, argyrophilic cells, and serotonin- and histamine-immunoreactive elements. Argentaffin and serotonin-immunoreactive cells were scattered, and argyrophilic cells were numerous, within the full thickness of the mucosa. Argyrophilic cell distribution was similar to that of histamine-immunoreactive elements. At the electron-microscopical level, the oxyntic mucosa of D. albiventris presented endocrine cells with secretory granules morphologically similar to those of the ECL cell of eutherian mammals. However, in this marsupial, the ECL cell exhibited a variable mixture of two distinct types of secretory granules: (1) granules with the morphological appearance of the eutherian ECL cell, and (2) granules morphologically similar to those of the eutherian enterochromaffin (EC) cells. Based on this morphological pattern of the ECL cell granules, it is proposed that in the oxyntic mucosa of the opossum D. albiventris, the EC and ECL cells represent distinct steps in the same line of cell differentiation; the ECL cell should also be a site of histamine storage.

The ultimobranchial gland of the dogfishScyliorhinus canicula L. immunofluorescence cross-reactivities with anti-sera to different calcitonins

SummaryCalcitonin in the ultimobranchial gland of the dogfishScyliorhinus canicula cross-reacts with anti-dogfish, anti-salmon and anti-porcine calcitonin, but not with anti-human calcitonin, using indirect immunofluorescence procedures. These results do not reflect completely expectations derived from the primary structures of the hormones, where known, and from their physiological interrelationships.