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The presence, localization and distribution of some regulatory peptides and serotonin were investigated by single and double immunohistochemical methods in the digestive system of two reptiles, Chalcides chalcides and Zonosaurus madascariensis. Both immunoreactive (IR) cells and nerve elements were demonstrated, showing different distributions according to the antisera tested. Similar results were observed in the two saurian species. Chromogranin-SPI-, serotonin-, somatostatin14-, pancreatic polypeptide (PP)-, and gastrin-IR cells were present along the gut epithelium. Cholecystokinin (CCK)-, and insulin-IR cells seemed to be more concentrated in the intestinal portion, while very few glucagon containing cells were observed. Bombesin-IR cells were found in the stomach and they constituted the only endocrine cells of the closed type in the gut. Vasoactive intestinal polypeptide (VIP)-, insulin-, and bombesin-IR nerve terminals were also seen. In the pancreatic duodenal portion of Z madascariensis, the insulin-, glucagon-, PP-, and somatostatin 14-IR cells were present as single elements or grouped in endocrine islets showing a typical topographical distribution. By double immunohistochemical techniques, chromogranin-SPI was found co-localized with the serotonin- and somatostatin-immunoreactivity, but CCK-IR cells were always negative to chromogranin-SP1 antiserum. The present work demonstrates that the chromogranin antiserum is not useful for identifying all the gut endocrine cell types; furthermore, the presence of insulin-immunoreactivity in the endocrine cells is confirmed, and, for the first time, insulin-immunoreactivity is shown in reptilian gut nerve fibres.

The presence, distribution, and interrelationships of the four typical pancreatic islet hormones were investigated in the digestive system of Protopterus annectens by single and double immunohistochemical methods. Insulin-, glucagon-, and somatostatin-immunoreactive (IR) elements were detected in both the pancreas and the gut. Pancreatic polypeptide (PP)-IR endocrine cells were always present in the gut, but were only present in the pancreas of a few specimens. Some of the latter cells also seemed to react with glucagon antiserum. In the pancreas the immunopositive cells were organized into islets of different sizes, and their organizations were studied by the double immunohistochemical techniques. In the few large islets insulin-IR cells were present in the central zone, glucagon- and PP-IR cells at the periphery, and somatostatin-IR cells intermingled with both the peripheral and the central endocrine cells. In the smaller islets, the number and the staining intensity of glucagon- and PP-IR endocrine cells varied markedly. In the gut, insulin-, somatostatin-, and PP-IR cells were of the open type; glucagon-containing cells were very few and had no luminal contact. They were differently distributed along the intestinal epithelium. Somatostatin-IR nerve fibers and somatostatin-IR neuron cell bodies were also observed in the intestinal wall. The organization of pancreatic endocrine cells in P. annectens is similar to that observed in the majority of teleosts even if a different topographical association can be found. Furthermore, islets of different sizes seem to display a different metabolic turnover, and the detection of pancreatic PP-immunoreactivity varied according to the specimens utilized. In the intestinal portion insulin-IR cells, in addition to PP-, glucagon- and somatostatin-IR cells are present: this suggests that intestinal insulin-like immunoreactivity may be more widespread than previously supposed.

The presence and distribution of eleven different types of immunoreactive endocrine cells and nine types of immunoreactive nerve elements were immunohistochemically identified in the gut and pancreas of the italian cave salamander, Hydromantes ambrosii. The majority of gastrointestinal endocrine cells were of open-type, often presenting basal cytoplasmic processes. Gastrin- and substance P-immunoreactive cells in the fundus and bombesin-immunoreactive cells in the intestinal portion were instead of closed type. Immunoreactive nerve fibres were particularly numerous in the muscular layers and blood vessel wall; bombesin- and substance P-immunoreactive nerve fibres were also abundant beneath gastro-intestinal epithelium. Besides substance P-, caerulein- and cholecystokinin-immunoreactive nerve fibres, all the other immunopositive nerve fibres seemed to be of intrinsic types. By the use of four different gastrin/cholecystokinin antisera three variously distributed subpopulations of endocrine cells and nerve elements were detected. Most of the pancreatic endocrine cells were organised in chord-like islets, polarized in the direction of blood vessels. A sparse network of bombesin-immunoreactive fibres was also found in the pancreas. The distribution of bombesin- and of the gastrin/cholecystokinin-immunoreactive material in the stomach and the presence of closed type endocrine cells indicate a more evoluted organization of the gastroenteropancreatic neuroendocrine system thus confirming the position of Hydromantes ambrosii among the higher urodeles.

The ultrastructural localization of glucagon in the presence of Scyliorhinus canicula was investigated. We used a post-embedding immunoelectron microscopy method on pancreatic samples fixed in glutaraldehyde and osmicated before embedding. Contrasting with uranyl acetate and lead citrate was also performed after immunolabelling, but best results were obtained with uranyl acetate only. Glucagon-like immunoreactivity was located in round granules (300-600 nm) surrounded by a limiting membrane. The matrix varied in electron density and exhibited a dense core surrounded by a less dense mantle. The granules were seen in two different cell types, which differed in the electron density of their cytoplasm. Glucagon-immunoreactive cells were the largest pancreatic cells types and were often localized near somatostatin-containing cells.

The presence of "embryonic" acetylcholinesterase activity, as described by Drews (1975) was investigated during early chick embryonic development, mainly in the following systems: a) primitive streak and Hensen's node during gastrulation movements; b) area opaca during blood islets and vessels differentiation; c) mesoderma of lateral laminae, during delamination movements. The demonstration of enzymic activity was performed with slightly modified histochemical methods. The enzyme was thus localized around the nuclei, in the cytoplasm and associated to plasma membrane of cells engaged in morphogenetic movements. The enzyme activity localized at the plasma membrane was supposed to be involved in the regulation of membrane functions concerning intercellular communications, such as inductive message, perhaps mediated by ion fluxes.

The presence of different kinds of cells in the gastric tubular glands of the Elasmobranchian Cartilaginous fish "Raja asterias" was investigated by histochemical and ultrastructural methods. Our study demonstrates that the gastric glands, unlike those of the other non-Mammalian Vertebrates, contain in the distal portion, near the neck zone, the oxyntic-like cells, and in the proximal portion the zymogenic-like cells. The former presents histochemical and ultrastructural patterns similar to the Mammalian oxyntic cells, the latter to the Mammalian peptic cells. Between these two types there are always cells with intermediate aspects. These features support the presence of two different stages in the secretory activity: at first the gastric tubular cell is implicated in the secretion of hydrochloric acid, later on, in the secretion of pepsinogen.

The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.

Using histological and histochemical methods for the identification of biogenic amins, phenolic and indolic groups, and of substances with a proteine and carbohydrate nature, it has been possible to identify at least five types of endocrine cells in the gastric mucose of Scyliorhinus stellaris and Scyliorhinus canicula. One of these, a type I cell, localised mainly in the piloric portion, shows morphological and histochemical characteristics which are perfectly comparable with those of the enterochromaffin type found in the gastro-intestinal tract of mammals. On the other hand, the four remaining cellular types are more difficult to define; three of these latter are present solely in the pyloric mucose, i.e., (a) type II cells with slightly fluorescent and metachromatic granules, with a positive reaction to the Grimelius test and to proteine test, and completely negative and argentaffin methods, (b) type III cells with non-fluorescent granules, but metachromatic and postive to argentaffin methods, and (c) type IV cells with slightly (argyrophilic) granules, strongly metachromatic and positive to the PAS test and to the tryptophan test, respectively. Types II and IV have been hypothetically correlated with the G and D cells, respectively, of mammals. The fifth cellular type, present solely in the central portion of the stomach has exclusively argyrophilic granules. This latter cellular type is considered similar to the ECL type, mammalian cell. No hypothesis, however, has yet been ventured with respect to the cells belonging to the third group.

The ontogeny and distribution of glucagon-like cells were studied in the gastrointestinal tract of embryos, neonates, and adults of the cartilaginous fish Scyliorhinus stellaris (L.) by immunocytochemistry. The results indicate that they appear early during embryonic development, and, in some portion of the gastrointestinal tract, even before the mucosa morphological differentiation. Immunoreactive glucagon-like cells were observed both in gastric and intestinal epithelium, being present in the pyloric portion only at a particular period of its differentiation. Some differences were observed between the embryonic and adult distributive pattern. They were more numerous in proliferative zone and sometimes were situated near other endocrine epithelial cells. These findings together with available information on trophic effects of some gastrointestinal hormonal peptides suggest a possible regulatory role of this peptide on the growth and differentiation of the gastrointestinal tract.