Endocrine Cell Differentiation Research Articles

Gastrointestinal hormones in birds: morphological, chemical, and developmental aspects.

Historically, the enterochromaffin cell was the first endocrine cell type detected in avian gut; subsequently, a number of types of such cells were distinguished on the basis of the ultrastructural features of the secretory granules. More recently, immunocytochemical procedures have revealed somatostatin-, pancreatic polypeptide (PP)-, polypeptide YY-, glucagon-, secretin-, vasoactive intestinal peptide (VIP)-, gastrin-, cholecystokinin-, neurotensin-, bombesin-, substance P-, enkephalin-, motilin-, and FMRFamide-like immunoreactivity in avian gastrointestinal endocrine cells. Most endocrine cells are located in the antrum; there are a number in the proventriculus and small intestine but few in the gizzard, cecum, and rectum. Several avian gastroenteropancreatic hormones, including glucagon, VIP, secretin, bombesin, neurotensin, and PP, have been isolated and sequenced. They resemble the equivalent mammalian peptides in terms of molecular size but differ in amino acid composition and sequence; some (e.g., VIP) differ only in minor respects, others (e.g., secretin) more radically. Gastrointestinal endocrine cells appear late in development; available data indicate that few types are recognized by either immunocytochemistry or electron microscopy before 16 days of incubation. Experimental evidence has shown that at least the majority of gut endocrine cells are of endodermal origin and are not derived from the neural crest or neuroectoderm as earlier proposed. In early embryos, the progenitors of gastrointestinal endocrine cells are more widespread than are the differentiated cells in chicks at hatching. This, along with other observations, raises the question of factors that might influence the differentiation of gut endocrine cells.

Human medullary thyroid carcinoma in culture provides a model relating growth dynamics, endocrine cell differentiation, and tumor progression.

We used an unique model, human medullary thyroid carcinoma (MTC) in culture (the TT line), to study features of neuroendocrine-related biochemistry in relationship to growth, differentiation, and tumor progression. Tumor tissues from patients with virulent MTC contain a very heterogeneous distribution of cells staining for calcitonin (CT) and have a high ratio of intracellular L-dopa decarboxylase activity (DDC) to CT. We found, in a culture line of MTC derived from a patient with virulent disease, that the degree of the inverse relationship between DDC and CT and the heterogeneous cellular distribution of CT probably relate to the rate of cellular growth and the biochemical set of individual cell clones. During exponential growth of the parent TT cell line, intracellular DDC and CT varied. DDC increased by 70% and CT decreased by 40%. Single time-point measurements in 54 cell clones or highly enriched cell populations revealed a more dramatic variability for CT (15-fold) than for DDC (5-fold). During growth of the clones having the highest and lowest CT measurements, respectively, inverse dynamics between DDC and CT were again found. However, each clone maintained a distinct range of CT during the entire growth curve, with a 2- to 4-fold difference in CT between the two clones throughout. In the low producing CT clone, ratios between DDC and CT rose to greater than 1.0 during growth, a very high value found before this study only in MTC tissues from patients with virulent disease. Immunohistochemical staining for CT of parent cells and clones grown on embryonic chick skin revealed increased cellular heterogeneity for CT distribution during growth. The TT line provides a powerful tool to study neuroendocrine related biochemical events in relationship to growth, differentiation, and tumor progression in MTC. Our in vitro findings in the TT line well explain observations made previously in patients. We conclude that: (1) DDC, a neural property of MTC, is an early differentiation marker as compared to CT and that the differentiation status of MTC cells varies inversely with cell growth rate; and (2) in patients with MTC, the virulence of the tumor probably varies inversely with differentiation status. The inverse ratio of DDC to CT is probably determined in MTC by the proportion of rapidly growing cells and numbers of cell clones which have a poor ability for maturation.

Gastrointestinal hormones in birds: Morphological, chemical and developmental aspects

Historically, the enterochromaffin cell was the first endocrine cell type detected in avian gut; subsequently, a number of types of such cells were distinguished on the basis of the ultrastructural features of the secretory granules. More recently, immunocytochemical procedures have revealed somatostatin-, pancreatic polypeptide (PP)-, polypeptide YY-, glucagon-, secretin-, vasoactive intestinal peptide (VIP)-, gastrin-, cholecystokinin-, neurotensin-, bombesin-, substance P-, enkephalin-, motilin-, and FMRFamide-like immunoreactivity in avian gastrointestinal endocrine cells. Most endocrine cells are located in the antrum; there are a number in the proventriculus and small intestine but few in the gizzard, cecum, and rectum. Several avian gastroenteropancreatic hormones, including glucagon, VIP, secretin, bombesin, neurotensin, and PP, have been isolated and sequenced. They resemble the equivalent mammalian peptides in terms of molecular size but differ in amino acid composition and sequence; some (e.g., VIP) differ only in minor respects, others (e.g., secretin) more radically. Gastrointestinal endocrine cells appear late in development; available data indicate that few types are recognized by either immunocytochemistry or electron microscopy before 16 days of incubation. Experimental evidence has shown that at least the majority of gut endocrine cells are of endodermal origin and are not derived from the neural crest or neuroectoderm as earlier proposed. In early embryos, the progenitors of gastrointestinal endocrine cells are more widespread than are the differentiated cells in chicks at hatching. This, along with other observations, raises the question of factors that might influence the differentiation of gut endocrine cells.

Preparation of rat islet B-cell-enriched fractions by light-scatter flow cytometry.

Flow cytometry has been examined as a method to separate islet cells into homogeneous subpopulations. Collagenase-isolated rat islets were dissociated into single cells and these were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. Light scatter histograms showed two peaks of viable cells. Radioimmunoassay of hormone content in cell fractions collected across the the two peaks showed that glucagon-containing cells were concentrated towards the left side of the left peak and somatostatin-containing cells were concentrated towards the right side of the left peak, whereas insulin-containing cells were clearly enriched in the right peak. The B-cell-enriched fraction (90% B cells, 3% A cells, 2% D cells) exhibited significant insulin secretory responses to glucose (16.7 mM), and 3-isobutyl-1-methylxanthine (0.1 mM), during a 24-h culture period, and these responses were slightly greater than those observed in the original mixed islet cell preparation (66% B cells, 14% A cells, and 4% D cells). These results indicate that flow cytometry can be applied to sort pancreatic islet cells into populations enriched in specific endocrine cell types for further study of the functions of individual cell types.

An immunocytochemical study of the cytogenesis of pancreatic endocrine cells in the lizard, Anolis carolinensis.

The differentiation of the pancreatic endocrine cells in the lizard Anolis carolinensis following oviposition was examined. Immediately postoviposition (PO) there was no apparent differentiation of epithelioid cells into endocrine or exocrine components. Individual subpopulations of the endocrine-like cells, which could not be identified during the early PO period on the basis of either their tinctorial properties at the light-microscopic level or their granule morphologies at the electron-microscopic level, exhibited specific hormonal localization by peroxidase-antiperoxidase complex immunocytochemistry. All four hormones searched for, insulin, glucagon, somatostatin, and pancreatic polypeptide (PP), were present in epithelioid cells shortly after oviposition. However, the immunostained secretory granules in the early PO period were smaller than those of the adult. Secretory granule morphologies that are typical of the adult were acquired at different times during development. Delta granules were observed first and were followed by alpha granules, and beta granules which appeared shortly before birth. The secretory granules of the PP-containing F cells could not be readily placed within this maturation sequence. Mosaic cells (containing more than one hormone) were not seen. Levels of immunoreactive insulin and glucagon in the pancreas increased several fold from day 10 to day 28 PO, but the attainment of adult beta-granule morphologies did not appear to be directly related to insulin itself. The results show that cytodifferentiation of the anolian endocrine pancreas occurs postoviposition and that immunocytochemical methods can be used to follow an organelle sequence during development. These findings suggest that subcellular organelles undergo structural remodeling during maturation which, at least in the case of secretory granules, may have functional significance.

Ultrastructural and cytochemical studies on the cytodifferentiation of duodenal endocrine cells

The development of cytodifferentiation of endocrine cells that produce the gastrointestinal hormones gastrin, cholecystokinin and secretin have been studied by a combined fluorescence-cytochemical, immunocytochemical and ultrastructural approach. The results show that, during development, several ultrastructurally distinct cell types exhibit COOH-terminal gastrin and cholecystokinin immunoreactivity. Furthermore, some cells simultaneously contain both gastrin- and cholecystokinin-specific antigenic determinants. Studies on the time course of development of gastrin and cholecystokinin cells, together with the above-mentioned data, suggest that gastrin cells may be converted into cholecystokinin cells in development. During this period, gastrin, cholecystokinin and secretin cells store the biogenic monoamine, 5-hydroxytryptamine a feature not displayed by the adult counter-parts of these cells. In the adult duodenum, characteristic enterochromaffin (EC) cells store 5-hydroxytryptamin for which, evidence for a possible hormonal role has been presented. Taken together, our data indicate that the differentiation of duodenal endocrine cells occurs in distinct steps, each involving a restriction in the biosynthetic repertoire of the cell.

Sequential differentiation of intestinal endocrine cells in the fetal mouse.

The duodenum and ileum of 15 to 18-day old Swiss ICR mouse embryos were studied with the electron microscope to follow the differentiation of endocrine cells. Endocrine cells were classified on purely morphological characteristics. EC cells appeared at 16 days of gestation in both segments. At the same stage G cells were seen in the duodenum and K cells in the ileum. ECL, and S cells were identified in the duodenum at 17 days. Finally, D cells were seen at 18 days of gestation in the duodenum and ileum. With the electron microscope, endocrine cells were not identified in the small intestine of the mouse before the formation of villi.

ISOLATION OF HIGHLY PURIFIED LEYDIG CELLS BY DENSITY GRADIENT CENTRIFUGATION

Purification of specific endocrine cells from mixed populations after dispersion of target tissues is important for detailed analysis of mechanisms of hormone action. A simple method for rapid isolation of endocrine cells with retention of biological integrity, has been developed by centrifugation in density gradients formed with Metrizamide. By this procedure, highly purified Leydig cells retaining morphological and biochemical characteristics were obtained. Such preparations bound 20, 300+/-3, 100 molecules of hCG per cell with affinity of 1.1+/-0.25 X 10(10) M-1. During incubation with hCG, cyclic AMP and testosterone responses of purified Leydig cells were considerably increased, and hCG concentrations as low as 0.2 pM caused activation of cAMP-dependent protein kinase.

The endocrine cells of the pancreas and related tumours

Up to seven endocrine cell types have been identified ultrastructurally in the pancreas, including glucagon A cells, insulin B cells, somatostatin D cells, pancreatic peptide F cells and 5-hydroxytryptamine EC cells. In addition, D1 cells, which have been proposed as the cell type producing VIP and possible P cells of unknown function are seen. Various patterns of endocrine cell differentiation have been found in 20 endocrine pancreatic tumours. Well and poorly differentiated B cells have been identified in 6 insulinomas, diagnostic G cells in 3 out of 7 gastrinomas, D1 and/or F cells in 7 diarrheogenic tumours. Moreover, cells apparently unrelated to the prevalent clinical syndrome have been noted in 8 of the 20 tumours. Granular non diagnostic cells (poorly diagnostic gastrin cells? D1 cells?) were particularly frequent in gastrinomas; agranular or poorly granular cells, either by "active" or "Stem cell" type, were present in nearly all tumours, particularly in diarrheogenic tumours, gastrinomas and malignant insulinomas. A cytological classification of pancreatic endocrine tumours is proposed.

Immunohistochemical localization of hypothalamic hormones.

Using an immunoperoxidase technique at the light and electron microscope levels, the localization of somatostatin and luteinizing hormone-releasing hormone (LH-RH) has been performed in the brain, as well as the pancreas and stomach. The following generalizations may be drawn on the localization of LH-RH and somatostatin. (a) LH-RH and somatostatin are contained in axons and nerve endings of the median eminence and organum vasculosum of the lamina terminalis. (b) LH-RH and somatostatin are present in different nerve endings. (c) In the subcommissural organ, subfornical organ and area postrema, the two neurohormones are present in the cytoplasm of ependyman and subependymal cells. (d) In the endocrine pancreas and gastric mucosa, somatostatin is localized within the secretory granules of specific endocrine cells.