Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of amphibians.

The occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretogranin II (SgII)-fragments (termed C23-3 and C26-3) has been investigated immunohistochemically in the endocrine pancreas of five amphibian species. Immunoreactivity for CgA was detected only in specimens of the genus Rana, whereas for SgII it was found in all the urodeles and anurans studied. Either CgA or the SgII-fragment displayed its own cellular distribution patterns in the endocrine pancreas of a given species. Moreover, immunoreactivity for both regions (C23-3 and C26-3) of the SgII-molecule exhibited by the same endocrine cell population have been encountered in newt and frog organs. Besides the interspecific heterogeneous distribution of CgA and of the two SgII-fragments in relation to the insular cell types, a striking heterogeneity of their immunostaining density among the endocrine cells of the same type was also revealed. The above findings entirely support the concept of a good conservation of granins during phylogeny; they do not support, however, the previously ascribed usefulness of these anionic glycoproteins as markers for all neuro-endocrine cells.

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of reptiles.

The endocrine pancreas of four reptile species belonging to the turtles, lizards and snakes was investigated immunohistochemically for the occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretonin II (SgII)-peptides (C23-3 and C26-3). CgA-immunoreactivity was found only in the turtle pancreas, whereas that for SGIIC23-3 appeared both in the turtle and snake. None of the species studied displayed immunoreactivity for SgIIC26-3. The two detected granins showed different distributions in relation to the endocrine cell types. Conspicuous variations of the immunostaining density for either granin in the same endocrine cell population and even complete lack of the immunoreaction were recorded. The findings suggest that, despite the restricted presence in the endocrine pancreas of the reptiles investigated, the granins are relatively well conserved during phylogeny; they do not confirm, however, the previously accepted usefulness of the granin protein family as common markers of neuroendocrine cells.

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the intestine of reptiles.

The occurrence, distribution and the possible cellular co-localizations of chromogranin A (CgA) and of two synthetic secretogranin II-peptides (SgIIC23-3 and SgIIC26-3) with several enteric neuropeptides and serotonin have been investigated immunohistochemically in turtles, lizards and snakes. The distribution of CgA-immunoreactivity was restricted only to the enteroendocrine cells in all the reptiles studied. SgII-immunoreactivity--absent in turtle--revealed nerve cells and fibers, besides enteroendocrine cells in lizard and snake guts. Moreover, the two antisera (C23-3 and C26-3) raised against the different regions of the SgII-molecule yielded distinct distribution patterns of immunoreactivity both in the lizard and snake organs. Small amounts of enteric serotonin cells co-stored CgA or SgIIC23-3 in lizards and snakes and only SgIIC26-3-peptide in snakes. CgA was found co-stored with somatostatin in a few enterocytes of the turtle duodenum. In the same gut segment of lizards and throughout the snake organ, neurotensin and the SgIIC23-3-peptide co-existed in a small number of endocrine cells. The pancreatic polypeptide-containing cells were devoid of immunoreactivity both for CgA and SgII. Bombesin immunopositive cells were absent throughout the intestines of the reptiles investigated. The above findings entirely support the heterogenous distribution of granins in neuroendocrine organs and tissues and also within the same neuroendocrine cell population. They further support the concept of a good conservation of granins during phylogeny.

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the intestine of amphibians.

The occurrence, distribution and possible cellular colocalizations of chromogranin A (CgA) and of two synthetic secretogranin II peptides (SgIIC23-3 and SgIIC26-3) with serotonin, somatostatin, neurotensin, pancreatic polypeptide and bombesin have been investigated immunohistochemically in the amphibian gut. CgA or SgIIC26-3-immunostained enterocytes were found throughout along the frog intestine, while no immunoreaction for any of the tested antisera against granins was seen in the same organ of newts. Variable amounts of serotonin-immunoreactive cells co-storing CgA or SgIIC26-3, but never both granins, were encountered in all intestinal segments of the frogs investigated. In addition, CgA was co-localized with somatostatin in a few endocrine cells of the frog (genus Rana) duodenum and small intestine. In the duodenum of another frog (genus Xenopus) several enterocytes co-stored SgIIC26-3 and neurotensin. Pancreatic polypeptide- and bombesin-immunoreactive cells, the latter detected only in the duodenum of Xenopus, did not contain and granin. The results suggest that, in spite of their relatively restricted occurrence in the intestine of frogs and even of their absence in that of newts, the granins are well conserved during phylogeny. On the other hand, the heterogeneous distributions of these anionic glycoproteins, related to the entero-endocrine cell types, make their previously assigned usefulness as markers of all neuro-endocrine cells unlikely.

Distribution and ultrastructure of somatostatin-immunoreactive cells in the pancreas of Rana esculenta.

Apart from a description of the general organization of the endocrine pancreas, the present study is focussed on the distribution and ultrastructural morphology of somatostatin-immunoreactive cells in the pancreas of the frog Rana esculenta. For light-microscopic histochemistry, the peroxidase-antiperoxidase technique was used. For the ultrastructural investigation, we employed the immunogold method. The endocrine pancreas of R. esculenta is composed of numerous islet-like structures, which contain several small somatostatin-immunoreactive cells arranged in the form of clusters. Often, however, single somatostatin cells are randomly distributed among the acinar tissue of the pancreas. These individually arranged elements possess long processes which terminate on exocrine pancreatic cells. The ultrastructural features of somatostatin-immunoreactive cells speak in favor of their endocrine and paracrine functions.

Somatostatin-immunoreactive cell in the gastrointestinal tract of the frog Rana esculenta.

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.

The entrapment of biological active substances into liposomes. II. Effects of oral administration of liposomally entrapped insulin in normal and alloxanized rats.

Insulin (I) was entrapped into liposomes of various lipid compositions. After oral administration, some of the normal rats treated with neutral liposomes (c) and (d) having egg yolk lecithin, prepared with 2 or 4 mg I/ml respectively, showed a decrease in blood glucose at 2 and 4 hrs. In alloxanized rats (75 mg alloxan/kg body weight) the positive liposomes (f) have induced in some animals a decrease in blood glucose at both time intervals after oral treatment. When neutral liposomes (c) were administered to diabetic rats (125 mg alloxan/kg body) the blood glucose decreased in 9 of 12 animals from 391 to 125 mg/100 ml blood, at 3 hrs after oral treatment. At 1 h, the effect had appeared in 3 of 14 animals. The neutral liposomes (g) having synthetic lecithin, induced a decrease in blood glucose in 5 of 10 animals. A good correlation was observed between decreased glucose and increased insulin levels with the same variability throughout the treated group. No increased insulin levels were observed in diabetic control and normal rats. By the RIA method, the insulin entrapment was 8%. Histological studies have shown that at 35 min after oral administration, neutral liposomes (c) penetrate deeply into the intestinal wall of alloxanized rats as compared both to the neutral liposomes with synthetic lecithin (g) or to the positive ones (f).

Lipovitellin-phosvitin crystals with orthorhombic features: thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates.

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.

Microperoxisomes and catalase peroxidatic activity in the pancreas of two amphibian species (Salamandra salamandra L. and Rana esculenta L.).

The fine structure of microperoxisomes (anucleoid peroxisomes) and catalase peroxidatic activity in salamander and frog pancreas have been investigated following glutaraldehyde fixation and incubation in a modified NOVIKOFF-GOLDFISCHER alkaline DAB-medium. Reactive microperoxisomes ranging in diameter from 0.16 to 0.76 micrometer were identified in both exocrine (acinar and ductular centro-acinar cells) and insular (B-, A-, D-cells) pancreatic tissue, as well as in the nerve fibers and endings. Their incidence was considerably higher in the exocrine parenchyma, where sometimes in frog they accumulated in clusters. Individual microperoxisomes showed close spatial relationship and occasionally membrane continuities with smooth surfaced endoplasmic reticulum and zymogen granules. They were also localized in the proximity or in direct contact with the nuclear envelope, mitochondria, GOLGI condensing vacuoles, lipid inclusions and plasma membrane. The morphological findings support the microperoxisomes origin from the agranular endoplasmic reticulum and their possible discharge into the extracellular spaces through an exocytosis-like mechanism. The cytochemical observations are discussed in relation with the concepts on catalase biosynthesis and peroxisomes functional significance.

The effects of short-term exposure to crowding stress on the pancreatic islets morphology and glycemia in mice.

Behavior, pancreatic islets morphology and plasma glucose levels of male mice exposed for 2, 4, 24 and 48 hours to crowding stress were investigated. The crowding induced an intense turmoil state associated with enhanced irritability and aggressiveness among the specimens of all experimental groups. Violent fights occurred especially in the first 4--6 hours, generally with the death of 1--2 individuals from each group. The changes recorded in the pancreatic islets affected first (2 hours) exclusively the insulin-producing cells, and in subsequent intervals they progressively expanded over all cell types. The changes occurred during the experiment in all islet cell types; however the B-cells showed by far the most pronounced alterations irrespective of the studied time interval. Most changes suggested the stimulation of the entire gland secretory activity, but particularly of B-cells, which was also proved by low glycemia values recorded at 3 of the 4 crowding time intervals. On the other hand, some alterations, occurring first at 24 hours, were regarded as signs of a moderate B-cells secretory hypoactivity; they may partly support the slight hyperglycemia obtained at this time interval. The significance of the above short-term observations in the induction of glycoregulation disturbances, diabetes included, as well as the presumably mediation role of adrenal-cortex and -medulla hormones under stress conditions are discussed and correlated with findings reported in literature.

The effects of crowding stress on the pancreatic islets morphology in golden hamsters.

Behavior and the pancreatic islets morphology of golden hamsters exposed for different periods (24 h, 15 and 30 d) to crowding stress were investigated. The crowding induced an intensive turmoil and enhanced irritability and aggressiveness, particularly among the female specimens. Within a week, a rank hierarchy seemed to establish and therefore, later the fighting incidence was more reduced. Marked morphological alterations were recorded in the endocrine pancreas, affecting in various degrees all cell types, but especially the insulin-producing cells. The B-cells showed increased sizes, higher incidence of mitotic divisions, a drastical reduction of secretory granules amounts, enlargement of GOLGI complexes, mitochondria swelling and extension of the ergastoplasm. On the other hand, after 30 d, part of these cells displayed pycnotic nuclei, large cytoplasmic vacuoles and increased number of lasosomes and lipid inclusions. Due to the B-cell hyperplasia, the relative number of both A1- and A2-cells per islet occurred diminished and their typical localization modified. The islets of 15 and 30 d crowded specimens showed enlarged sinusoids and clear peri-insular spaces. The above morphological modifications, which suggest as a whole the global stimulation of gland secretory activity, are presented in relation with other authors findings. The presumably involvements of adrenal-cortex and -medulla hormones in the mediation of stress-induced glycemic and pancreatic alterations are discussed.

Electron microscopic localization of Mg2+ -dependent adenosine triphosphatase activity in the amphibian pancreas (Salamandra salamandra L. and Rana esculenta L.).

The ultrastructural distribution of Mg2+ -dependent adenosine triphosphatase (ATPase) activity has been investigated in the salamander and frog pancreas by using glutaraldehyde fixations and a modified Wachstein-Meisel reaction medium. In both species the reaction product (lead phosphate) was found associated with the plasma membrane external side of all islet cell types (B-, A- and D-cells) and of acinar and ductular/centro-acinar cells. Except the apical pole of salamander acinar and centro-acinar cells, usually devoid of reaction, no preferential distribution of enzyme activity depending on endocrine or exocrine cell aspects could be observed. Other specific enzyme localizations included the mitochondria matrices, nucleoles, condensed nuclear chromatin, periaxolemmal spaces in nerve bundles and sometimes the cleft of neuro-glandular junctions. The occurrence of reaction deposits in connective tissue, in the cytoplasm of both islet and exocrine cells and in the nerve fiber axoplasm was considered as a possible diffusion artifact. The reaction intensity, but not its distribution, varied sensibly with the incubation period. 2-iodoacetamide and p-chlormercuribenzoic acid decreased the amount of reaction deposits at the level of all reactive sites and especially in mitochondria. The specificity of Mg2+ -ATPase demonstration in this paper is analysed taking into account several inherent shortcomings of the Wachstein-Meisel incubation medium and of the fixative. The different enzyme localizations, as well as their functional significances are discussed in relation with the findings of other authors.

The effects of increased Waler salinity on the fine structure and acid phosphatase activity in frog pancreas (Rana temporaria L.).

Morphological alterations and AcPase activity in frog pancreas under the influence of high salinity water were investigated by electron microscopy. The 1% increase in the sodium chloride concentration of the water in which the animals were kept induced severe degranulation of all islet cell types and the stimulation of the autophagocytosis process. The latter was particularly obvious in the acinar cells, in which the occurrence of various morphological types of lysosomes was recorded. Other involutional changes in both endocrine and exocrine pancreas included lipid accumulation, mitochondria shrinkage, nuclear pycnosis and plasmalemma lysis. The ultrastructural modifications, mainly ascribed to the disturbance of the ionic balance of the body fluid, were accompanied by a general increase of the histochemically demonstrable AcPase activity. The enzyme was exclusively detected in lysosomes, GOLGI complex, and in different types of islet secretory granules. Several hypotheses concerning the functional significance of the enzym distribution are discussed.

Fine structural localization of glucose-6-phosphatase activity in the pancreatic islets of two amphibian species (Salamandra salamandra L. and Rana esculenta L.).

The ultrastructural distribution of glucose-6-phosphatase activity has been investigated in the salamander and frog pancreas. In the pancreas of salamander the enzyme was located in the A-cells, while in frog it occurred in all main types of islet cells B-, A-, and D-cells). As a rule, the reaction intensity was higher in the frog islet cells. No reaction was recorded in the exocrine pancreatic tissue of both species. The glucose-6-phosphatase activity was constantly detected in the lumen of rough endoplasmic reticulum and between the nuclear envelopes. Other enzyme localizations, observed especially in the A-cells of the salamander pancreas, were considered possible diffusion artifacts ro remnants of other phosphatase activity. The enzyme distribution in different types of islet cells, as well as its functional significance are discussed in relation to the findings of other authors.

Ultrastructural localization of adenyl cyclase activity in the pancreas of two amphibian species (Salamandra salamandra L. and Rana esculenta L.).

The distribution of adenyl cyclase activity in the pancreas of one species of urodeles and anura has been investigated using electron microscopy. In both islet and acinar tissues the reaction product was located at the outer cell surfaces, while in nerve bundles it occurred in the cleft between axolemma and Schwann cell-extensions. The functional signification of the enzyme localization is discussed in relation to the findings of other authors.

The terminal autonomic nervous system in the pancreas of newts. Electron microscopic investigations using acetylcholinesterase and zinc iodide-osmium tetroxide reactions.

The intrinsic innervation in the pancreas of newts Triturus vulgaris, Tr. montandoni and Notophthalmus. All the investigated species display: cholinergic innervation better developed than adrenergic, no qualitative differences regarding the fine structure of nerve fibers and terminals as compared to that previously described in the pancreas or in other glands, axo-axonal contacts. Their functional signification as peripheric synapses is suggested. Frequently neuro-glandular junctions with or without membrane specializations. The occurrence of the latter is discussed in connection with peripheral transmission of nervous impulse.

Immunohistological demonstration of insulin and glucagon in islet tissue of reptiles, amphibians and teleosts using epoxy-embedded material and antiporcine hormone sera.

Islet tissue from 2 reptilian, 5 amphibian and 7 teleost species was fixed in glutaraldehyde, embedded in epoxy-resin, stored for up to 3 years, cut in ultrathin sections and stained with the indirect immunofluorescent technique for insulin and glucagon following removal of the plastic. The antisera were directed against porcine insulin and glucagon, or FITC labeled and directed against the globulin fraction of the producer species. Positive results were obtained in species from all vertebrate classes investigated. Insulin demonstration in teleosts was particularly difficult.