Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog,Rana catesbeiana,demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.     

An immunohistochemical study of the pancreatic endocrine cells of the nude mouse, Balb/c-nu/nu

The regional distribution and frequency of the pancreatic endocrine cells in the nude mouse, Balb/c-nu/nu were studied by immunohistochemical (peroxidase anti-peroxidase; PAP) methods using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas of the mouse was divided into two lobes, the splenic and duodenal lobes, and each lobe was subdivided into three regions, the pancreatic islets (central and peripheral regions), the exocrine region and the pancreatic duct region (consisting of duct epithelium and surrounding connective tissue--sub-epithelial connective tissue). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions compared to those of the duodenal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion as compared to those of the splenic portion. In the exocrine parenchyma of the splenic lobe, only insulin-, glucagon- and somatostatin-IR cells were detected.. Here, the insulin- and glucagon-IR cells formed cell clusters, while somatostatin-IR cells were present as solitary cells. In the exocrine region of the duodenal portion, only insulin-, somatostatin- and hPP-IR cells were observed, with the same distributional pattern as that found in the splenic lobe. However, clusters of cells consisting only of hPP-IR cells were distributed in the pancreas parenchyma as small islets. In the pancreatic duct region, only solitary hPP-IR cells were demonstrated in the sub-epithelial connective tissue regions of the splenic portion. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells, especially of the hPP-IR cells, were found in the nude mouse. In addition, somewhat different distributional patterns were found between the two pancreatic lobes.     

Distribution and frequency of endocrine cells in the pancreas of the ddY mouse: an immunohistochemical study

The regional distribution and frequency of pancreatic endocrine cells in ddY mice were studied by an immunohistochemical (peroxidase anti-peroxidase; PAP) method using four types of specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central portion. Most of glucagon- and somatostatin-IR cells were observed in peripheral regions although a somewhat smaller number of cells were also located in the central regions. HPP-IR cells were randomly distributed throughout the entire islets. In the exocrine pancreas, insulin-, glucagon-, somatostatin- and hPP-IR cells were detected; they occurred mainly among the exocrine parenchyma as solitary cells. Cell clusters consisted of only insulin- or only glucagon-IR cells and were distributed in the pancreas parenchyma as small islets. In addition, insulin- and glucagon-IR cells were also demonstrated in the pancreatic duct regions. Insulin-IR cells were located in the epithelium and sub-epithelial connective tissue regions as solitary cells and/or clusters (3-4 cells), and glucagon-IR cells were mainly located in the epithelium as solitary cells. Overall, there were 63.89+/-5.39% insulin-, 26.52+/-3.55% glucagon-, 7.25+/-2.83% somatostatin- and 1.90+/-0.58% hPP-IR cells. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddY mouse.     

An immunohistochemical study of pancreatic endocrine cells in SKH-1 hairless mice

The regional distribution and frequency of the pancreatic endocrine cells in the SKH-1 hairless mouse were studied by an immunohistochemical (peroxidase anti-peroxidase; PAP) method using four types of specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (PP). The pancreas of mice were divided into three portions; pancreatic islets, exocrine and pancreatic ducts. The pancreatic islets were further subdivided into three regions (central, mantle and peripheral region) according to their located types of immunoreactive cells. In the pancreatic islet portions, insulin-immunoreactive cells were located in the central and mantle regions with 84.60 +/- 7.65 and 33.00 +/- 12.45/100 cells frequencies, respectively, but most of somatostatin-, glucagon- and PP-immunoreactive cells were detected in the mantle and peripheral regions. In the mantle region, somatostatin-, glucagon- and PP-immunoreactive cells were demonstrated with 28.70 +/- 9.91, 52.00 +/- 14.05 and 2.60 +/- 1.51/100 cells frequencies, respectively, and showed 6.20 +/- 2.86, 15.30 +/- 5.31 and 21.50 +/- 10.28/100 cells frequencies, respectively in peripheral regions. However, glucagon-immunoreactive cells were also demonstrated in the central regions with 4.00 +/- 2.83/100 cells frequency. In the exocrine portions, insulin-, glucagon-, somatostatin- and PP-immunoreactive cells were demonstrated in the SKH-1 mouse with 0.90 +/- 0.74, 0.80 +/- 0.79,4.90 +/- 3.54 and 2.70 +/- 1.34/100 cells frequencies, respectively. In the pancreatic duct portions, insulin-, glucagon- and somatostatin-immunoreactive cells were demonstrated in the subepithelial connective tissues and showed islet-like appearances with 30.30 +/- 14.67, 2.70 +/- 3.13 and 5.90 +/- 4.23/100 cells frequencies, respectively. However, no PP-immunoreactive cells were demonstrated in these regions. In conclusion, some peculiar distributional patterns of pancreatic endocrine cells were found in the SKH-1 hairless mouse.     

Immunohistochemical identification of pancreatic hormones,neuropeptides and cytoskeletal proteins in pancreas of the camel (Camelus dromedarius)

The patterns of distribution of insulin (INS), glucagon (GLU), atrial natriuretic peptide (ANP), neuropeptide-Y (NPY), cholecystokinin-octapeptide (CCK-8), neurofilament-200 protein (NF), S-100 protein (S-100), and vimentin (VIM) in the pancreas of the one-humped camel (Camelus dromedarius) were investigated using immunohistochemical techniques. INS-immunoreactive cells were observed in the central and peripheral parts of the islets of Langerhans, but some solitary INS-positive cells were found outside the islets. INS-positive cells constituted 44.26–90.91% [mean ± standard deviation (std): 67.34 ± 14.20] of the total number of islet cells. GLU-immunopositive cells were located mainly in the peripheral region of the islets, and they constituted 11.43–44.44% [mean ± std: 23.54 ± 8.27] of the total number of islet cells. ANP and CCK-8 immunoreactivity was observed in neurons and perivascular nerves fibers. NPY was identified in pancreatic neurons and in some peripheral and central cells of the islets of Langerhans. VIM immunoreactivity was observed in the endothelial cells of blood vessels and the nerves located in the perivascular, interlobular and periacinar regions. VIM was also detected immunohistochemically in the periductal nerves of the pancreas. NF occurred only in nerves. S-100 was discerned mainly in the nerves of the interlobular connective tissue and in nerves lying close to blood vessels and acinar tissue. It is concluded that INS, GLU, ANP, NPY, CCK-8, NF, S-100, and VIM are well distributed in the pancreas of the camel. J. Morphol. 231:185–193, 1997. © 1997 Wiley-Liss, Inc.     

Natural purified porcine gastric inhibitory polypeptide (GIP) stimulates exocrine pancreas secretion due to contamination with a cholecystokinin-like substance

The effects of purified natural gastric inhibitory polypeptide-enterogastrone III (GIP-EG III) and a fraction which is further purified by high pressure liquid chromatography (GIP-HPLC) were investigated on the endocrine and exocrine isolated perfused pancreas of rats. At the dose of 5 ng/ml used for both GIP preparations, only GIP-EG III significantly stimulated volume and amylase secretion of the exocrine pancreas. The response of insulin release to stimulation by GIP-EG III or GIP-HPLC was not significantly different. In the presence of cholecystokinin-octapeptide (CCK-8) at a concentration which gave half-maximal stimulation of amylase secretion, GIP-EG III almost doubled the response of the exocrine pancreas, whereas GIP-HPLC had no additional effect. CCK-8 alone significantly increased total insulin output under hyperglycemic conditions. We conclude that porcine GIP purified by gel chromatography contains a CCK-like substance which can be removed by further purification on high pressure liquid chromatography without affecting the insulinotropic activity. Some of the reported effects of GIP could be due to contamination.     

An immunohistochemical study of the pancreatic endocrine cells of the ddN mouse

The regional distribution and frequency of the pancreatic endocrine cells in the ddN mouse were studied using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions as compared with the duo-denal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion. In the exocrine parenchyma of the splenic lobe, only insulin- and glucagon-IR cells were detected but all four kinds of IR cells were observed in the duodenal portion. In addition, insulin and hPP-IR cells were also demonstrated in the pancreatic duct regions. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddN mouse with somewhat different distributional patterns between the two pancreatic lobes.     

Localization of GABA high-affinity binding sites in the pancreas of neonatal rat

Summary The localization of gamma-aminobutyric acid (GABA) high-affinity binding sites was investigated in the exocrine and endocrine pancreas of neonatal rats by means of ³H-GABA autoradiography. GABA-binding was identified on Schwann cells and on the cells of the intralobular excretory ducts. In the endocrine part of the pancreas, no labelling was observed except in peripheral islet cells which, on the basis of their scarcity and distribution, could be somatostatin cells. Furthermore, peri-insular innervation showed considerable labelling.     

Distribution of microtubules and microfilaments in exocrine (Ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (Cells of the adenohypophysis and islets of Langerhans)

Summary We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against -tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against -tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogeneously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.     

Distribution of neuropeptides in the porcine stellate ganglion

The localization and distribution of neuropeptides including neuropeptide Y (NPY), [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P and somatostatin (SOM) were analyzed in the stellate ganglion of the pig by use of the indirect immunofluorescence technique. NPY, MEAGL, SOM, VIP and CGRP immunoreactivities were found to exist in subpopulations of neuronal cell bodies of the stellate ganglion. A population of the small intensely fluorescent (SIF) cells showed MEAGL immunoreactivity. In addition, the presence of NPY-, MEAGL-, CGRP-, SP-, SOM- and VIP-immunoreactive nerve fibers and axonal varicosities were observed in the stellate ganglion. The localization and pattern of distribution of these peptides in the porcine stellate ganglion were compared with studies carried out on stellate ganglia of other mammalian species.     

The Leu 7+ cell population of the human pancreas: preferential distribution and morphology

Anti-Leu 7 is a monoclonal antibody which recognizes an antigenic determinant present on the surface of a subset of human large granular lymphocytes and on central and peripheral neural elements. Furthermore it cross-reacts with an intracellular protein of secretory granule matrix in neuroendocrine cells. The presence of Leu 7+ cells has been studied in lymphoid and non-hemopoietic organs. We have analyzed the Leu 7 positivity in six pancreata from cadaver donors by means of immunocytochemical methods. Leu 7+ cells were found to be also present in the exocrine portion of the organ in which they represent a nonhomogeneous cellular population. In fact, two different types of Leu 7+ elements populate the exocrine pancreas: a, Leu 7+ cells showing an intracellular granule positivity; b, Leu 7+ cells showing surface positivity. The endocrine pancreas, in contrast, contains the majority (85%) of Leu 7+ elements, belonging to the intracellular positive type only.     

Phylogenetic study of serotonin-immunoreactive structures in the pancreas of various vertebrates

Summary The distribution pattern of serotonin (5HT) in the pancreas was studied immunohistochemically by using a 5HT monoclonal antibody in various vertebrates including the eel, bullfrog, South African clawed toad, turtle, chicken, mouse, rat, guinea-pig, cat, dog and human. In all species examined, except the bullfrog, 5HT-like immunoreactivity was observed in nerve fibers, in endocrine cells, or in both. Positive nerve fibers were found in the eel, turtle, mouse, rat and guinea-pig. These fibers ran mainly along the blood vessels and partly through the gap between the exocrine glands. In the eel and guinea-pig, positive fibers invaded the pancreatic islet. Occasionally, these positive fibers were found adjacent to the surface of both exocrine and endocrine cells, suggesting a regulatory role of 5HT in pancreatic function. 5HT-positive endocrine cells were observed in the pancreas of all species except for the bullfrog and rat. In the eel and in mammals such as the mouse, guinea-pig, cat, dog and human, 5HT-positive cells were mainly observed within the pancreatic islet. In the South African clawed toad, turtle and chicken, the positive cells were mainly in the exocrine region. The present study indicates that the distribution patterns of 5HT in the pancreas varies considerably among different species.     

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.     

Mammalian pancreas development: Regeneration and differentiation in vitro

Studies of the temporal sequence of mammalian pancreas development have demonstrated the existence of a protodifferentiated state that is characterized by low constant levels of amylase specific activity. In order to investigate this state further, guts from 9-day mouse embryos were cultured for 3 days, during which a protodifferentiated dorsal pancreas formed, consisting of a neck region or duct that extended from the intestinal wall and terminated in a large bulb of tissue. The pancreas bulb was then removed, leaving only the duct. This day corresponded to Day 12 of gestation. During 5 days of additional culture, 37 of 43 ducts regenerated a pancreas with an amylase specific activity equal to that of control pancreas (2.4 μg of maltose hydrate released/min/μg of protein at 37°C). Guts placed in culture at 12 days, with removal of the pancreas the same day, gave similar results. Other combinations of starting embryonic age plus culture time, prior to pancreas removal, totaling 12 or 13 days also underwent regeneration. Only of 18 pancreases regenerated from ducts equivalent to 14 days of gestation. Removal of the stomach, the intestine, and the ventral pancreas did not affect the ability of the dorsal pancreas to regenerate. However, removal of the duct at the presumptive intestinal wall did not result in regeneration. Regenerated pancreas differentiation was further confirmed by electron microscopic demonstration of zymogen granules in exocrine cells and of at least two types of secretory granules in endocrine cells. The results demonstrate that the protodifferentiated duct can regenerate a new pancreas including both exocrine and endocrine tissue, and that the regenerated pancreas is not retarded in its development when compared with the control pancreas.     

The distribution and frequency of endocrine cells in the splenic lobe of grass lizard (Takydromus wolteri). An immunohistochemical study

The regional distribution and frequency of the pancreatic endocrine cells in the splenic lobe of grass lizard, Takydromus wolteri, were studied by immunohistochemical (PAP) method using six types of specific mammalian antisera against bovine Sp-1/chromogranin (bCG), serotonin, insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas was subdivided into two regions--islet kike and exocrine regions. The frequency of each immunoreactive (IR) endocrine cells was calculated as mean number/total 100 islet cells and as mean number/total 1,000 cells (including exocrine and endocrine cells) using automated image analysis process. In addition, the percentage of each IR cell was also calculated. All of six endocrine cells were demonstrated. They were dispersed in the whole pancreatic parenchyma between exocrine acinar cells, or they were also observed as islet like clusters. In islet-like regions, bCG-, insulin- and glucagon-IR cells were detected as one or two cell layer cords and they were located between this cell-cords with 14.30+/-5.62, 61.50+/-9.76 and 26.50+/-9.31/100 cells frequencies, respectively. However, somatostatin-IR cells were mainly located in the peripheral parts not in cell-cords with 12.40+/-4.86/100 cells, and no serotonin- and hPP-IR cells were demonstrated. In exocrine regions, all of bCG-, serotonin-, insulin-, glucagon-, somatostatin- and hPP-IR cells were detected and they occurred mainly among the exocrine parenchyma as solitary cells with 10.30+/-2.54, 0.80+/-0.63, 15.50+/-5.30, 5.80+/-2.66, 3.10+/-1.29 and 11.00+/-3.33/1000 cells frequencies, respectively. In addition, serotonin-IR cells were mainly located between epithelia and connective tissue of pancreatic duct. Overall, there were 0.58+/-0.49% serotonin-, 56.44+/-9.35% insulin-, 23.73+/-8.22% glucagon-, 11.28+/-3.03% somatostatin- and 7.97+/-2.02% hPP-IR cells.     

Immunoreactivity to glial cell line-derived neurotrophic factor and its receptors in the trout pancreas: a further endocrine-exocrine relationship?

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting the survival of several neuronal populations in the central, peripheral and autonomous nervous system. Outside the nervous system, GDNF functions as a morphogen in kidney development and regulates spermatogonial differentiation. GDNF exerts its roles by binding to glial cell line-derived neurotrophic factor receptor (GFR) a1, which forms a heterotetramic complex with rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor. In this study we report the presence of GDNF-, RET- and GFRa1-like immunoreactivity in the pancreas of juvenile trout. GDNF immunoreactivity was observed in the islet cells, while GFRa1- and RET- immunoreactivity was observed in the exocrine portion. These findings suggest a paracrine role of GDNF towards exocrine cells showing GDNF receptors GFRa1 and RET. The relationship could reflect physiological interactions, as previously indicated in mammalian pancreas, and/or a trophic role by endocrine cells on exocrine parenchyma, which shows a conspicuous increase during animal growth.     

Postnatal development of the pancreas in the opossum. Light microscopy.

The pancreas of the newborn opossum consists of a central region of forming islets surrounded by primitive tubules that end in proacinar cells. Paratubular buds, which are outgrowths from the tubular epithelium, characterize the newborn pancreas and eventually give rise to both exocrine and endocrine units. 4 days after birth, definite intralobular ducts, acini and centroacinar cells are observed. In addition to the central expanding islets (primary islets), endocrine cells are observed singly or in small groups in the ductal epithelium. The endocrine cells are believed to originate from the terminal cells of the ductal epithelium and, throughout the entire postnatal period, retain a close association with the exocrine epithelium. With the simultaneous proliferation of both endocrine and exocrine components from the ductal system, the majority of the islets observed at 24 days (5.0 cm) appear to be surrounded by a single layer of acinar cells. As acini develop and the ducts expand toward the periphery, this layer of acinar cells separates from the developing islets, the majority of which have become localized within the centers of lobules to form the secondary islets by the 10.0-cm stage (59 days). A marked development of lobules is observed by the 13.0-cm stage and the majority of acinar cells now are filled with zymogen granules. Acinar cells continue to proliferate late into the postnatal period and the majority of acini exhibit a tubular form in the juvenile and adult opossum.     

Distribution of microtubules and microfilaments in exocrine (ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (cells of the adenohypophysis and islets of Langerhans). The relationship between cytoskeletons and epithelial-cell polarity

We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against beta-tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against beta-tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogenously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.