Regional distribution of immunoreactive prolactin-releasing peptide in the human brain

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.     

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.     

Immunohistochemical study on the ontogenetic development of the regional distribution of peptide YY, pancreatic polypeptide, and glucagon-like peptide 1 endocrine cells in bovine gastrointestinal tract

The regional distribution and relative frequency of peptide YY (PYY)-, pancreatic polypeptide (PP)-, and glucagon-like peptide 1 (GLP-1)-immunoreactive (IR) cells were determined immunohistochemically in the gastrointestinal tract at seven ontogenetic stages in pre- and postnatal cattle. Different frequencies of PYY-, PP-, and GLP-1-IR cells were found in the intestines at all stages; they were not found in the esophagus and stomach. The frequencies varied depending on the intestinal segment and the developmental stage. The frequencies of PYY- and PP-IR cells were lower in the small intestine and increased from ileum to rectum, whereas GLP-1-IR cells were more numerous in duodenum and jejunum, decreased in ileum and cecum, and increased again in colon and rectum. The frequencies also varied according to pre- and postnatal stages. All three cell types were most numerous in fetus, and decreased in calf and adult groups, indicating that the frequencies of these three types of endocrine cells decrease with postnatal development. The results suggest that these changes vary depending on feeding habits and adaptation of growth, secretion, and motility of intestine at different ontogenetic stages of cattle.     

Distribution of enteric neural peptide YY in the dog gastrointestinal tract

Various regions of the dog gastrointestinal tract were investigated for the distribution of peptide YY (PYY) neurons using immunocytochemistry and radioimmunoassay. PYY neurons that encircled non-PYY-immunoreactive neurons were mainly observed in the myenteric plexus from the stomach to the colon. There was more PYY-like immunoreactivity in the muscle layer of the stomach and ileum than in the other intestines. The results of high performance liquid chromatography revealed that neural PYY-immunoreactive substance is identical to authentic PYY. PYY was not localized in the cholinergic neurons. These results indicate that PYY, as a neuropeptide, is involved in the regulation of gastrointestinal function.     

Identification and characterization of the emetic effects of peptide YY

Emesis was noted following intravenous bolus injections into dogs of a chromatographic subfraction derived from porcine small intestinal tissue extracts. The active agent was isolated from this subfraction using sequential ion-exchange and reverse-phase HPLC and demonstrated to be the recently identified regulatory peptide PYY. The threshold dose for PYY-induced emesis in the dog is less than 120 pmol/kg. Emesis was sometimes seen following large IV bolus doses of neuropeptide Y (NPY), but none was seen following IV injection of pancreatic polypeptide (PP). Dogs prepared with discrete, bilateral lesions of the area postrema were refractory to a suprathreshold emetic dose of PYY. PYY is the most potent, circulating emetic peptide identified to date.     

Regional distribution and plasma concentration of peptide YY in sheep

Peptide YY (PYY)-positive cells are distributed in the mucosa of the ileum, cecum, colon, and rectum of sheep, but not in other layers of these regions. By radioimmunoassay, mucosal content of PYY in the ovine large intestine was much less than that in the rat intestine. The plasma concentration of immunoreactive PYY did not significantly fluctuate over a 48-h period in conscious sheep, even after ingestion of roughage and concentrate. Intraluminal nutrients into the ileum and i.v. CCK8 also did not raise the plasma level of PYY. Therefore, PYY seems unlikely to play a role as "ileal brake" in sheep.     

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas

Summary In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit>guinea pigpig and cat> >dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4°C, but not at 37°C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproductible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.     

Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract

Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.     

Effects of peptide YY on intestinal blood flow distribution and motility in the dog

Previous investigators in our laboratory have demonstrated that peptide YY (PYY), a putative gut hormone, exerts a potent emetic effect when administered intravenously to conscious dogs. The current study was carried out to examine the effects of an emetic dose of PYY on cardiovascular status, splanchnic blood flow distribution (estimated using 15 micron microspheres) and small intestinal motility in anesthetized dogs. PYY, infused i.v. at a dose of 25 pmol/kg/min led to a localized significant reduction in small intestinal muscularis externa blood flow both 15 and 30 min after the start of PYY infusion in both jejunum and ileum. This decreased muscularis perfusion was not accompanied by any significant change in whole gut wall blood flow and was explained on the basis of an observed significant redistribution of blood flow away from the muscularis towards the mucosa/submucosa. Similar, although non-significant, effects of PYY on colonic blood flow distribution were also observed. Despite the effects on jejunum and ileum, PYY exerted minimal effects on duodenal blood flow. The decrease in ileal and jejunal muscularis blood flows was accompanied by a significant increase in the amplitude of intestinal contractions in these regions. Frequency of contractions was unaltered however. These results demonstrate that PYY infusion leads to concurrent changes in small intestinal blood flow and motility.     

Distribution and characterisation of immunoreactive somatostatin in human gastrointestinal tract

Immunoreactive somatostatin (IRS) was measured in acid extracts of human gastrointestinal tissue. The highest levels were found in the duodenum, pancreas, jejunum and stomach with lower levels in the ileum and colon. In the antrum, pylorus, duodenum and pancreas the main peak of IRS (1.6K IRS) coeluted with synthetic somatostatin-14 on both gel filtration chromatography and HPLC. In the body of stomach, jejunum, ileum and colon, a large peak coeluting with synthetic somatostatin-28 (3.5K IRS) on both chromatographic systems was also identified, while minor peaks of IRS assigned molecular weights of 6000 (6K) and greater than 15 000 (15K) were seen in some extracts. The total IRS content and pattern of molecular forms were similar in tissues obtained from adults at surgery or rapid post mortem, and in tissue taken from human fetuses after prostaglandin termination of pregnancy. When tissues were divided into mucosal and muscle layers, greater than 90% of the IRS was in the mucosa with less than 10% in the muscle layer. In the muscle layer the IRS was almost entirely the 1.6K form in all tissues. Immunohistochemical studies showed the IRS in the mucosa to be localised in endocrine-type cells, while in the muscle layer the IRS is present in nerve fibres and neurones of the myenteric plexus. It is suggested that (1) different mechanisms may control the biosynthesis of somatostatin-14 and somatostatin-28 in mucosal cells in different parts of the gut, (2) different biosynthetic controls may operate in endocrine-like and neuronal cells in the same region of the gut.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS

Antibodies directed against the C-terminus of cholecystokinin octapeptide (CCK8) and caerulein were used to study immunoreactive peptides in pig brain. One antibody, a mouse monoclonal raised to caerulein (c.MAb), reacts equally with heptadecapeptide gastrin (G17), CCK8 and caerulein, the other raised to CCK8 (L48) shows 10 times lower immunoreactivity with caerulein compared with G17 and CCK8. Extracts were purified by adsorption to alginic acid, gel filtration chromatography and reversed phase HPLC. In addition to material with the expected properties of CCK33, 39 and 58 a novel peptide was identified that reacted 10 times better with c.MAb compared with L48. This material emerged in a similar position to CCK58 on Sephadex G50 but had a greater retention time on reversed phase HPLC. It had CCK-like bioactivity and digestion with trypsin gave a fragment showing a pattern of immunoreactivity similar to that of the parent compound. This pattern of activity is distinct from other known mammalian CCKs; the material may represent an addition to the gastrin-CCK family in mammals.     

Ghrelin cell density in the gastrointestinal tracts of animal models of human diabetes

Ghrelin cell density in the gastrointestinal tract of animal models of human diabetes type 1 and 2 was investigated. The animals used were non-obese diabetic (NOD) mice and obese diabetic mice. Ghrelin cells were detected by immunohistochemistry and quantified by computerized image analysis. Ghrelin-immunoreactive cells were detected in all animals studied. They were abundant in the oxyntic mucosa, patchy and few in the duodenum and rare in the colon. The density of ghrelin-immunoreactive cells decreased in diabetic, pre-diabetic NOD mice and in obese diabetic mice as compared to controls, though not statistically significant. It was concluded that the reduced density of ghrelin-immunoreactive cells in animal models of human diabetes type 1 and 2 might explain the slow gastric emptying and slow intestinal transit found in diabetes gastroenteropathy.     

Distribution of obestatin and ghrelin in human tissues: immunoreactive cells in the gastrointestinal tract, pancreas, and mammary glands.

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immunoreactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.     

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.     

Immunocytochemical identification of polypeptide YY (PYY) cells in the human gastrointestinal tract

Various parts of the human gastrointestinal tract were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). PYY-immunoreactive cells were observed in the lower part of the ileum, in the colon and in the rectum, and PP-immunoreactive cells were found in the colon and rectum. Both cell types were of the open type, i.e. they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were seen to emit cytoplasmic processes to the neighbouring goblet cells. This latter observation suggests that PYY cells may exert a paracrine action on the mucus-secreting goblet cells. Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immunoreactive cells. On the ultrastructural level PYY-immunoreactivity was localized in basal granulated endocrine cells. These cells contained round or slightly oval electron dense granules with a mean diameter of 150 nm (range 100-300 nm).     

Immunohistochemical distribution of neuropeptide Y,peptide YY,pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y₁, Y₂, Y₄ and Y₅ receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y₁ and Y₄ receptor-like ir in all epidermal layers, while the Y₂ and Y₅ subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y₁-like and Y₄-like ir. These data have shown the presence and distribution of PYY, PP and Y₁ and Y₄ receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.     

The chemical identity of the immunoreactive LHRH-like peptide biosynthesized in the human placenta

Freshly obtained human placental trophoblasts were minced and pulselabeled for 30 min at 37°C with tritiated L-Tyrosine. After homogenisation, the crude extract was centrifuged and deproteinized with 10% TCA. The supernatant was defatted and the peptides concentrated through hydrophobic binding on ODS-silica cartridges. The bound, crude peptide extract was eluted and subjected to gradient, reverse-phase High Performance Liquid Chromatography. The fractions corresponding to the absorption peak of reference, synthetic LHRH were collected and extensively purified to radioactive homogeneity by further multiple HPLC. After digestion with pyroglutamate aminopeptidase, the resulting nonapeptide was manually sequenced by dansyl-Edman degradation. All the incorporated radioactivity was found to reside exclusively in residue number 4 of the nonapeptide; thus establishing for the first time the primary sequence of biosynthetic placental LHRH as: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, identical to its hypothalamic counterpart.     

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.