Characterization and distribution of bombesin-like peptides in the rat brain and gastrointestinal tract

Extracts of rat brain and gastrointestinal tract, analyzed by reverse-phase high-performance liquid chromatography and radioimmunoassay, contained two bombesin-like immunoreactivity peaks with similar retention times as porcine gastrin-releasing peptide (GRP) and its COOH-terminal decapeptide, neuromedin C or GRP(18-27). However, the GRP-like peptide peak did not elute with exactly the same retention time as porcine GRP. The highest concentration of bombesin-like immunoreactivity was found in extracts of antrum, whereas the lowest was found in whole brain. Neuromedin C was present at lower concentrations than the GRP in antrum, duodenum, and ileum, while similar amounts of each were found in brain.     

Characterisation of bombesin-like immunoreactivity in human fetal lung

A sensitive radioimmunoassay for bombesin-like immunoreactivity (BLI) was developed and utilised in conjunction with G50 gel chromatography and reverse-phase HPLC, to study the content and molecular characteristics of bombesin-like peptides in acid extracts of human fetal lung. The antiserum, (B5), is directed towards the C-terminal region of the bombesin molecule and cross-reacts 70% with synthetic porcine GRP and the synthetic GRP fragment, GRP (14-27). Specimens of lung were collected from fetuses of gestational ages 15-22 weeks, following prostaglandin termination of pregnancy. The tissue was extracted into 0.1 N HCl at 90 degrees C. The mean BLI content was 50.2 pg/mg wet weight of tissue (range 15.5-136 pg/mg; n = 13). No correlation between gestational age and BLI content could be established. G50 gel chromatography of acid extracts, under dissociating conditions, revealed two peaks of BLI, one in the position of synthetic porcine GRP and the second, constituting greater than 90% of the immunoreactivity, eluting with synthetic amphibian bombesin. Reverse-phase ODS silica HPLC of this major G50 peak, utilising a methanol/trifluoroacetic acid gradient, indicated that this peptide was similar to the GRP C-terminal fragment, GRP (14-27). We have therefore (1) confirmed the presence and heterogeneity of BLI in human fetal lung, and (2) shown, for the first time, that the majority of this BLI more closely resembles a fragment of GRP than amphibian bombesin itself.     

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.     

Expression of the ammonia transporter proteins Rh B glycoprotein and Rh C glycoprotein in the intestinal tract

Ammonia metabolism is important in multiple aspects of gastrointestinal physiology, but the mechanisms of ammonia transport in the gastrointestinal tract remain incompletely defined. The present study examines expression of the ammonia transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) in the mouse gastrointestinal tract. Real-time RT-PCR amplification and immunoblot analysis identified mRNA and protein for both RhBG and RhCG were expressed in stomach, duodenum, jejunum, ileum, and colon. Immunohistochemistry showed organ and cell-specific expression of both RhBG and RhCG. In the stomach, both RhBG and RhCG were expressed in the fundus and forestomach, but not in the antrum. In the forestomach, RhBG was expressed by all nucleated squamous epithelial cells, whereas RhCG was expressed only in the stratum germinativum. In the fundus, RhBG and RhCG immunoreactivity was present in zymogenic cells but not in parietal or mucous cells. Furthermore, zymogenic cell RhBG and RhCG expression was polarized, with apical RhCG and basolateral RhBG immunoreactivity. In the duodenum, jejunum, ileum, and colon, RhBG and RhCG immunoreactivity was present in villous, but not in mucous or crypt cells. Similar to the fundic zymogenic cell, RhBG and RhCG expression in villous epithelial cells was polarized when apical RhCG and basolateral RhBG immunoreactivity was present. Thus the ammonia transporting proteins RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they function cooperatively to mediate gastrointestinal tract ammonia transport.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig

Summary Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Regional distribution and molecular forms of rat islet amyloid polypeptide

Using a highly sensitive and specific radioimmunoassay (RIA) for rat islet amyloid polypeptide (IAPP), we clarified regional distribution and molecular forms of rat IAPP. IAPP[1-37] and IAPP[19-37] were identified in normal rat pancreas by sequence analyses IAPP[19-37], accounting for 57% of IAPP-immunoreactivity in rat pancreas, is a major molecular form of rat IAPP moiety. In human, however, IAPP[1-37] is the major component, with IAPP[17-37] composing as little as 2-6% of IAPP-immunoreactivity in pancreas. This indicates that processing of IAPP in pancreas differs in species. A large amount of IAPP (328.5 +/- 25.0 pmol/g wet weight) was found in rat pancreas and the peptide was also detected in pyloric antrum of the stomach, duodenum, jejunum, ileum, and colon at 0.1-0.8% of the level of pancreas. It was not detected in central nervous system. The content of rat IAPP in pancreas fell to 54% of control after 4 day fasting. The distribution of IAPP suggests its possible endocrine or paracrine function in pancreas and gastrointestinal tract.     

Isolation and primary structure of gastrin-releasing peptide from a teleost fish, the trout (Oncorhynchus mykiss).

Immunohistochemical studies have established that fish gastrointestinal tissues contain peptides with gastrin-releasing peptide (GRP)/bombesin-like immunoreactivity, but the molecular nature of this material is unclear. In this study, the most abundant peptide that was immunoreactive towards an antiserum raised against pig GRP was isolated in pure form from an extract of the stomach of the rainbow trout (Oncorhynchus mykiss). The primary structure of the peptide was established as: Ser-Glu-Asn-Thr-Gly-Ala-Ile-Gly-Lys-Val10- Phe-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly20-His-Leu-Met-NH2. Although this amino acid sequence is shorter than those of mammalian GRPs by four residues, the COOH-terminal dodecapeptide is identical to the corresponding region in pig GRP. The data indicate, therefore, that the predominant molecular form of GRP in the stomach of a teleost fish is structurally more similar to mammalian GRP than to the amphibian skin peptide, bombesin.     

Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract

 Colocalisation of synaptophysin has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum and colon) using double-immunofluorescence stainings. Numerous synaptophysin immunoreactive cells were seen in the antrum, while a smaller number were found in the intestinal tract. Synaptophysin immunoreactivity was strong in the antrum but weak in the intestine. In the intestinal colocalisation studies the synaptophysin immunoreactivity was enhanced by using the tyramide amplification method. Synaptophysin and chromogranin A were colocalised but the latter occurred mainly basally, whereas synaptophysin was found to occur diffusely throughout the cytoplasm. Synaptophysin immunoreactivity occurred in the serotonin cells throughout the gastrointestinal tract, and in the antral gastrin and somatostatin cells. In the intestinal tract only a small fraction of somatostatin, gastrin, cholecystokinin, enteroglucagon, enteroglucagon/ peptide tyrosine tyrosine displayed synaptophysin immunoreactivity. In the gastrointestinal tract (except the antrum), chromogranin A is a better general neuroendocrine marker than synaptophysin. The functional role of synaptophysin is unclear but it may be involved in the intracellular transport and release of hormones. Based on the distribution background of synaptophysin, it seems to be of greater importance in the antrum than in the intestinal tract as a whole. Accepted: 3 September 1998     

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.     

Distribution of bombesin- and cholecystokinin-like immunoreactivity in rat and dog brain and gastrointestinal tract

Using a specific bombesin radioimmunoassay and an immunoassay for cholecystokinin which sees all C-terminal fractions, the distribution of bombesin-like (BLI) and cholecystokinin-like (CCK-LI) immunoreactivity in the brain and gastrointestinal tract of the rat and dog has been studied. Both peptides are found in the brain and gut but the rat contains more CCK and BLI than the dog; this is particularly noted in the stomach, colon and cerebral cortex whereas the small intestine of both species contains equivalent amounts of peptides. This contrasts with other comparative studies, mainly on nervous system CCK, which find no major distribution differences in man, monkey, pig and rat. This finding suggests that CCK-LI and BLI peptides may have a more predominant role in the rat than in the dog.     

Identification, characterization and release of GRP gene-associated peptides from the normal porcine and human gastro-intestinal tract

Using a radioimmunoassay directed towards human proGRP (42-53) on acetic acid extracts, immunoreactivity was measured throughout the porcine GI-tract in concentrations that were parallel to those of GRP (gastrin-releasing peptide or 'mammalian bombesin'). Gel filtration and HPLC studies of human and porcine tissue extracts revealed that the immunoreactivity was mainly due to a peptide with a molecular size of 8-9 kDa. The peptide did not contain the GRP sequence, making it a major fragment of the GRP C-flanking part of proGRP. Furthermore, a peptide of similar size with proGRP (42-53) immunoreactivity was released from isolated, perfused preparations of porcine antral and non-antral stomach and pancreas in parallel with GRP in response to electrical stimulation of the vagus nerves. Our results suggest that a processing of preproGRP occurs in normal, adult human and porcine tissues, that is similar to that previously demonstrated in small cell lung carcinomas and human fetal lungs. The finding that the immunoreactive proGRP fragment is released from the tissues upon appropriate stimulation raises the question of a possible physiological role for proGRP products other than GRP.     

The intestinal mucosa preferentially releases somatostatin-28 in pigs

We studied the molecular forms of somatostatin-like immunoreactivity (SLI), newly released from isolated perfused preparations of the porcine antrum, stomach, pancreas and upper small intestine: Perfusion effluents were concentrated by Sep-Pak C-18 adsorption, eluted with ethanol, dessicated, and subjected to gel filtration with subsequent radioimmunoassays for somatostatin-14 and N-terminal somatostatin-28 immunoreactivity. All the SLI newly released from the stomach and antrum eluted at the position of somatostatin-14, and such was also the case for more than 95% of the SLI newly released from the pancreas, while 68 -/+ 7% and 75 -/+ 8% of the SLI newly released from the isolated perfused jejunum and ileum, respectively, corresponded to somatostatin-28. By reverse phase HPLC the identity of these peptides with synhetic somatostatin-14 and -28 was established.     

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 localization of pancreatic spasmolytic polypeptide (PSP) in the pig.

Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Islet amyloid polypeptide (IAPP) in the gastrointestinal tract and pancreas of man and rat

Summary An immunohistochemical study for islet amyloid polypeptide (IAPP) was made on the gastrointestinal (GI) tract and pancreas of man and rat, using antisera raised against a synthetic peptide of C-terminal human IAPP (24–37) and a synthetic peptide of rat IAPP (18–37). A large number of IAPP-immunoreactive cells were found in the pyloric antrum, and a small number in the body of the stomach in both man and rat. Cytoplasmic processes extended out from the bipolar peripheral region of the immunoreactive cells, rather like neuronal processes, and some appeared to make contact with other immunoreactive cells. In addition, small numbers of immunoreactive cells were also seen in the duodenum and rectum, whereas they were absent from the jejunum, ileum and large intestine. An examination was made for evidence of colocalization of IAPP-immunoreactive material with material immunoreactive for gastrin, somatostatin, vasoactive intestinal polypeptide, pancreatic polypeptide, insulin, and glucagon, but none was found. IAPP-immunoreactive cells were also found in the pancreas of non-diabetic and non-insulin-dependent diabetic patients, but they were completely absent from a patient with insulin-dependent diabetes mellitus despite the presence of IAPP in the plasma. The results of these studies suggest that the peptide may have a biological role in situ in the GI tract and, in addition to the pancreas, may be a possible source of plasma IAPP.     

Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract.

Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C-terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the procine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, beta- and/or gamma-preprotachykinin, in the same neurons.     

Distribution of bombesin-like peptides in the alimentary canal of several vertebrate species

The objective of this study was to quantitate and characterize the variants of bombesin-like immunoreactivity in the alimentary canal of the rat, rabbit, hawk, owl, dog, monkey and human. Bombesin-like immunoreactivity was found throughout the entire gastrointestinal tract of all species studied. In the rat, the highest concentration of bombesin-like immunoreactivity was found in the colon. Gel chromatography showed that bombesin-like immunoreactivity corresponded to gastrin-releasing peptide (GRP-27) and GRP-10. In the dog, the greatest concentration of bombesin-like immunoreactivity was observed in the mucosal layer of the fundus, whereas the concentration of bombesin-like immunoreactivity in the muscle layer of the dog did not vary significantly from region to region. Gel chromatography showed that bombesin-like immunoreactivity in the dog corresponded to GRP-27, bombesin, GRP-10, and a smaller fragment. In the human, the concentration of bombesin-like immunoreactivity did not vary significantly from region to region in the mucosal and muscular layers. Gel chromatography of human fundal mucosa showed that bombesin-like immunoreactivity peaks occur in the regions of GRP-27, bombesin and GRP-10. These findings substantiate the observation that bombesin-like peptides play a variety of roles in the regulation of gut function.     

Effect of synthetic neuromedin C, a decapeptide of gastrin-releasing peptide (GRP [18-27]), on blood flow and exocrine secretion of the pancreas in dogs

Neuromedin C, the smaller molecular form of gastrin releasing peptide (GRP [18-27]), has been recently identified from canine intestinal muscle and porcine spinal cord. This study was conducted to determine if this newly identified peptide retains biological activity on canine pancreas in vivo. Intravenous injection of graded doses of synthetic Neuromedin C caused a marked increase of systemic blood pressure and initial reduction of pancreatic blood flow in eleven anesthetized dogs, as measured by Laser Doppler Flowmetry. Flow volume and protein output of pancreatic juice were also increased by Neuromedin C in a dose-related manner in six dogs. These results suggest that this peptide is one of the biologically active forms of mammalian bombesin-like peptides and may possess physiological significance as a novel neuropeptide.     

Pancreastatin distribution and plasma levels in the pig

Pancreastatin is a peptide isolated from porcine pancreas which has insulin-suppressive actions in vitro and sequence homology with chromogranin A. Using radioimmunoassay and immunocytochemistry we investigated whether pancreastatin has a more widespread distribution and a possible endocrine role in the pig. Pancreastatin immunoreactivity was found in plasma, adrenal gland, pancreas, anterior pituitary and throughout the gastrointestinal tract. The immunoreactivity was colocalized with chromogranin immunoreactivity in endocrine cells and ultrastructurally (in the pancreas) to storage granules. Characterization of pancreastatin-like immunoreactivity, using gel permeation and high performance liquid chromatography, separated 3 different pancreastatin-like immunoreactive forms: one molecular form, indistinguishable from synthetic pancreastatin 1-49, was predominant in pancreas and thyroid and released into the circulation postprandially. However, a high dose (greater than 1 nmol/l) infusion of pancreastatin 33-49 (the biologically active moiety in vitro) into conscious pigs had no effect on either basal or glucose-stimulated insulin secretion.