Bombesin-like immunoreactivity in human gastrointestinal tract

In the present study the distribution and molecular characteristics of bombesin-like immunoreactivity (BLI) were studied in acid extracts of human gastrointestinal tract. The highest levels were found in the fundus, antrum, pylorus and pancreas with lower levels in the duodenum, jejunum, terminal ileum and colon. BLI was also detected in both the muscle and mucosal layers of the antrum and colon. Sephadex G-50 gel chromatography under acid dissociating conditions revealed two peaks of immunoreactivity, one in the position of synthetic porcine gastrin releasing peptide (GRP) and the second eluting with synthetic amphibian bombesin. Variations in the proportions of the two molecular forms were seen in different regions of the gut. In the stomach and pancreas greater than 70% of the BLI eluted with the GRP marker while in pylorus, jejunum and terminal ileum only 20% was present in this form. Reverse-phase ODS silica HPLC of the major antral BLI peak, utilising a methanol/trifluoroacetic acid gradient indicated that this peptide was similar to porcine GRP. We have therefore (1) demonstrated the presence and heterogeneity of bombesin-like immunoreactivity throughout the human gastrointestinal tract and (2) shown for the first time that a proportion of this BLI closely resembles porcine GRP.     

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.     

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).     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.     

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.     

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.     

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

Neuromedin B is a major bombesin-like peptide in rat brain: regional distribution of neuromedin B and neuromedin C in rat brain, pituitary and spinal cord

Neuromedin B and neuromedin C are the novel mammalian bombesin-like peptides isolated from porcine spinal cord. We have developed highly specific and sensitive radioimmunoassays for neuromedin B and neuromedin C, and determined their regional distribution in rat central nervous system. Prior to measurements of the tissue contents, immunoreactive neuromedin B and C were characterized by gel-filtration and high performance liquid chromatography. Neuromedin B and C immunoreactivities have similar regional distribution in rat brain, but the content of immunoreactive neuromedin B is 2-6 times higher than that of immunoreactive neuromedin C in every region. These results indicate that neuromedin B is a major endogenous bombesin-like peptide in rat brain and has specific functions of physiological importance.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

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.     

Development and function of bombesin-like peptides and their receptors

Amphibian bombesin and its related peptides consist a family of neuropeptides in many vertebrate species. Bombesin and two major bombesin-like peptide in mammals, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to elicit various physiological effects. These include inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. Receptors for GRP and NMB (GRP-R and NMB-R), as well as third subtype of bombesin-like peptide receptor (BRS-3) have been cloned. These receptors are G-protein-coupled receptors and are expressed in various brain regions and in the digestive tract. In this paper, we will summarize studies on these peptides and their receptors, with special reference to research using gene-knockout mice. These studies clearly demonstrated the role of three receptors in vivo and in vitro. We will also discuss the phylogeny of these receptors.     

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.     

Primary structures of the bombesin-like neuropeptides in frog brain show that bombesin is not the amphibian gastrin-releasing peptide

Peptides displaying gastrin-releasing peptide/bombesin-like immunoreactivity were isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the more abundant peptide was established as: Gly-Ser-His-Trp-Ala-Val-Gly-His-Leu-Met. NH2. This sequence shows one substitution (Ser for Asn) compared with mammalian gastrin-releasing peptide (18-27) (neuromedin C). The extract also contained gastrin-releasing peptide but bombesin was absent. The data indicate that bombesin is not the amphibian counterpart of gastrin-releasing peptide.     

Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry

Summary Five anti-gastrin releasing peptide (GRP) sera have been characterized against GRP, bombesin and related polypeptides spotted on cellulose acetate discs. Antibodies reacting with the C-terminal G-14 sequence of bombesin and the 19–27 sequence of GRP, were detected in all sera. Antibodies directed exclsively against the bombesin unrelated 1–17 sequence of GRP were found only in one serum (R-6902). With parallel immunohistochemical tests only the C-terminal immunoreactivity was detected in endocrine-paracrine cells of the chicken proventriculus, while both immunoreactivities were present in nerve fibres and a few nerve cell bodies of the mammalian gut. The distribution of GRP- and bombesin-like immunoreactive nerves in the gastric mucosa of both pyloric and oxyntic type the submucosal and myenteric plexus along the whole gastrointestinal wall and at sphincter regions is detailed.     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

Immunocytochemical localization of gastrin-releasing peptide/bombesin-like immunoreactive neurons in insects

Summary GRP/bombesin-like immunoreactive material was immunocytochemically detected in neurons of seven insect species belonging to seven orders, while such neurons were not found in three insect species belonging to two other orders. In some insect species certain neurons were found in corresponding places and approximately the same numbers. It seems likely that such neurons have a common evolutionary origin and are homologous. The fact that the GRP-antiserum reveals such homologous neurons in species belonging to different orders, suggests that the part of the GRP/bombesin-like peptide recognized by the antiserum has been relatively stable during evolution. As the GRP-antiserum had to be used in much higher concentrations on insect tissue than for GRP endocrine cells in chicken proventriculus, the chemical resemblance of the insect peptide(s) to GRP and bombesin may be limited.     

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.     

Bombesin-like peptides in small cell lung cancer: biochemical characterization and secretion from a cell line

High intracellular levels of BN-like peptides are present in tumors and cell lines of small cell carcinoma of the lung (SCCL) as well as the putative precursor cells of this tumor, the pulmonary endocrine cell. In cell line NCI-H209 the density of bombesin-like peptides was 8.9 +/- 1.1 pmol/mg total protein. Gel filtration chromatography of an extract of these cells revealed one major peak of immunoreactivity which coeluted with synthetic bombesin (1620 daltons). Also, high pressure liquid chromatography revealed one major peak of immunoreactivity was present which eluted before synthetic peptide. Therefore, SCCL bombesin-like peptides may be of similar size but are more hydrophilic than synthetic peptide. Cells maintained in culture continuously release bombesin-like peptides into the growth medium. Also, high concentrations of K+ stimulated the secretion of immunoreactive bombesin from cell lines in a Ca++-dependent manner. These SCCL bombesin-like peptides may function as important regulatory agents in the malignant lung.     

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.     

NPY-like peptides occur in the nervous system and midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata

The distribution of the NPY-like substances in the nervous system and the midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata was determined by immunocytochemistry using an antiserum directed against synthetic porcine NPY. The peroxidase-antiperoxidase procedure revealed that NPY immunoreactive cell bodies and nerve fibers were observed in the brain, optic lobes, corpora cardiaca, suboesophageal ganglion and ventral nerve cord of the locust and in the brain, optic lobes and suboesophageal ganglion of the fleshfly. In the locust midgut, numerous endocrine cells and nerve fibers penetrating the outer musculature contained NPY-like immunoreactivity. The concentrations of NPY immunoreactive material in acetic acid extracts of locust brain, optic lobes, thoracic ganglia, ovaries and midguts was measured using a specific radioimmunoassay technique. The dilution curves of the crude tissue extracts were parallel to the standard curve. The highest amount of NPY-like immunoreactivity was found in the locust ovary and midgut. Reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay were used to characterize the NPY-like substances in the locust brain and midgut. HPLC-analysis revealed that NPY-immunoreactivity in the locust brain eluted as three separate peaks. The major peak corresponded to a peptide less hydrophobic than synthetic porcine NPY. RP-HPLC analysis of midgut extracts revealed the presence of an additional NPY-immunoreactive peak which had a retention time similar to the porcine NPY standard. The present data show the existence of a widespread network of NPY immunoreactive neurons in the nervous system of the locust and the fleshfly. Characterization of the immunoreactive substances indicates that peptides similar but not identical to porcine NPY are present in the central nervous system and midgut of insects.