Bombesin-like immunoreactivity in skates and the in vitro effect of bombesin on coronary vessels from the longnose skate, Raja rhina.

Bombesin-like immunoreactivity is present in nerve fibers projecting to the cardiovascular system, including the coronary arteries, and to the gastrointestinal canal, and in endocrine cells of the gut of skates belonging to the family Rajidae. Synthetic bombesin contracted isolated coronary rings from the longnose skate, Raja rhina, in a cumulative fashion. The contractile response was 84% of that of 60 mM potassium chloride. The pD2-value for bombesin was 8.83 (S.E.M. = 0.33; n = 15). Phentolamine, atropine and two substance P-antagonists increased the sensitivity to bombesin, while atenolol, sotalol, nifedipine, tetrodotoxin and two bombesin antagonists were devoid of significant effects. We conclude from this study that a bombesin-like peptide is present in nerves innervating the cardiovascular system and the gastrointestinal canal of skates of the family Rajidae, and that bombesin contracts coronary vessels in vitro via a direct mechanism and/or via mechanisms involving alpha-adrenergic and muscarinergic receptors.     

Immunohistochemical localization of bombesin-like peptides in the digestive tract of the bowfin,Amia calva

1. The distribution of bombesin-like immunoreactivity was determined in the gastrointestinal tract of the primitive holostean fish, the bowfin (Amia calva) using immunocytochemistry.2. Immunostaining using two different antisera raised against frog skin bombesin revealed a population of apparent endocrine cells containing bombesin-like immunoreactivity in the epithelium of the antral mucosa in the stomach.3. No bombesin-containing endocrine cells were present in any other segment of the gut. Bombesinergic nerves were not observed anywhere in the bowfin gastrointestinal tract.4. The antral bombesin endocrine cells were of the open type and were distributed diffusely from the base to the tips of antral glands, with some tendency to cluster near the base of the glands.5. These results suggest that a bombesin-like peptide may play an endocrine role in control of gastric functions such as regulation of acid secretion and gastric motility. These results support the hypothesis that bombesin serves a role in bony fish analogous to the role of antral cholecystokinin in amphibia and antral gastrin in amniotes.     

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.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

Bombesin potentiates the effect of acetylcholine on isolated strips of fish stomach.

The interaction between bombesin and acetylcholine acting on smooth muscle of the stomach wall was investigated in two species of teleost fish. Oncorhynchus mykiss (rainbow trout) and Gadus morhua (Atlantic cod). Acetylcholine or bombesin alone has an excitatory effect on the stomach muscle. The effect on contraction amplitude of acetylcholine (10(-6)-10(-5) M) alone is about 10-times greater than the effect of bombesin (10(-9)-10(-7) M). In molar terms however, bombesin is more potent than acetylcholine. Bombesin (10(-8)-10(-7) M) added 0.5-3 min prior to acetylcholine potentiates the effect of acetylcholine in a dose-dependent manner. The potentiation is most pronounced in circular muscle preparations, but is present also in longitudinal muscle preparations. Bombesin affects the response to carbachol (10(-6) M) with a similar potentiation, indicating that the potentiation is not caused by inhibition of choline esterase activity. Atropine (10(-6)-10(-5) M) abolishes the response to bombesin plus acetylcholine as well as the response to acetylcholine alone. Tetrodotoxin (10(-6) M) does not block the effect of acetylcholine, bombesin or the combination acetylcholine plus bombesin. Substance P (10(-9)-10(-7) M) which has a similar excitatory effect on the stomach muscle as bombesin, does not potentiate the effect of acetylcholine. Immunohistochemistry has shown the presence of strong bombesin-like immunoreactivity in stomach nerves of the cod and weak bombesin-like immunoreactivity in rainbow trout nerves. In addition, bombesin-like immunoreactivity was demonstrated in endocrine cells in the gastric and intestinal mucosa of both species. It is concluded that bombesin, contained either in nerve fibres or in mucosal endocrine cells, specifically potentiates the effect of acetylcholine in the fish stomach.     

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.     

Bombesin-like peptides in mammals.

Acid extracts of rat gut and brain contain substances that cross-react in a radioimmunoassay for the amphibian skin tetradecapeptide bombesin. Highest concentrations are present in the fundic part of the stomach, but there are significant amounts throughout the small and large intestine. Concentrations in the brain are highest in the hypothalamus. On gel filtration the rat bombesin-like immunoreactivity eluted as two major peaks. Fractionation of the second peak on cation exchange chromatography resolves this material into two further components. Intravenous infusions of partially purified preparations of the two components separated on gel filtration cause increases in serum gastrin in rats that are similar to those produced by immunochemically comparable amounts of synthetic bombesin.     

VIP-, substance P-, gastrin/CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout,Salmo gairdneri

Summary The presence of peptides in the gastrointestinal tract of the rainbow trout, Salmo gairdneri, was investigated immunocytochemically. VIP-like immunoreactivity was demonstrated in nerves in all layers of the stomach and the intestine, whereas substance P-like immunoreactivity was localized to endocrine cells, predominantly in the mucosa of the stomach, and to nerves mainly concentrated in the myenteric plexus throughout the gut. Endocrine cells reactive to gastrin/CCK antiserum were demonstrated in the intestinal mucosa, while no immunoreactivity was found in the stomach. Bombesin-immunoreactive and somatostatin-immunoreactive cells were localized in the stomach mucosa, and cells reactive to glucagon antiserum in the intestinal mucosa. Radioimmunoassay of stomach mucosa and muscle confirmed the presence of VIP-like and substance P-like immunoreactivity in these tissues, while gastrin/CCK-like immunoreactivity was low and bombesin-like immuno-reactivity was insignificant. In conclusion, molecules resembling the mammalian brain-gut peptides may be involved in the neuronal and hormonal control of gut function in fish.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Release of bombesin-like immunoreactivity from the isolated perfused rat stomach

In the present study the release of bombesin-like immunoreactivity (BLI), somatostatin and gastrin was determined form the isolated perfused rat stomach. Gastric inhibitory polypeptide (GIP, 2 X 10(-9) M) had no effect on BLI while stimulating somatostatin and gastrin release. In these experiments the luminal pH of the stomach was kept at pH 7. Reduction of the luminal pH to 2 resulted in an inhibition of BLI secretion by GIP while gastrin release was abolished and somatostatin remained unaffected compared to luminal pH 7. Acetylcholine (10(-6) and 2 X 10(-6) M) elicited a dose-dependent stimulation of BLI secretion while gastrin was stimulated and somatostatin secretion suppressed independent of the administered dose. The present data demonstrate that release of bombesin-like immunoreactivity can be modulated by intestinal hormones and neurotransmitters and is integrated into the complex system of gastrointestinal neuroendocrine regulation.     

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.     

Intramural distribution of immunoreactive vasoactive intestinal polypeptide (VIP), substance P,somatostatin and mammalian bombesin in the oesophago-gastro-pyloric region of the human gut

Summary The intramural distribution of vasoactive intestinal polypeptide (VIP), substance P, somatostatin and mammalian bombesin was studied in the oesophago-gastro-pyloric region of the human gut. At each of 21 sampling sites encompassing this entire area, the gut wall was separated into mucosa, submucosa and muscularis externa, and extracted for radioimmunoassay. VIP levels in the mucosa were very high in the proximal oesophagus (1231±174 pmol/g, mean±SEM) and showed varied, but generally decreasing concentrations towards the stomach, followed by a clear-cut increase across the pyloric canal (distal antrum: 73±16 pmol/g, proximal duodenum: 366±62 pmol/ g); consistent levels were found in submucosa and muscle (200–400 pmol/g) at most sites, the stomach again showing lower concentrations. By contrast, substance P was present in small amounts as far as the proximal stomach, but sharply increased across the pyloric canal, especially in mucosa and submucosa (distal antrum: 20±6.5 and 5.5±1.3 pmol/g; proximal duodenum: 62±8.5 and 34±11 pmol/g, respectively). Somatostatin concentrations were very low in the mucosa of the oesophagus and stepwise increased in the cardiac, mid-gastric and pyloric mucosa (cardia: 224±72 pmol/g; distal antrum: 513±152 pmol/g; proximal duodenum: 1013±113 pmol/g); concentrations in the submucosa and muscularis were generally low, with the exception of antrum and duodenum. Mammalian bombesin was comparatively well represented throughout the oesophageal muscularis (5–8 pmol/g), but most abundant in the stomach in all layers (oxyntic mucosa: 24±2.7 pmol/g; submucosa: 20±5.7 pmol/g; muscle: 28±5.0 pmol/g). In conclusion, a distinct differential distribution of the four peptides studied was revealed, indicating a diffuse, but highly differentiated peptide-containing innervation of the proximal human gut.     

Stimulation of canine pancreatic polypeptide, gastrin, and gastric acid secretion by ranatensin, litorin, bombesin nonapeptide and substance P

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.     

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.     

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.     

Gastrin/CCK-like peptides in the spiny dogfish, Squalus acanthias; concentrations and actions in the gut

1. The presence of gastrin/CCK-like immunoreactive material in both muscle and mucosal layers of the whole gut, except the cardiac stomach, in Squalus acanthias has been confirmed by radioimmunoassay. The highest levels were measured in rectum and spiral intestine respectively. 2. Fractionation of the spiral intestine mucosal extract on DEAE 52 columns indicated the presence of multiple forms of gastrin/CCK in the elasmobranch gut. 3. Synthetic mammalian gastrin- and CCK-like peptides, when effective, increased the rhythmic activity and sometimes the basal tonus in smooth muscle preparations from the intestine or rectum. 4. The irregular effects of mammalian peptides on the motility indicated an essential difference from the mammalian counterpart in the structure of the elasmobranch peptide present in the gastrointestinal nerves.     

Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust,Schistocerca gregaria (Forskal)

Summary The endocrine cells of the midgut epithelium of the desert locust are found dispersed among the digestive cells and are similar to those of the vertebrate gut. According to their reactivity to silver impregnation techniques and the ultrastructural features of the secretory granules (shape, electron-density, size, and structure) 10 types of endocrine cell have been identified, of which seven are located in the main segment of the midgut or in the enteric caeca, and the other three seem to be present only in the ampullae through which the Malpighian tubules drain into the gut. The endocrine cells have a slender cytoplasmic process that reaches the gut lumen, a feature that supports the receptosecretory nature postulated for this cellular type in insects as well as vertebrates. Antisera directed against mammalian gastrin, CCK, insulin, pancreatic polypeptide and bombesin reacted with some of the endocrine cells. This is the first time that insulin- and bombesin-like immunoreactive cells have been described in the midgut of an insect.     

Biological activity of a bombesin-like peptide extracted from the intestine of the ratfish, Hydrolagus colliei.

1. Ratfish (Hydrolagus colliei) intestines were boiled in water to inactivate proteases and then treated with cold 4% trifluoroacetic acid to extract bombesin-like peptides. 2. The extract was fractionated in several steps using reverse-phase and ion exchange HPLC, and bombesin-like immunoreactive peptides were detected by radioimmunoassay using an antiserum specific for the bioactive C-terminal region of bombesin. 3. A highly purified bombesin-like peptide-containing fraction stimulated amylase release in a dose-responsive fashion from rat pancreatic acini; the dose-response curve was parallel to a bombesin standard, and the ratfish peptide stimulated the same maximal rate of amylase secretion as the bombesin standard. 4. A potent, highly selective bombesin receptor antagonist completely abolished the stimulation of amylase release caused by the ratfish peptide, demonstrating the specificity of the response. 5. Estimates of the bombesin-like peptide concentration of this fraction by radioimmunoassay and by bioassay were nearly identical, indicating that ratfish bombesin is very similar biologically and antigenically to frog skin bombesin.     

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.     

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.