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.     

Pharmacology and neurochemistry of bombesin-like peptides

The pharmacology and neurochemistry of bombesin-like peptides was investigated. Synthetic analogues which had modifications near the N-terminal inhibited specific binding of (125I-Tyr4)BN with high affinity in rat brain and these peptides were potent hypothermic agents after central injection. In comparison, BN-like peptides with modifications near the C-terminal bound with low affinity and were not potent hypothermic agents. These data indicate that the C-terminal of BN is required for central high affinity binding and biological potency. Because substitution of D for L-amino acids at the 8, 10, 13 or 14 positions greatly reduced receptor binding affinity and ability to induce hypothermia, central receptors for BN show marked stereospecificity. Also, the pharmacology of BN in the periphery was investigated using dispersed guinea pig pancreatic acini and found to be similar to that of the brain. Because endogenous BN-like peptides extracted from brain tissue possess appreciable biological activity, these receptors are likely activated by endogenous BN-like peptides in vivo.     

Isolation and sequence analysis of human bombesin-like peptides

The decapeptide form of human gastrin releasing peptide was isolated from acid extracts of liver tissue containing a metastatic human bronchial carcinoid tumor. A larger form also was isolated and partially characterized. During gel permeation chromatography the major immunoreactive peak eluted in the same region as synthetic gastrin releasing decapeptide while a second minor immunoreactive peak eluted near gastrin releasing peptide. Bombesin-like immunoreactivity (BLI) was purified by successive applications to reverse phase high pressure liquid chromatography (HPLC) columns. After four successive HPLC purifications a single peak of bombesin-like immunoreactivity was detected. Amino acid analysis, microsequence analysis and coelution with synthetic peptide indicated that the predominant form present in metastatic tumor tissue was identical to the decapeptide form of canine gastrin-releasing peptide. The less abundant form was purified by cation exchange chromatography followed by reverse phase high pressure liquid chromatography. Partial microsequence analysis of this peptide, through the first 11 residues, was Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu. This sequence differed from that of hog heptacosapeptide gastrin releasing peptide at positions 1,3,4 and 5 and from the canine peptide as positions 1,3,5, and 7.     

Presence of ranatensin-like and bombesin-like peptides in amphibian brains

Antibodies specific for the carboxyl-terminal regions of bombesin and of ranatensin were used to study the tissue distribution of substances immunochemically similar to these two peptides in three amphibian species. Brain extracts of Rana catesbeiana, Rana pipiens, and Xenopus laevis all contained considerable quantities of both types of immunoreactivity, with measured concentrations as high as several hundred pmol per g tissue. The two antibodies used in this study had very low crossreactivity (less than 1% by RIA) with the other peptide. In addition, gel filtration revealed different elution profiles for the two immunoreactive substances extracted from amphibian brains. Immunocytochemistry revealed differences in localization within nerve fibers and cell bodies and specific absorption by the appropriate peptide. Ranatensin-like peptides were also present in high concentrations in skin of the two Rana species but not in that of Xenopus. Bombesin-like peptides were more abundant in the stomach of all three species. Significant amounts of substance P/phylasaemin-like immunoreactivity also were detected in the brains of all three species. It is concluded that ranatensin-like peptides are not confined to the skin and can be included as central nervous system neuropeptides in amphibians. These two groups of peptides are not species-specific since both are found in brain and stomach of amphibians whose skin contains only ranatensin-like peptide or neither.     

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).     

Tobacco smoke exposure and bombesin-like peptides in guinea pigs

Bombesin-like peptides (BLPs) are associated with tobacco smoke (TS)-induced diseases. We sought to determine if acute TS exposure releases BLPs into the pulmonary circulation. Sensitized and non-sensitized guinea pigs were chronically exposed to TS or compressed air. Thereafter, the lungs were acutely challenged with TS while perfused. Perfusates were analyzed for BLPs. TS increased BLPs in non-sensitized guinea pigs. A separate study determined daily bombesin exposure increased lung cell counts but not airway hyperresponsivensess. TS exposure releases BLPs into the pulmonary circulation but can be modified by host factors and bombesin itself does not induce airway hyperresponsiveness.     

Lung carcinoid cell lines have bombesin-like peptides and EGF receptors

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.     

Sensitization of pulmonary chemosensitive neurons by bombesin-like peptides in rats

Small cell lung cancer (SCLC) patients suffer from pulmonary stresses such as dyspnea and chest pain, and the pathogenic mechanisms are not known. SCLC cells secrete a variety of bioactive neuropeptides, including bombesin-like peptides. We hypothesize that these peptides may enhance the sensitivity of the pulmonary chemosensitive nerve endings, contributing to the development of these pulmonary stresses in SCLC patients. This study was therefore carried out to determine the effects of bombesin and gastrin-releasing peptide (GRP), a major bombesin-like peptide, on the sensitivities of pulmonary chemoreflex and isolated pulmonary vagal chemosensitive neurons. In anesthetized, spontaneously breathing rats, intravenous infusion of bombesin or GRP significantly amplified the pulmonary chemoreflex responses to chemical stimulants such as capsaicin and ATP. The enhanced responses were completely abolished by perineural capsaicin treatment of both cervical vagi, suggesting the involvement of pulmonary C-fiber afferents. In isolated pulmonary vagal chemosensitive neurons, pretreatment with bombesin or GRP potentiated the capsaicin-induced Ca(2+) transient. This sensitizing effect was further demonstrated in patch-clamp recording studies; the sensitivities of these neurons to both chemical (capsaicin and ATP) and electrical stimuli were significantly enhanced by the presence of either bombesin or GRP. In summary, our results have demonstrated that bombesin and GRP upregulate the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro.     

Multiple bombesin-like peptides with opposite functions from skin of Odorrana grahami

Bombesin-like peptides (BLPs) are a family of neuroendocrinic peptides that mediate a variety of biological activities. Three mature BLPs from the skin secretions of the frog Odorrana grahami were purified. Several bombesin-like peptide cDNA sequences encoding precursors of BLPs were identified from the skin cDNA library of O. grahami. This is the maximal diversity of BLPs ever found in animals. Five mature BLPs (B1-B5) based on the amino acid sequences derived from the cDNA cloning were synthesized. In the in vitro myotropic contraction experiment, all synthesized BLPs displayed a stimulating effect toward rat stomach strips, except B4 and B5 which showed the opposite effect, suggesting that certain BLPs may act as antagonists of bombesin receptors while most other BLPs act as agonists. This finding will facilitate the finding of novel bombesin receptors and novel ligands of bombesin receptors. The diversity of amphibian BLPs and their precursors were also analyzed and results suggest that amphibian BLPs and corresponding precursors of various sizes and processing patterns can be used as markers of taxonomic and molecular phylogenetics. The remarkable similarity of preproregions gives rise to very different BLPs and 3'-terminal regions in distantly related frog species, suggesting that the corresponding genes form a multigene family originating from a common ancestor. The diversification of BLP loci could thus be part of an evolutionary strategy developed by amphibian species as a result of shifts to novel ecological niches when environmental factors change rapidly.     

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.     

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.     

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.     

Peptide transmitters of primary sensory neurons: similar actions of tachykinins and bombesin-like peptides

Previous studies have demonstrated that two peptides, substance P (SP) and substance K (SK), are contained in a common prohormone--beta-preprotachykinin. Both peptides are cleaved from the prohormone and appear to coexist throughout the brain. This study evaluated the behavioral activity of SK and compared it to the activities of SP, bombesin (BN), and structurally related peptides. After intraspinal injection, all of the peptides induced "bite/scratch" behaviors, which differed in durations of action. The specific rank order of these durations of action were: BN greater than gastrin releasing peptide (GRP) = ranatensin (RT) greater than neuromedin B (NMB) greater than kassinin (KASS) = SK = SP and ranged from dose-dependent maxima of approximately 2 min (SP) to approximately 100 min (BN). To examine the possibility that differences in durations of action are due to differences in rates of proteolytic degradation, each peptide was incubated in spinal cord homogenates at 37 degrees C, and the degradation rates were monitored by radioimmunoassay (RIA) and by bioassay. The lengths of incubation time required to produce approximately 90% degradation of peptide immunoreactivity varied across peptides from less than 5 min (SP) to more than 60 min (BN and RT). Degradation of bioactivity generally paralleled degradation of immunoreactivity. The results of this study suggest that durations of nociceptive effects produced by the peptides tested are due, in part, to their resistance to proteolytic degradation.     

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

CCK-antagonist L-364,718: influence on rat pancreatic growth induced by caerulein and bombesin-like peptides

The present study investigates the inhibitory effect of the novel potent benzodiazepine-related CCK-antagonist L-364,718 on pancreatic growth in the rat induced by chronic administration of caerulein and bombesin-like peptides. Caerulein, injected s.c. twice daily at a dose of 1 microgram/kg body weight, and bombesin (10 micrograms/kg) induced a similar increase (1.5-3-fold) in pancreatic wet weight, total protein, amylase, trypsin, putrescine and spermidine content after 14 days of treatment. Growth induced by caerulein showed a significant increase in total DNA content suggesting cellular hyperplasia, whereas bombesin-like peptides led to cellular hypertrophy. In comparison to bombesin the decapeptide neuromedin C (10 micrograms/kg) was found to be 30-50% less potent. In the same dose range, neuromedin B and the tachykinins neurokinin A and B, all structurally related to bombesin, had no significant trophic effect on the rat pancreas. Administration of the CCK-antagonist L-364,718 twice daily at a dose of 0.1 mg/kg or at 1.0 mg/kg, either s.c. or orally, led dose-dependently to a near-complete inhibition of the caerulein-induced trophic effect. In contrast, L-364,718 administered at identical dosages, did not affect pancreatic hypertrophy induced by bombesin and neuromedin C. It is concluded that both peptides mediate their effect on the rat pancreas directly and not via release of endogenous cholecystokinin. Tachykinins are not involved in the regulation of pancreatic growth. Caerulein- and bombesin-like peptides have comparable effects on the stimulation of protein and polyamine synthesis.     

Inhibition of gastric emptying by bombesin-like peptides is dependent upon cholecystokinin-A receptor activation.

The amphibian peptide bombesin (BN) and the related mammalian peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) inhibit gastric emptying in rats. Exogenous administration of BN stimulates the release of cholecystokinin (CCK), a gastrointestinal peptide that also potently inhibits gastric emptying. To determine whether the inhibition of gastric emptying by BN-like peptides is mediated by a CCK-dependent mechanism, we examined the ability of the CCK-A receptor antagonist, devazepide, to block the inhibition of saline gastric emptying produced by BN, GRP18-27 and NMB. Using the same dosages as in the gastric emptying experiment, we also evaluated the effect of devazepide on feeding suppression produced by systemically administered BN. Our results showed that devazepide completely blocked the suppression of gastric emptying produced by BN, GRP18-27 and NMB but had no effect on BN-induced suppression of food intake. These results suggest that BN-like peptides inhibit gastric emptying through an indirect mechanism that is dependent upon CCK-A receptor activation. In contrast, the suppression of food intake by BN, in this experimental paradigm, is independent of CCK-A receptors.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

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.