Neuromedin B-preferring Research Articles

Overexpression of gastrin-releasing peptide receptor induced layer disorganization in brain

Gastrin-releasing peptide-preferring and neuromedin B-preferring receptors, members of the bombesin-like peptide receptor subfamily, are reported to regulate proliferation, migration and differentiation. Since they are expressed in developing brain, we postulated that the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors might be involved in normal brain development. Here we examined the effects of the overexpressions of the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors on chick brain development in vivo using a retrovirus. In the overexpressed exogenous gastrin-releasing peptide-preferring receptor brain, we found laminar disorganization in the telencephalon, tectum and particularly in the cerebellum with severe atrophy. Processes of the radial glial cells in the telencephalon and optic tectum, as well as the projections of the Bergmann glia in the cerebellum were distorted, which might disturb normal cell migration. Despite the atrophy of the cerebellum, densely-stained proliferating cell nuclear antigen- and phospho-histone H3-positive cells increased in number. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells also increased in the cerebellum, suggesting that the ectopically proliferating cells were subjected to apoptosis. Glial fibrillary acidic protein-positive cells also increased in the hyperpallium accessorium and in the outer layers of the tectum. We also found smaller and spindle-shaped cells which resembled undifferentiated embryonic tumor cells. On the other hand, the layer structures of the neuromedin B-preferring receptors overexpressed brain were well organized and developed, and the size of brain was generally enlarged. These results indicated that although the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors are involved in normal brain development, both receptors contribute and exert their effects differently.

Brain phospholipase C/diacylglycerol lipase are involved in bombesin BB 2 receptor-mediated activation of sympatho-adrenomedullary outflow in rats

Bombesin receptors are mainly divided into two subtypes: BB 1 receptor (neuromedin B-preferring receptor) and BB 2 receptor [gastrin-releasing peptide (GRP)-preferring receptor]. Previously, we reported that intracerebroventricularly (i.c.v.) administered bombesin elevates plasma noradrenaline and adrenaline by production of brain arachidonic acid in rats. Arachidonic acid is released mainly by phospholipase A 2 (PLA 2)-dependent pathway or phospholipase C (PLC)/diacylglycerol lipase-dependent pathway. In the present study, bombesin and GRP elevated plasma catecholamines in a dose-dependent manner (1 and 5 nmol/animal, i.c.v.), while neuromedin B (1, 5 and 10 nmol/animal, i.c.v.) had no effect in urethane-anesthetized rats (bombesin = GRP ≫ neuromedin B). The bombesin (1 nmol/animal, i.c.v.)-induced response was dose-dependently attenuated by [ d-Phe 6, des-Met 14]-bombesin (6–14) ethylamide (bombesin BB 2 receptor antagonist) (15.3 and 30.6 nmol/animal, i.c.v.) and also by U-73122 (PLC inhibitor) (10 and 100 nmol/animal, i.c.v.) and RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 μmol/animal, i.c.v.). However, d-Nal-cyclo[Cys-Tyr- d-Trp-Orn-Val-Cys]-Nal-NH 2 (bombesin BB 1 receptor antagonist) (30 and 100 nmol/animal, i.c.v.), mepacrine (PLA 2 inhibitor) (1.1 and 2.2 μmol/animal, i.c.v.) and U-73343 (inactive analog of U-73122) (100 nmol/animal, i.c.v.) had no effect. These results suggest the involvement of brain PLC/diacylglycerol lipase in the brain bombesin BB 2 receptor-mediated activation of sympatho-adrenomedullary outflow in rats.

Characterization of the bombesin-like peptide receptor family in primates

In mammals, bombesin-like peptides mediate a broad range of physiological functions through binding to three highly conserved G-protein-coupled receptors: the neuromedin B-preferring, the gastrin-releasing peptide-preferring, and the bombesin-receptor subtype 3. Selective modulation of these receptors presents opportunities for the development of novel therapeutics. To ascertain if rhesus monkey could serve as a surrogate animal model for the development of modulators of bombesin-like receptor function, we undertook a search for additional receptor family members and studied the expression profiles of the three known bombesin-related receptors. We found no evidence for additional receptor family members in mammals, suggesting that the expression of the previously described bombesin-receptor subtype 4 is limited to amphibians. We studied the distribution of the three receptors in a broad array of human and rhesus monkey tissues. Based on the similarity between the human and the rhesus expression profiles, we conclude that the rhesus monkey may be a suitable animal model to evaluate the clinical efficacy and potential side effects of bombesin-like peptide ligands.

Disruptions in feeding and body weight control in gastrin-releasing peptide receptor deficient mice.

Bombesin (BN) interacts with two mammalian receptor subtypes termed gastrin-releasing peptide (GRP)-preferring (GRP-R) and neuromedin B (NMB)-preferring (NMB-R) that may mediate the satiety action of BN. We examined the feeding behavior of mice that were deficient in the GRP-R (GRP-R KO) to assess the overall contribution of this receptor subtype in the feeding actions of BN-related peptides. GRP-R KO mice failed to suppress glucose intake in response to systemically administered BN and GRP(18-27), whereas both peptides elicited a potent reduction of intake in wild-type (WT) mice. Neither GRP-R KO nor WT mice suppressed glucose intake following NMB administration. Unlike the impaired responses to BN-like peptides, the feeding inhibitory action of cholecystokinin was enhanced in GRP-R KO mice. Consistent with behavioral results, GRP-R KO mice also exhibited a reduction in c-Fos immunoreactivity in the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN) following peripheral administration of BN. An evaluation of meal patterns showed that GRP-R KO mice ate significantly more at each meal than WT mice, although total 24 h food consumption was equivalent. A long-term analysis of body weight revealed a significant elevation in GRP-R KO mice compared with WT littermates beginning at 45 weeks of age. These data suggest that the GRP-R mediates the feeding effects of BN-like peptides and participates in the termination of meals in mice.

GRP-receptor-mediated signal transduction, gene expression and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF

Bombesin-like peptides have been implicated as growth factors in various human cancers. Human adenocarcinoma cell lines (Capan-1, Capan-2, MiaPaCa-2 and HPAF) were tested to determine whether they express the gastrin-releasing peptide-preferring bombesin receptor (GRPR) and neuromedin B-preferring bombesin receptor (NMBR). Using RT-PCR the highest level of GRP receptor mRNA was found in HPAF cells. NMB receptor mRNA expression moderate in all cell lines investigated. We therefore selected the HPAF cell line to investigate whether bombesin treatment affects intracellular Ca 2+ ([Ca 2+] i), cAMP level, DNA synthesis as a measure of cell proliferation, and expression of three transcription factors: c-fos, c-myc and high mobility group protein IY (HMG-I(Y)). Bombesin administration led to an immediate increase in free intracellular Ca 2+ concentration ([Ca 2+] i) but did not change cAMP levels. The peptide also enhanced [ 3H]thymidine incorporation in HPAF cells (but not in the other cell lines), an effect that was concentration dependent, reaching 36 ± 5% stimulation over control values at 24 h with an EC 50 of 2.27 × 10 −12 M. Furthermore, bombesin stimulated c-fos, c-myc and HMG-I(Y) expression in a time-dependent manner: the c-fos mRNA level increased dramatically in the first 30 min of exposure, then returned to basal level within 2 h, while the c-myc and HMG-I(Y) mRNA levels peaked at 2 h and 4h, respectively. All actions of bombesin were blocked by BME (D-Phe 6-bombesin-(6–13)-methylester), a selective GRP receptor antagonist, but not by the NMB receptor antagonist BIM-23127 (D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH 2). We conclude that HPAF cells express mRNA for GRP receptors and that functional receptors are present in the cell membrane. The occupation of these receptors leads to a sequence of intracellular events involving rapid mobilization of intracellular Ca 2+, expression of c-fos, c-myc and HMG-I(Y) mRNA, and stimulation of cell proliferation. Conversely, although NMB receptor mRNA can be detected, its actual translation to functional receptors does not reach a detectable level.

Functional Properties of Two Bombesin-Like Peptide Receptors Revealed by the Analysis of Mice Lacking Neuromedin B Receptor

The neuromedin B-preferring receptor (NMB-R) is one of the members of the bombesin (BN)-like peptide receptor subfamily in mammals. Previously, we have generated and characterized mice with targeted disruption of the two other BN-like peptide receptors, bombesin receptor subtype-3 (BRS-3) and gastrin-releasing peptide-preferring receptor (GRP-R). Here we describe the generation and analysis of NMB-R-deficient mice to investigate how NMB-R differs from BRS-3 and GRP-R. Compensation for NMB-R deficiency by overexpression of GRP-R and/or BRS-3 was not detected. Although the hypothermic effect of NMB was reduced by 50% in NMB-R-deficient mice, the effect of GRP infusion was comparable to the wild-type mice. In contrast, fundic smooth muscle contraction on stimulation with NMB or GRP was normal in NMB-R-deficient mice. Administration of GRP but not NMB suppressed glucose intake in both normal and NMB-R-deficient mice. These results suggest that the NMB-R has an essential role in thermoregulation, but not for smooth muscle contraction of the fundus or for the suppression of feeding behavior. In addition, the behavioral phenotypes of GRP-R-deficient mice were not observed in NMB-R-deficient mice. These data show that the functions of NMB-R and GRP-R are distinct, with only partial overlap.

PD 176252 — The first high affinity non-peptide gastrin-releasing peptide (BB 2) receptor antagonist

In this paper we describe the development of a novel series of non-peptide, “balanced” neuromedin-B preferring (BB 1)/gastrin-releasing peptide preferring (BB 2) receptor ligands as exemplified by PD 176252. PD 176252, which exhibits nanomolar affinity for both the BB 1 (K i=0.15nM) and BB 2 (K i=1.0nM) receptors, has been demonstrated to be a competitive antagonist at these bombesin receptor subtypes.

Caudal hindbrain neuromedin B-preferring receptors participate in the control of food intake.

Recent studies have identified two subtypes of bombesin (BN) receptors in the rat central nervous system: gastrin releasing-peptide (GRP) preferring and neuromedin B (NMB) preferring. To investigate a role for the NMB-preferring receptor subtype in feeding suppression elicited by fourth ventricular (4V) BN administration, we evaluated the ability of a selective NMB-preferring receptor antagonist, BIM-23127, to block suppression of glucose intake produced by 4V BN (10 pmol). Our results showed that 4V administration of BIM-23127 dose dependently antagonized the suppression of glucose intake produced by 4V BN. In addition, 4V administration of BIM-23127 alone increased glucose intake above that observed in the baseline condition. These results support a role for the NMB-preferring BN receptor subtype in the suppression of intake produced by 4V BN administration and suggest that endogenously released NMB participates in ingestive control.

Comparison of the peptide structural requirements for high affinity interaction with bombesin receptors

Recently it has been established that both a gastrin-releasing peptide (GRP)-preferring bombesin receptor and a neuromedin B-preferring bombesin receptor mediate the mammalian actions of bombesin-related peptides. Because many tissucs used for studies of the structure-activity relationship of these peptides possess both receptor subtypes and none possess only the neuromedin B-preferring subtype, there is minimal information on the peptide structural features determining receptor selectivity and it is unknown whether the determinants of agonism at both bombesin receptor subtypes are similar. In the present study we have used native cells either possessing only one bombesin receptor subtype or stably transfected with one subtype to study in detail the peptide structural requirements for interacting and activating each receptor subtype. For the naturally occurring agonists, at the GRP-preferring bombesin receptor the relative affinities were litorin = ranatensin = bombesin > GRP > neuromedin B, phyllolitorin and at the neuromedin B-preferring bombesin receptor were litorin = neuromedin B = ranatensin > bombesin, phyllolitorin > GRP. For the GRP-preferring bombesin receptor the heptapeptide and for the neuromedin B-preferring bombesin receptor the octapeptide was the minimal car☐yl fragment interacting with the receptor/or causing biologic activity, and the nonapeptide and full decapeptide, respectively, were the minimal required for full affinity. Making neuromedin B more bombesin- or GRP-like by replacing amino acids in position 3, 6, and 9 demonstrated that position 3 was the most important, followed by position 9 for receptor subtype selectivity. A conformationally restricted GRP analogue. [ d-Cys 6, d-Ala 11,Cys 14]bombesin-(6–14) had a significantly higher affinity for GRP-preferring bombesin receptor than NMB receptor. These results demonstrate that: (1) the structure-function relations for the two mammalian bombesin receptors have important differences; (2) suggest that the active conformation of neuromedin B must differ markedly from the β-sheet model proposed for GRP; and (3) suggest that one important function of the NH 2 terminus of GRP and neuromedin B is determining receptor subtype selectivity.

Bombesin receptor subtype mediation of gastroenteropancreatic hormone secretion in rats

Bombesin is a potent releaser of many gut and pancreatic hormones including gastrin, glucose-dependent insulinotropic polypeptide (GIP), pancreatic polypeptide (PP), cholecystokinin (CCK), enteroglucagon, and insulin. Three mammalian bombesin-like peptides, gastrin-releasing peptide (GRP), neuromedin C (NMC or GRP-10), and neuromedin B (NMB), and two bombesin receptor subtypes, GRP preferring and NMB preferring, have been characterized. We used a highly potent, selective antagonist of the GRP-preferring receptor, [ d-Phe 6]bombesin(6–13)-methylester {[ d-Phe 6]Bn(6–13)OMe}, to determine the receptor subtype mediating bombesin-induced secretion of gastrin, GIP, PP, peptide YY (PYY), and insulin, as well as the importance of endogenous bombesin-like peptides in controlling basal secretion of these hormones. Unanesthetized rats received femoral vein infusion of saline, bombesin (10 nmol/kg/h), [ d-Phe 6]Bn(6–13)OMe (1000 nmol/kg/h), or bombesin plus [ d-Phe 6]Bn(6–13)OMe. Blood was withdrawn from jugular vein catheters before and 30 min after the start of infusions. Plasma gastrin, GIP, PP, PYY, and insulin were measured by specific radioimmunoassays. [ d-Phe 6]Bn(6–13)OMe alone reduced basal insulin levels by 28% ( p < 0.05) but did not alter basal levels of plasma PP, GIP, PYY, or gastrin ( p > 0.05 for each). Bombesin infusion significantly increased plasma levels of each hormone ( p < 0.0001 for each). [ d-Phe 6]Bn(6–13)OMe completely blocked bombesin-induced increases in PP, insulin, and gastrin, and almost completely blocked increases in GIP and PYY ( p < 0.01 for each). Our results suggest that (a) exogenous bombesin significantly stimulates PP, insulin, GIP, PYY, and gastrin secretion, (b) bombesin-induced secretion of these hormones is primarily mediated by the GRP-preferring receptor, and (c) an endogenous bombesin-like peptide acting at this receptor subtype plays an important physiological role in control of basal secretion of insulin but not PP, GIP, PYY, or gastrin.

Structure-activity requirements of bombesin for gastrin-releasing peptide- and neuromedin B-preferring bombesin receptors in rat brain

The pharmacological profile of [ 125I][Tyr 4]bombesin binding to gastrin-releasing peptide- and neuromedin B-preferring sites has been investigated in rat cerebral cortex and olfactory bulb membranes, respectively. [ 125I][Tyr 4]bombesin specific binding to cerebral cortex membranes was displaced biphasically by gastrin releasing peptide and [D-Phe 6]bombesin-(6–13)-ethyl amide. In the presence of 10 nM neuromedin B, displacement curves for bombesin-related peptides were monophasic with gastrin releasing peptide displaying approximately 100-fold higher affinity than neuromedin B. In olfactory bulb membranes, [ 125I][Tyr 4]bombesin binding was also displaced biphasically by gastrin releasing peptide, [D-Phe 6]bombesin-(6–13)-ethyl amide and neuromedin B. In the presence of 10 μM [D-Phe 6]bombesin-(6–13)-ethyl ester, displacement curves were monophasic with neuromedin B possessing approximately 10-fold higher affinity than gastrin-releasing peptide. Under these conditions, successive deletion of N-terminal amino acids from bombesin-(1–14) was well tolerated at both sites, with little loss in affinity up to bombesin-(5–14). A 5- to 10-fold drop in affinity was observed at both sites with bombesin-(6–14), whilst the octapeptide acetyl-bombesin-(7–14) displayed similar affinities to bombesin-(1–14). Bombesin-(8–14), -(9–14) and -(10–14) were essentially inactive (IC 50 > 10 μM). C-terminal deletion of Met 14 (bombesin-(1–13)) resulted in 100-fold loss of affinity at the gastrin-releasing peptide site and complete loss of affinity at the neuromedin B site. Fragments smaller than bombesin-(1–13) were virtually inactive at either site. Replacement of consecutive amino acids in the minimal active fragment, acetyl-bombesin-(7–14), with L-alanine revealed the critical importance of Trp 8 and Leu 13 for binding to both sites.

Bombesin/GRP-preferring and neuromedin B-preferring receptors in the rat urogenital system

Bombesin binding sites were localized in the rat urogenital system by autoradiography of 125I-Tyr 4-bombesin binding to frozen tissue sections. Saturable binding was observed in the bladder, seminal vesicle, uterus, and oviduct. In all organs, the binding sites corresponded to layers of smooth muscle. Radioligand binding studies were performed on homogenized membrane preparations from bladder, uterus, and seminal vesicle. Membrane binding was saturable, reversible, time- and temperature-dependent, and specific for bombesin and related peptides. Analysis of saturable equilibrium binding from all three organs yielded a best fit to a one-site model of high affinity binding with apparent K ds of 720 pM for bladder, 470 pM for uterus, and 700 pM for seminal vesicle. Neuromedin B was potent in displacing saturable 125I-Tyr 4-bombesin binding from bladder and seminal vesicle but not uterus membranes. In order to characterize these binding sites further, the ability of these membranes to interact with a specific bombesin receptor antagonist, [Leu 13-psi-CH 2NH-Leu 14]-bombesin, and with GTP-gamma-S was determined. [Leu 13-psi-CH 2NH-Leu 14]-bombesin was much more potent in displacing saturable 125I-Tyr 4-bombesin binding from uterus than from bladder and seminal vesicle membranes, further supporting the distinction between the uterus and the bladder/seminal vesicle binding sites as bombesin receptor subtypes. GTP-gamma-S inhibited saturable 125I-Tyr 4-bombesin binding to membranes from all three organs, indicating that both receptor subtypes are linked to GTP-binding proteins. We conclude that smooth muscle in the rat urogenital system expresses bombesin receptors and that endogenous GRP and neuromedin B may regulate some reproductive and excretory functions. The bladder and seminal vesicle express the neuromedin B-preferring subtype and the uterus expresses the bombesin/GRP-preferring subtype of bombesin receptor.

Ligand binding, internalization, degradation and regulation by guanine nucleotides of bombesin receptor subtypes: A comparative study

Recent cloning studies confirm two subtypes of Bn receptors exist, a neuromedin B-preferring receptor (NMB-R) and a gastrin-releasing peptide-preferring receptor (GRP-R). Both subtypes occur widely in GI tract and the CNS; however, in contrast to the GRP-R subtype little is known about the ligand-receptor interactions for the NMB-R. Therefore, in the present study we explored the ligand-receptor interactions including kinetics, stoichiometry, internalization, degradation and regulation by guanine nucleotide binding proteins with the NMB-R and compared it to the GRP-R. The rat glioblastoma C-6 cell line which possess functional NMB-R and 3T3 cells which possess functional GRP-R were used. 125I-[ d-Tyrg̊]NMB and 125I-[Tyr 4]Bn were prepared using Iodogen and purified on HPLC. At 37g̊C binding of 125I-[ d-Tyrg̊]NMB to NMB-R or 125I-[Tyr 4]Bn to GRP-R was maximal by 5–15 min and decreased to 60–70% after 60 min. HPLC analysis of the 60 min supernatant showed that > 80% of each tracer was degraded. Addition of proteinase inhibitors had a varied inhibitory effect on degradation with the relative order of potency in C-6 cells being leupeptin > bacitracin > chymostatin > phosphoramidon ⪢ bestatin and amastatin and 3T3 cells being bacitracin = phosphoramidon > leupeptin = bestatin > chymostatin > amastatin in 3T3 cells. By HPLC analysis addition of bacitracin prevented the degradation in both cell types. With both receptor subtypes dissociation of bound radioligands was slow, with 70–80% of either 125I-[ d-Tyrg̊]NMB or 125I-[Tyr 4]Bn remained cell-associated after 60 min suggesting possible peptide internalization. With an acid wash procedure to remove surface bound radioligands, 60% of the C-6 cell-associated 125I-[ d-Tyrg̊]NMB and 52% of the 3T3 cell-associated 125I-[Tyr 4]Bn were internalized after 30 min at 37°C. With membranes from cells possessing either receptor subtype, the stable guanine nucleotide GPP(NH)P inhibited in a dose-dependent fashion binding of ligands. Computer analysis demonstrated that GPP(NH)P decreased receptor affinity for ligands to both receptor subtypes. These results demonstrated that NMB receptors, similar to GRP receptors and rapidly internalize bound agonists and rapidly degrade agonists. The ligand-receptor interaction is regulated by a guanine nucleotide binding protein for both Bn receptor subtypes.

Activation of neuromedin B-preferring bombesin receptors on rat glioblastoma C-6 cells increases cellular Ca2+ and phosphoinositides.

Recent cloning studies confirm the presence of two subtypes of bombesin (Bn) receptors. In contrast to the gastrin-releasing peptide (GRP)-preferring subtype, which has been widely studied, nothing is known about the cellular mechanisms of the neuromedin B (NMB)-preferring subtype, which occurs widely in the central nervous system and gastrointestinal tissues, partially because of the lack of a cell line with functional receptors. In the present study we have investigated Bn receptors on the rat glioblastoma cell line C-6, reported to contain mRNA of the NMB receptor subtype. Binding of 125I-[D-Tyr0]NMB to these cells was time- and temperature-dependent, saturable, reversible, and only inhibited by Bn receptor agonists or antagonists. For Bn receptor agonists the relative potencies were: NMB (1.7 nM) approximately equal to litorin (3 nM) greater than ranatensin (8 nM) greater than Bn (19 nM) greater than neuromedin C (NMC) (210 nM) greater than GRP (500 nM). These relative affinities were almost identical to those for the NMB receptor subtype on rat oesophageal tissue and for Balb 3T3 cells stably transfected with the NMB receptor subtype. These potencies differed from those for the GRP receptor subtype on rat pancreatic acini [Bn approximately equal to litorin (4 nM) greater than ranatensin, NMC, GRP (15-20 nM) much greater than NMB (351 nM)]. The relative potencies of four different classes of Bn receptor antagonists were compared. Results from C-6 tumour cells agreed closely with those for binding to the NMB receptor subtype on rat oesophageal tissue and in Balb 3T3 cells stably transfected with this receptor, and differed markedly from those for binding to the GRP receptor subtype on rat pancreatic acini. Four Bn receptor antagonists had a higher affinity for the GRP subtype ([D-Phe6]Bn-(6-13)ethyl ester (500 x), [D-Phe6][psi 13-14,Cpa14]Bn- (6-14) (70 x) (where psi 13-14 refers to the replacement of the -CONH- peptide bond between Leu13 and Met14 by -CH2NH2) [psi 13-14,Leu14]Bn, [D-Phe6]Bn-(6-13) propylamide (30 x)] and two had a higher affinity for the NMB subtype on C-6 cells and transfected cells ([D-Pro4,D-Trp7,9,10] substance P-(4-11) (9 x) and [Tyr4,D-Phe12]Bn (18 x)]. In C-6 tumour cells, Bn receptor agonists caused an increase in cytosolic Ca2+ and the generation of inositol phosphates. For both responses, NMB was more than 50-fold more potent than GRP. Neither NMB nor GRP increased cyclic AMP. These results demonstrate that the rat glioblastoma cell line C-6 possesses functional NMB-preferring Bn receptors, and agonist occupation activates phospholipase C, thus increasing cytosolic Ca2+ and inositol phosphate formation. Because the interaction of Bn-related peptides with C-6 cell receptors is identical with that reported in other tissues containing the mRNA for the NMB subtype, this cell line should prove useful in exploring further the cellular basis of action of the peptides that interact with this receptor in the central nervous system and various other tissues.