Identification of bombesin receptor subtype-specific ligands: effect of N-methyl scanning, truncation, substitution, and evaluation of putative reported selective ligands.

Mammalian bombesin (Bn) receptors include the gastrin-releasing peptide receptor, neuromedin B receptor, and bombesin receptor subtype 3 (BRS-3). These receptors are involved in a variety of physiological/pathologic processes, including thermoregulation, secretion, motility, chemotaxis, and mitogenic effects on both normal and malignant cells. Tumors frequently overexpress these receptors, and their presence is now used for imaging and receptor-mediated cytotoxicity. For these reasons, there is an increased need to develop synthetic, selective receptor subtype-specific ligands, especially agonists for these receptors. In this study, we used a number of strategies to identify useful receptor subtype-selective ligands, including synthesizing new analogs (N-methyl-substituted constrained analogs, truncations, and substitutions) in [d-Tyr(6),betaAla(11),Phe(13),Nle(14)]Bn(6-14), which has high affinity for all Bn receptors and is metabolically stable, as well as completely pharmacologically characterized analogs recently reported to be selective for these receptors in [Ca(2+)](i) assays. Affinities and potencies of each analog were determined for each human Bn receptor subtype. N-Methyl substitutions in positions 14, 12, 11, 10, 9, and 8 did not result in selective analogs, with the exception of position 11, which markedly decreased affinity/potency. N-Terminal truncations or position 12 substitutions did not increase selectivity as previously reported by others. Of the four shortened analogs of [d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn(6-14) reported to be potent selective BRS-3 ligands on [Ca(2+)](i) assays, only AcPhe,Trp,Ala,His(tauBzl),Nip,Gly,Arg-NH(2) had moderate selectivity for hBRS-3; however, it was less selective than previously reported Apa(11) analogs, demonstrating these are still the most selective BRS-3 analogs available. However, both of these analogs should be useful templates to develop more selective BRS-3 ligands.

Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors.

Bombesin receptors are overexpressed on a variety of human tumors like prostate, breast, and lung cancer. The aim of this study was to develop radiolabeled (Indium-111, Lutetium-177, and Yttrium-90) bombesin analogues with affinity to the three bombesin receptor subtypes for targeted radiotherapy. The following structures were synthesized: diethylenetriaminepentaacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH1) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH2). [111In]-BZH1 and in particular [90Y]-BZH2 were shown to have high affinity to all three human bombesin receptor subtypes with binding affinities in the nanomolar range. In human serum metabolic cleavage was found between beta-Ala11 and His12 with an approximate half-life of 2 hours. The metabolic breakdown was inhibited by EDTA and beta-Ala11-His12 (carnosine) indicating that carnosinase is the active enzyme. Both 111In-labeled peptides were shown to internalize into gastrin-releasing peptide-receptor-positive AR4-2J and PC-3 cells with similar high rates, which were independent of the radiometal. The biodistribution studies of [111In]-BZH1 and [111In]-BZH2 ([177Lu]-BZH2) in AR4-2J tumor-bearing rats showed specific and high uptake in gastrin-releasing peptide-receptor-positive organs and in the AR4-2J tumor. A fast clearance from blood and all of the nontarget organs except the kidneys was found. These radiopeptides were composed of the first pan-bombesin radioligands, which show great promise for the early diagnosis of tumors bearing not only gastrin-releasing peptide-receptors but also the other two bombesin receptor subtypes and may be of use in targeted radiotherapy of these tumors.

Inhibition of growth of OV-1063 human epithelial ovarian cancers and c- jun and c- fos oncogene expression by bombesin antagonists.

Receptors for bombesin are present on human ovarian cancers and bombesin-like peptides could function as growth factors in this carcinoma. Therefore, we investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonists RC-3940-II and RC-3095 on the growth of human ovarian carcinoma cell line OV-1063, xenografted into nude mice. Treatment with RC-3940-II at doses of 10 microg and 20 microg per day s.c. decreased tumour volume by 60.9% (P< 0.05) and 73.5% (P< 0.05) respectively, after 25 days, compared to controls. RC-3095 at a dose of 20 microg per day reduced the volume of OV-1063 tumours by 47.7% (P = 0.15). In comparison, luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix at a dose of 100 microg per day caused a 64.2% inhibition (P< 0.05). RT-PCR analysis showed that OV-1063 tumours expressed mRNA for bombesin receptor subtypes BRS-1, BRS-2, and BRS-3. In OV-1063 cells cultured in vitro, GRP(14-27) induced the expression of mRNA for c- jun and c- fos oncogenes in a time-dependent manner. Antagonist RC-3940-II inhibited the stimulatory effect of GRP(14-27) on c- jun and c- fos in vitro. In vivo, the levels of c- jun and c- fos mRNA in OV-1063 tumours were decreased by 43% (P< 0.05) and 45% (P = 0. 05) respectively, after treatment with RC-3940-II at 20 microg per day. Exposure of OV-1063, UCI-107 and ES-2 ovarian carcinoma cells to RC-3940-II at 1 microM concentration for 24 h in vitro, extended the latency period for the development of palpable tumours in nude mice. Our results indicate that antagonists of bombesin/GRP inhibit the growth of OV-1063 ovarian cancers by mechanisms that probably involve the downregulation of c- jun and c- fos proto-oncogenes.

Inhibition of growth of MDA-MB-468 estrogen-independent human breast carcinoma by bombesin/gastrin-releasing peptide antagonists RC-3095 and RC-3940-II.

BACKGROUND: The growth of breast carcinoma is promoted by autocrine growth factors such as the bombesin (BN)-like peptides and epidermal growth factor (EGF). The stimulatory action of BN-like peptides can be blocked by the use of BN/gastrin-releasing peptide (GRP) antagonists. METHODS: The authors investigated the effects of synthetic BN/GRP antagonists RC-3095 and RC-3940-II on tumor growth and the expression of mRNA for EGF receptors and three BN receptor subtypes in MDA-MB-468 human breast carcinoma. Athymic nude mice with xenografts of MDA-MB-468 human breast carcinoma were injected subcutaneously for 6 weeks with RC-3940-II at doses of 20 or 40 microg/day. In another study, the effects of RC-3940-II and RC-3095 were compared. RESULTS: RC-3940-II caused a significant and dose-dependent growth inhibition of MDA-MB-468 tumors in nude mice; therapy with either dose of RC-3940-II significantly (P<0.01) reduced the mean final tumor volume and weight compared with controls. RC-3940-II induced a persistent regression of > 50% of all tumors. One of 3 tumors treated with 20 microg of RC-3940-II and 3 of 5 tumors treated with 40 microg were found to have regressed completely by the end of the study. When RC-3940-II and RC-3095 were compared at the dose of 20 microg/day, both powerfully suppressed growth of MDA-MB-468 tumors, with RC-3940-II causing a complete regression of 2 tumors and RC-3095 a complete regression of 1 tumor. Receptor analyses of untreated MDA-MB-468 tumors revealed an overexpression of EGF receptors and two classes of binding sites for BN/GRP. mRNAs for receptors of GRP, neuromedin B, and BN receptor subtype-3 were detected by reverse transcriptase-polymerase chain reaction. CONCLUSIONS: A virtual arrest of growth or regression of MDA-MB-468 human breast carcinoma after therapy with RC-3940-II and RC-3095 indicates that these BN/GRP antagonists could provide a new treatment modality for breast tumors expressing BN and EGF receptors.

Bombesin receptors in distinct tissue compartments of human pancreatic diseases.

Overexpression of receptors for regulatory peptides in various human diseases is reportedly of clinical interest. Among these peptides, bombesin and gastrin-releasing peptide (GRP) have been shown to play a physiological and pathophysiological role in pancreatic tissues. Our aim has been to localize bombesin receptors in the human diseased pancreas to identify potential clinical applications of bombesin analogs in this tissue. The presence of bombesin receptor subtypes has been evaluated in specimens of human pancreatic tissues with chronic pancreatitis (n = 23) and ductal pancreatic carcinoma (n = 29) with in vitro receptor autoradiography on tissue sections incubated with 125I-[Tyr4]-bombesin or the universal ligand 125I-[D-Tyr6, beta-Ala11, Phe13, Nle14]-bombesin(6-14) as radioligands and displaced by subtype-selective bombesin receptor agonists and antagonists. GRP receptors were identified in the pancreatic exocrine parenchyma in 17 of 20 cases with chronic pancreatitis. No measurable bombesin receptors were found in the tumor tissue of ductal pancreatic carcinomas, however, GRP receptors were detected in a subset of peritumoral small veins in 19 of 29 samples. Moreover, residual pancreatic islets in these tissues were shown to express the BB3 receptor subtype. These data demonstrate the presence of bombesin receptors in three distinct tissue compartments of the pancreas, namely GRP receptors in the exocrine parenchyma in chronic pancreatitis and in peritumoral vessels around ductal pancreatic carcinomas, and BB3 receptors in residual pancreatic islets. Such a selective expression of bombesin receptor subtypes in pancreatic tissues may not only be of pathophysiological significance but may represent the basis for potential diagnostic and therapeutic clinical applications of bombesin analogs, including GRP receptor scintigraphy to differentiate chronic pancreatitis from ductal pancreatic carcinoma.

Comparative pharmacology of the nonpeptide neuromedin B receptor antagonist PD 168368.

The mammalian peptide neuromedin B (NMB) and its receptor are expressed in a variety of tissues; however, little is definitively established about its physiological actions because of the lack of potent, specific antagonists. Recently, the peptoid PD 168368 was found to be a potent human NMB receptor antagonist. Because it had been shown previously that either synthetic analogs of bombesin (Bn) or other receptor peptoid or receptor antagonists function as an antagonist or agonist depends on animal species and receptor subtype studied, we investigated the pharmacological properties of PD 168368 compared with all currently known Bn receptor subtypes (NMB receptor, gastrin-releasing peptide receptor, Bn receptor subtype 3, and Bn receptor subtype 4) from human, mouse, rat, and frog. In binding studies, PD 168368 had similar high affinities (K(i) = 15-45 nM) for NMB receptors from each species examined, 30- to 60-fold lower affinity for gastrin-releasing peptide receptors, and >300-fold lower affinity for Bn receptor subtype 3 or 4. It inhibited NMB binding in a competitive manner. PD 168368 alone did not stimulate increases in either intracellular calcium concentration or [(3)H]inositol phosphates in any of the cells studied but inhibited NMB-induced responses with equivalent potencies in cells containing NMB receptors. PD 168368 was only minimally soluble in water. When hydroxypropyl-beta-cyclodextrin rather than dimethyl sulfoxide was used as the vehicle, both the affinity and the antagonist potency of PD 168368 were significantly greater. The results demonstrate that PD 168368 is a potent, competitive, and selective antagonist at NMB receptors, with a similar pharmacology across animal species. PD 168368 should prove useful for delineating the biological role of NMB and selectively blocking NMB signaling in bioassays and as a lead for the development of more selective nonpeptide antagonists for the NMB receptor.

The bombesin receptor subtypes have distinct G protein specificities.

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Galphaq and mouse Galphaq but not bovine retinal Galphat or bovine brain Galphai/o. The GRP-R- and NMB-R-catalyzed activations of Galphaq were dependent upon and enhanced by different betagamma dimers in the same rank order as follows: bovine brain betagamma > beta1gamma2 >> beta1gamma1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain betagamma, beta1gamma1, and beta1gamma2 and squid retinal Galphaq. In addition, GRP-R showed higher catalytic activity on squid Galphaq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgammaS binding to Galphai/o or Galphat. However, BRS-3 showed little, if any, coupling with squid Galphaq but clearly activated mouse Galphaq. GRP-R and NMB-R catalyzed GTPgammaS binding to both squid and mouse Galphaq, with GRP-R activating squid Galphaq more effectively, and NMB-R also showed slight preference for squid Galphaq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galphaq family.

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes.

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.

Role of different bombesin receptor subtypes mediating contractile activity in cat upper gastrointestinal tract.

Mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) are known to increase the motility of different segments in the gut. The present study was carried out to identify the bombesin receptor subtypes mediating the contractions induced by exogenous bombesin-like peptides in muscle strips isolated from cat esophagus, fundus, and duodenum. Both GRP-10 and NMB evoked concentration-dependent contractions in circular strips of esophagus and fundus and in longitudinal strips of the duodenum. These contractions were tetrodotoxin- and atropine-resistant. The potency of NMB in esophageal strips was 33 times higher than that of GRP-10. The NMB-preferring receptor antagonists D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2 (SSocta) and D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH2 (BIM-23127) shifted the NMB and GRP concentration-response curves to the right, while the GRP-preferring receptor antagonist [D-Phe6]Bombesin(6-13)-methyl-ester (BME) did not affect the response to the peptides. Isolated muscle strips from the cat fundus and duodenum showed a higher sensitivity to GRP-10 than to NMB. In both segments, BME shifted the GRP-10 and NMB concentration-response curves to the right, while SSocta had no effect. The antagonism of BME was competitive on duodenal but not competitive on fundic muscle. We conclude that the direct myogenic action of GRP-10 and NMB in the esophagus is mediated mainly via NMB-preferring receptors, while GRP-preferring receptors are responsible for the contractile responses to bombesin-like peptides in feline fundus and duodenum. Our data suggest that the GRP receptor population located on fundic muscle might be nonhomogeneous.

Bombesin-like peptide receptor subtypes promote mitogenesis, which requires persistent receptor signaling.

Bombesin-like peptides (BLPs) can regulate the growth of normal and transformed cells. To compare the relative activities of the three known human BLP receptor subtypes [i.e., the gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, or BLP receptor subtype 3] in growth regulation, we expressed each receptor in a receptor-deficient host, Balb/3T3 cells. None of the receptor agonists used in our study promoted DNA synthesis by quiescent parental, nontransfected Balb/3T3 cells. Using clones stably transfected with the NMB receptor however, we found that NMB stimulated the incorporation of [3H]thymidine 2.5- to 8-fold over basal levels. The greatest net stimulation of [3H]thymidine incorporation occurred when the medium contained insulin. In quiescent Balb/3T3 cells transfected with the GRP receptor, GRP promoted a 15-fold increase in DNA synthesis in the absence of insulin or other growth factors. GRP also induced the labeling of a large percentage (53%) of the cells with bromodeoxyuridine. To determine the length of time that GRP receptor signaling was required to drive quiescent cells into the S phase of the cell cycle, we blocked GRP receptor signaling by addition of a competitive GRP receptor antagonist at different times after stimulating cells with GRP. Our data demonstrate that persistent GRP receptor signaling throughout a large part of the G1 phase of the cell cycle is important in the mitogenic effects of GRP in these cells. Hitherto uncharacterized GRP receptor signaling pathways may be important in this process. BLPs also stimulated a mitogenic response by transfectants expressing the BLP receptor subtype 3 if insulin was contained in the medium. Taken together, these studies indicate that all three BLP receptor subtypes may contribute to growth regulation in vivo.

Discovery of high affinity bombesin receptor subtype 3 agonists.

Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.

Bombesin receptor structure and expression in human lung carcinoma cell lines.

Mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory neuropeptides involved in numerous physiologic processes, and have been implicated as autocrine and/or paracrine growth factors in human lung carcinoma. Three structurally and pharmacologically distinct bombesin receptor subtypes have been isolated and characterized: the gastrin releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The three receptors are structurally related, sharing about 50% amino acid identity. They are members of the G-protein coupled receptor superfamily with a seven predicted transmembrane segment topology characteristic of receptors in this family. The signal transduction pathway for GRP-R and NMB-R involves coupling to a pertussis-toxin insensitive G-protein, activation of phospholipase C (PLC), generation of inositol trisphosphate (IP3), release of intracellular calcium, and activation of protein kinase C. While all three bombesin receptors are activated by bombesin agonists, GRP-R, NMB-R, and BRS-3 have very different affinities for the mammalian bombesin-like peptides GRP and NMB, as well as bombesin receptor antagonists. The three bombesin receptor subtypes are expressed in an overlapping subset of human lung carcinoma cell lines. Any therapeutic strategy based on modulation of bombesin growth responses in human lung carcinoma would be well served to take into account the pharmacologic heterogeneity of the relevant receptors.

Peptide structural requirements for antagonism differ between the two mammalian bombesin receptor subtypes.

Recently it has been established that both a gastrin-releasing peptide (GRP) receptor and a neuromedin B (NMB) receptor mediate the actions of bombesin-related peptides in mammals. Five different classes of peptides that function as GRP receptor antagonists have been identified; however, it is unknown whether similar strategies will yield antagonists for the closely related NMB receptor. In the present study we have used either native cells possessing only one bombesin (Bn) receptor subtype or cells stably transfected with one subtype to determine whether using the strategies that were used successfully for GRP receptors would allow NMB receptor antagonists to be identified. [DPhe12]Bn analogs; des Met14 amides, esters and alkylamides; psi 13-14 Bn pseudopeptides; and D-amino acid-substituted analogs of substance P (SP) or SP(4-11) were all synthesized and each functioned as a GRP receptor antagonist. All of these antagonists had low affinity for the NMB receptor. Application of similar strategies to NMB by formation of [DPhe8]NMB, [psi 9-10]NMB pseudopeptides, des-Met10 NMB amides, alkylamide or esters did not result in any potent NMB receptor antagonists. D-Amino acid SP and SP(4-11) analogs were weakly selective NMB receptor antagonists. No COOH-terminal fragments of NMB or GRP functioned as a GRP or NMB receptor antagonist. These results demonstrate that none of the known strategies used to prepare peptide GRP receptor antagonists are successful at the NMB receptor, suggesting that a different strategy will be needed for this peptide, such as the formation of somatostatin octapeptide or D-amino acid-substituted substance P analogs. These results suggest that even though there is a close homology between GRP and NMB and their receptors, their structure-function relations are markedly different. These results indicate that the development of receptor subtype-specific peptide agonists or peptide antagonists for newly discovered receptor subtypes of gastrointestinal hormones/neurotransmitters may be difficult because the strategies developed for one well-studied subtype may not apply to the other even though it is structurally closely related.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

Different types of bombesin receptors on neurons in the dorsal raphe nucleus and the rostral hypothalamus in rat brain slices in vitro.

The actions of the peptides bombesin (BN), gastrin releasing peptide (GRP), neuromedin C (NMC), litorin and neuromedin B (NMB) were studied on neurons in slices of rat brain maintained in vitro to determine the BN receptor type present in different brain areas. Intracellular and extracellular recordings were made from hypothalamic neurons on the border of the periventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) and from mesencephalic 5-HT sensitive neurons in the dorsal raphe nucleus. In the region of the brain containing the SCN and PVN, BN and the BN-related peptides excited 31 out of 77 neurons on which they were tested. There was little difference in the potency of the BN-related peptides as excitants of neurons, the EC50 being about 10 nM. The response to the peptides usually lasted between 5 and 15 min with little sign of desensitization. Using NMC, GRP and NMB as agonists, the equilibrium constant for the GRP receptor antagonist [D-Phe6]-BN-(6-13)-ethylamide was approximately 10 nM. The response to the peptides fully recovered on washout of the antagonist. The CCKB/gastrin receptor antagonist CI-988 (1 microM) had no effect on either GRP- or NMC-mediated excitation. In the dorsal nucleus 40 of 75 neurons were sensitive to the BN-related peptides. BN, [Tyr4]-BN, NMB and litorin, were 10-20 times more potent than GRP and NMC. The responses to the BN-related peptides were not blocked by the selective GRP receptor antagonists [D-Phe6]-BN-(6-13)-methylester, [DF5Phe6][D-Ala11]-BN-(6-13)-methylester and [D-Phe6]-BN-(6-13)- ethylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin receptors in a human duodenal tumor cell line: binding properties and function.

The bombesin family of peptides elicit numerous biological responses in the gut, including stimulation of cell proliferation, and have been implicated as growth factors in a variety of gastrointestinal tumors. Even though these peptides and their receptors are distributed throughout the gastrointestinal tract, there are few cell lines available as model systems to study bombesin action in gastrointestinal cells. In this study, we have characterized functional bombesin receptors in a human duodenal cancer cell line, HuTu-80. The binding of [125I-Tyr4]bombesin to intact cells at 4 degrees C reached equilibrium by 6 h. Scatchard analysis of [125I-Tyr4]bombesin binding showed that HuTu-80 cells contained a single class of high affinity binding sites (5900 +/- 1960/cell; Kd = 80 +/- 20 pM). [125I-Tyr4]bombesin binding was inhibited by bombesin receptor agonists and antagonists with the following order of potencies: gastrin-releasing peptide (GRP) = GRP-(14-27) = bombesin > [DPhe6]bombesin(6-13)ethylamide > [Leu13 psi-(CH2NH)Leu14]bombesin > neuromedin B. Photoaffinity cross-linking studies, in which N-5-azido-2-nitrobenzoyloxysuccinimide was used to covalently couple [125I]GRP(14-27) to cells at 4 degrees C, resulted in the specific labeling of a broad band with an apparent molecular mass of 66,000 daltons. Consistent with the presence of high affinity receptors, bombesin increased the formation of inositol phosphates in HuTu-80 cells in a dose-dependent manner (concentration eliciting half-maximal effect, 290 +/- 70 pM). However, under conditions where both insulin and serum increased [3H]thymidine incorporation into DNA, 10 nM bombesin had no effect either alone or in the presence of insulin. Bombesin also had no effect on colony formation by HuTu-80 cells in soft agar. Furthermore, the bombesin receptor antagonist, [Leu13 psi(CH2NH)Leu14]bombesin, did not inhibit [3H]thymidine incorporation or clonal growth either in the absence or in the presence of serum. Together, these results show that HuTu-80 cells contain high affinity bombesin receptors of the GRP subtype. These receptors are functionally coupled to second messenger production but do not stimulate cell proliferation.

GRP-preferring bombesin receptor subtype mediates contractile activity in cat terminal ileum.

Mammalian bombesin-like peptides, such as gastrin-releasing peptide and neuromedin B, are known to increase motility of different segments in the gut. The present study was carried out to identify the receptor subtype mediating these contractions of ileal longitudinal muscles in cats, in vitro. Both gastrin-releasing peptide (GRP) and neuromedin B (NMB) evoked concentration-dependent contractions of the strips. [D-Phe6]Bombesin(6-13)-methyl-ester, a highly selective GRP-preferring receptor antagonist, competitively inhibited contractions induced by either agonist. On the other hand, D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2, a selective NMB receptor antagonist, did not affect the actions of either gastrin-releasing peptide or neuromedin B. Our results suggest that bombesin-like peptides contract cat terminal ileum via activating GRP receptors.

Receptor-activated currents in mouse fibroblasts expressing transfected bombesin receptor subtype cDNAs.

BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M). N-acetyl-GRP-(20-26) amide, [Leu13-psi(CH2NH)-Leu14]Bn, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also blocked GRP Rt responses but at higher concentrations (10(-5) M). However, at these concentrations, these four antagonists had little effect on NmB Rt responses, thereby showing a specificity of these antagonists for the GRP receptors.