Search

p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases, which are divided into two groups: group-I (PAKs1-3) and group-II (PAKs4-6). In various tissues, Group-II PAKs play important roles in cytoskeletal dynamics and cell growth as well as neoplastic development/progression. However, little is known about Group-II PAK's role in a number of physiological events, including their ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth factors (GFs). We used rat pancreatic acini to explore the ability of GI hormones/neurotransmitters/GFs to activate Group-II-PAKs and the signaling cascades involved. Only PAK4 was detected in pancreatic acini. PAK4 was activated by endothelin, secretagogues-stimulating phospholipase C (bombesin, CCK-8, and carbachol), by pancreatic GFs (insulin, insulin-like growth factor 1, hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor), and by postreceptor stimulants (12-O-tetradecanoylphobol-13-acetate and A23187 ). CCK-8 activation of PAK4 required both high- and low-affinity CCK 1 -receptor state activation. It was reduced by PKC-, Src-, p44/42-, or p38-inhibition but not with phosphatidylinositol 3-kinase-inhibitors and only minimally by thapsigargin. A protein kinase D (PKD)-inhibitor completely inhibited CCK-8-stimulated PKD-activation; however, stimulated PAK4 phosphorylation was only inhibited by 60%, demonstrating that it is both PKD-dependent and PKD-independent. PF-3758309 and LCH-7749944, inhibitors of PAK4, decreased CCK-8-stimulated PAK4 activation but not PAK2 activation. Each inhibited ERK1/2 activation and amylase release induced by CCK-8 or bombesin. These results show that PAK4 has an important role in modulating signal cascades activated by a number of GI hormones/neurotransmitters/GFs that have been shown to mediate both physiological/pathological responses in acinar cells. Therefore, in addition to the extensive studies on PAK4 in pancreatic cancer, PAK4 should also be considered an important signaling molecule for pancreatic acinar physiological responses and, in the future, should be investigated for a possible role in pancreatic acinar pathophysiological responses, such as in pancreatitis. NEW & NOTEWORTHY This study demonstrates that the only Group-II p21-activated kinase (PAK) in rat pancreatic acinar cells is PAK4, and thus differs from islets/pancreatic cancer. Both gastrointestinal hormones/neurotransmitters stimulating PLC and pancreatic growth factors activate PAK4. With cholecystokinin (CCK), activation is PKC-dependent/-independent, requires both CCK 1 -R affinity states, Src, p42/44, and p38 activation. PAK4 activation is required for CCK-mediated p42/44 activation/amylase release. These results show PAK4 plays an important role in mediating CCK physiological signal cascades and suggest it may be a target in pancreatic acinar diseases besides cancer.

A synthetic peptide, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transfected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein kinase phosphorylation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mitogen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 activation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased c-fos as well as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold increase in c-fos mRNA caused by 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), Pro(26), Psi(26,27), Phe(27))GRP-(20-27) (BW2258U89), a GRP receptor antagonist. These results indicate that (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased nuclear oncogene expression and upstream events include mitogen activated protein kinase phosphorylation and Elk-1 activation.

The effects of some oncogenes, growth factors and neuropeptides are mediated by tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and paxillin cytoskeletal proteins. In this study the ability of bombesin/gastrin releasing peptide (BB/GRP) to stimulate tyrosine phosphorylation of p125(FAK) and paxillin in non-small cell lung cancer (NSCLC) H1299 cells was investigated. BB, 100 nM caused increased p125(FAK) and paxillin tyrosine phosphorylation maximally after 1 min. The effect of BB on p125(FAK) and paxillin tyrosine phosphorylation was concentration-dependent, being half maximal at 4-8 nM. Also, 100 nM GRP, GRP(14-27) but not GRP(1-16) increased p125(FAK) and paxillin tyrosine phosphorylation indicating that the C-terminal of GRP is essential. BW2258U89, a GRP receptor antagonist, caused a dose-dependent inhibition of BB-stimulated p125(FAK) and paxillin tyrosine phosphorylation with an IC50 value of 3 microM. Cytochalasin D (0.3 microM), which inhibits actin polymerization, reduced the ability of BB to stimulate tyrosine phosphorylation of p125(FAK) and paxillin. Genistein (50 microM) and H-7 (50 microM), which are kinase inhibitors, reduced the tyrosine phosphorylation of p125(FAK) and paxillin stimulated by BB. Also, treatment of NCI-H1299 cells with FAK antisense resulted in decreased FAK tyrosine kinase activity and proliferation. These results suggest that p125(FAK) is an important enzyme for NSCLC proliferation.

Recently, a fourth member of the bombesin (Bn) receptor family (fBB4-R) was isolated from a cDNA library from the brain of the frog, Bombina orientalis. Its pharmacology and cell biology are largely unknown, and no known natural cell lines or tissues possess sufficient numbers of fBB4-R's to allow either of these to be determined. To address these issues, we have used three different strategies. fBB4-R expression in cells widely used for other Bn receptor subtypes was unsuccessful as was expression in two frog cell lines. However, stable fBB4-R cell lines were obtained in CHO-K1 cells which were shown to faithfully demonstrate the correct pharmacology of the related Bn receptor, the GRP receptor, when expressed in these cells. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) was found to have high affinity (Ki = 0.4 nM) for the fBB4 receptor and 125I-[DTyr6,betaala11,Phe13,Nle14]Bn(6-14) to be an excellent ligand for this receptor. The fBB4-R had a unique pharmacology for naturally occurring Bn-related agonists, with the presence of a penultimate phenylalanine being critical for high-affinity interaction. It also had a unique profile for six classes of Bn antagonists. The fBB4-R was coupled to phospholipase C with activation increasing [3H]inositol phosphates and mobilizing Ca2+ almost entirely from cellular sources. There was a close correlation between agonist the receptor occupation and the receptor activation. Three of the five classes of Bn receptor antagonists that interacted with higher affinity with the fBB4-R functioned as fBB4-R antagonists and two as partial agonists. fBB4-R activation stimulated increases in phospholipase D (PLD) over the same range of concentrations at which it activated phospholipase C. These results demonstrate that the fBB4 receptor has a unique pharmacology for agonists and antagonists and is coupled to phospholipase C and D. The availability of these cell lines, this novel ligand, and the identification of three classes of antagonists that can be used as lead compounds should facilitate the further investigation of the pharmacology and cell biology of the BB4 receptor.

The gastrin-releasing peptide receptor (GRP-R) is one of three members of the mammalian bombesin subfamily of seven-transmembrane G protein-coupled receptors that mediate diverse biological responses including secretion, neuromodulation, chemotaxis, and growth. The X chromosome-linked GRP-R gene is expressed widely during embryonic development and predominantly in gastrointestinal, neuronal, and neuroendocrine systems in the adult. Surprisingly, gene-targeted mice lacking a functional GRP-R gene develop and reproduce normally and show no gross phenotypic abnormalities. However, peripheral administration of bombesin at dosages up to 32 nmol/kg to such mice had no effect on the suppression of glucose intake, whereas normal mice showed a dose-dependent suppression of glucose intake. These data suggest that selective agonists for the GRP-R may be useful in inducing satiety.

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.

We characterized the bombesin receptor population in the rat stomach and determined the receptor subtype mediating the contractile effect of bombesin in the gastric fundus. Using in vitro receptor autoradiography, we evaluated the ability of the specific gastrin-releasing peptide-preferring receptor antagonist [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester to inhibit binding of 125I-[Tyr4]bombesin to the gastric fundus, corpus and antrum. Binding to these regions was completely inhibited by [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester suggesting that these receptors are the gastrin-releasing peptide-preferring subtype. We found that the rank order of potency for the contractile effect of bombesin, and the related mammalian peptides neuromedin C and neuromedin B, was bombesin > neuromedin C > neuromedin B. [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester was equipotent in antagonizing contractions produced by all three peptides. Furthermore, receptor tachyphylaxis to either neuromedin C or neuromedin B abolished the subsequent contractile response elicited by neuromedin C and neuromedin B, suggesting that one bombesin receptor subtype mediates rat gastric fundal contractions. Together, these results demonstrate that the bombesin receptor subtype in the rat stomach is gastrin-releasing peptide-preferring subtype and that this subtype is responsible for the effects of bombesin-like peptides on fundal smooth muscle contraction.

The cellular basis of down-regulation and desensitization in phospholipase C-linked receptors is unclear. Recent studies with some receptors suggest that elements in the carboxyl terminus of the receptor are important in mediating these processes. Three mutant gastrin-releasing peptide receptors (GRP-R) were studied: one whose last 37 carboxyl-terminal amino acids were eliminated (construct MGT346); one that replaced all of the carboxyl-terminal Ser and Thr eliminated in MGT346 with Ala, Asn, or Gly (construct JF1); and one that selectively replaced the Ser and Thr of the protein kinase C consensus sequence (PKC-CS) located within the same region with alanine (construct TS360AA). Desensitization was assessed by measuring the ability to activate phospholipase C and increase cellular [3H]inositol phosphates, or increase [Ca2+]i, after pre-exposure to 3 nM bombesin for 24 h. Wild-type GRP-R was maximally desensitized and down-regulated after a 24-h exposure to 3 nM bombesin, and removal of the PKC-CS alone markedly attenuated each process. Elimination of additional serines and threonines by truncation (MGT346) or replacement (JF1) did not decrease down-regulation or desensitization further. To confirm the necessity of second messenger activation in mediating down-regulation, we further investigated two additional mutant GRP-R that bound agonist with high affinity but fail to activate phospholipase C (constructs R139G and A263E). Neither construct underwent significant down-regulation. Removal of all GRP-R carboxyl-terminal Ser or Thr, either by MGT346 or JF1, reduced internalization by > 80%, whereas elimination of the PKC-CS in TS360AA only attenuated internalization by 21 +/- 2%. These data suggest that activation of the distal carboxyl-terminal PKC-CS is essential for chronic desensitization and down-regulation of the GRP-R, and provide no evidence for involvement of second messenger-independent processes. In contrast, internalization is equally regulated by both second messenger-dependent and independent processes.

Bombesin (BN)-related peptides, such as gastrin-releasing peptide (GRP), have been shown in vivo to stimulate release of pepsinogen. However, whether this is due to a direct interaction with chief cells is not clear. To clarify this we prepared isolated chief cells (> 90% pure) from guinea pig stomach. BN, GRP, or neuromedin B (NMB), at concentrations up to 1 microM, did not stimulate pepsinogen release or affect the stimulation caused by vasoactive intestinal peptide (VIP) (100 nM) or CCK-8 (10 nM), respectively. In addition, BN, GRP, or NMB at a concentration of 1 microM did not increase cAMP nor did they alter the increase in cAMP caused by VIP or secretin. BN (1 microM) did not alter basal cytosolic calcium [Ca2+]i or affect the increase in [Ca2+]i caused by CCK-8 (1 microM). Furthermore, BN, GRP, or NMB at a concentration of 1 microM did not increase the generation of inositol phosphates (IP) or alter the increase in [3H]IP1, [3H]IP2, or [3H]IP3, caused by CCK-8 (1 microM) or carbachol (1 mM). Binding studies demonstrated no saturable binding of either [125I][Tyr4]BN or [125I][D-Tyr0]NMB using experimental conditions where binding with other peptide ligands to other receptors on chief cells is seen. We conclude that BN-related peptides do not interact directly with specific receptors on chief cells to stimulate or alter stimulated pepsinogen secretion, increase the breakdown of inositol phosphates, or alter [Ca2+]i or cAMP.

Analogues of the amphibian neuropeptide, bombesin, and of the mammalian homologue, gastrin-releasing peptide, have been synthesized and their biological activity studied in small cell lung carcinoma and rat pancreatic acinar cells. The compounds are truncated sequences of the active tetradecapeptide BN(1-14) or GRP(20-27). Peptides were cyclized between position 5 or 7 and the carboxyl end of the des-Met14 fragment with D and L Ala11 and Lys5 substitutions, as well as various N-terminal groups attached. The smallest cyclic peptide, BN(7-13), bound to SCLC membranes with microM potency and inhibited BN stimulation of intracellular Ca++ levels. The most potent inhibitor is N-chloroambucil-[His7,D-Ala11]BN(7-13)ethyl ester, which antagonized BN function in SCLC and acinar cells with nM potency and also inhibited clonal growth of carcinoma cell lines.

Most seven-transmembrane G-protein-coupled receptors are rapidly internalized after binding agonist, but the general amino acid recognition sequences mediating this phenomenon have not been identified. In this study, components of the gastrin-releasing peptide receptor (GRP-R) regulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoylation sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and internalized agonist similarly to wild type receptor. Progressively larger truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affinity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341AA), one with the carboxyl-terminal protein kinase C-consensus sequence converted to Ala (TS360-361AA), and one with all Ser and Thr distal to Cys341 converted to Ala, Asn, or Gly (JF1). All constructs bound agonist similarly to wild type receptor. CC340-341AA internalized similarly to native receptor (93 +/- 3% of wild type by 60 min), whereas internalization of TS360-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). JF1, however, internalized as poorly as T346, with only 16 +/- 2% of the wild type receptors internalized by 60 min. To assess G-protein coupling, selected receptor constructs were stably transfected into Balb fibroblasts, and phosphoinositol hydrolysis was determined. The largest GRP-R truncation, T346, increased total inositol phosphates (EC50 = 2.9 +/- 0.9 nM) similarly to wild type receptor (EC50 = 5.1 +/- 2.2 nM), as did CC340-341AA (EC50 = 5.4 +/- 1.5 nM) and TS360-361AA (EC50 = 3.1 +/- 1.2 nM). These data demonstrate that the multiple Ser and Thr located within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-consensus sequence, specifically regulate GRP-R internalization rates independent of receptor-G-protein coupling.

Bombesin (Bn)-related peptides affect numerous cell functions; however, receptor characterization by radiolabeled ligands is limited because only radiolabeled agonists exist. In the present study we demonstrate that [D-Tyr6]Bn(6-13)methyl ester [[D-Tyr6]Bn(6-13)ME] functions as a Bn receptor antagonist with high affinity. The binding of both the radiolabeled agonist 125I-[Tyr4]Bn and the radiolabeled antagonist 125I-[D-Tyr6]Bn(6-13)ME to AR42J cells, murine 3T3 cells, and dispersed guinea pig pancreatic acini was time and temperature dependent, saturable, and reversible. Binding of the antagonist more rapidly reached equilibrium and was more rapidly reversible. Guanine nucleotides did not affect binding of the radiolabeled antagonist, whereas guanosine-5'-(beta,gamma-imido)triphosphate decreased agonist binding by decreasing Bn receptor affinity. Acid stripping studies demonstrated that the radiolabeled agonist, but not the antagonist, was internalized in each cell system. Bn receptor affinities for various Bn receptor agonists or antagonists in each cell system were identical when computed from an analysis of inhibition curves for binding of radiolabeled agonist or antagonist. However, with AR42J cells and 3T3 cells the radiolabeled agonist demonstrated a > 2-fold higher number of Bn receptors than did the radiolabeled antagonist. Binding studies using cell membranes, in contrast to cells, showed equal numbers of Bn receptors with either radiolabeled ligand. The radiolabeled agonist demonstrated high affinity binding to both rat pancreatic acinar and esophageal muscularis mucosa membranes, whereas the radiolabeled antagonist interacted with high affinity only with the gastrin-releasing peptide-preferring subtype of Bn receptors on pancreatic tissue. These results demonstrate that 125I-[D-Tyr6]Bn(6-13)ME is a high affinity radiolabeled antagonist that interacts specifically with Bn receptors. In contrast to the radiolabeled agonist, binding of the antagonist is not affected by guanine nucleotides and it is not internalized, which allows quantitation of only Bn cell surface receptors. Furthermore, the radiolabeled antagonist can distinguish Bn receptor subtypes, whereas the radiolabeled agonist does not. This ligand should prove useful for characterizing Bn receptors as well as studying their regulation.

Previous studies have shown that various bombesin receptor antagonists can distinguish between bombesin receptor subtypes in peripheral tissues. To determine whether these antagonists would be useful in differentiating bombesin receptor subtypes in the rat central nervous system, we used in vitro receptor autoradiography to examine the binding affinities of several bombesin receptor antagonists for two brain regions we had characterized as possessing distinct bombesin receptor subtypes. Our results demonstrate that, consistent with peripheral bombesin receptors, bombesin receptor subtypes in the rat brain can be differentiated by various bombesin receptor antagonists.

Bombesin/gastrin releasing peptide (BN/GRP) receptors were solubilized and purified from human glioblastoma (U-118) and lung carcinoid cell lines (NCI-H720). The U-118 cells, when extracted with CHAPS/cholesterol hemisuccinate (CHS), bound (125I-Tyr4)BN with high affinity (Kd = 2 nM) to a single class of sites (Bmax = 150 fmol/mg protein). Specific (125I-Tyr4)BN binding was inhibited with high affinity by BN, GRP, GRP14-27, and receptor antagonists such as (D-Phe6)BN6-13methylester(ME) and (D-Phe6)BN6-13 propylamide(PA) (IC50 = 2, 22, 3, 1 and 2 nM, respectively) but not GRP1-16 or BN1-12. The solubilized and cellular receptor bound peptides with similar affinity. The solubilized receptor was purified using (Lys0, Gly1-4, D-Ala5)BN and (Lys3, Gly4,5, D-Tyr6)BN3-13 PA affinity resins. When eluted from the affinity resins by NaCl, the receptor bound (125I-D-Tyr6)BN6-13ME with high affinity. The NCI-H720 BN/GRP receptor was purified 86,000-fold after extraction with CHAPS/CHS and purification using both affinity resins. SDS-PAGE analysis indicated that major 65 and 115 kDa proteins were purified. These data indicate that BN/GRP receptors can be solubilized from human cells and purified using affinity chromatography techniques with retention of ligand binding activity.

We have previously demonstrated the presence of two distinct bombesin receptor subtypes in the rat CNS and distinguished them as bombesin/gastrin-releasing peptide (BBS/GRP) and neuromedin B (NMB)-preferring binding sites. In the present study, we conducted a complete evaluation of the distribution of these binding sites throughout the rat brain using in vitro receptor autoradiography. The BBS/GRP-preferring binding sites were characterized as those that bound 125I-(Tyr4)BBS but not 125I-(D-Tyr0)NMB. At these sites 125I-(Tyr4)BBS binding was inhibited in the presence of 100 nM BBS but not by the same concentration of NMB. In contrast, NMB-preferring sites bound both radioligands and binding at these sites was inhibited in the presence of 100 nM NMB. Our results indicate that the distributions of BBS/GRP and NMB-preferring binding sites are widespread and distinct at all levels of the rat brain suggesting these peptides mediate separate functions in the rat central nervous system.

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.

Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.

Recent studies suggest that different subtypes of receptors may mediate the action of various bombesin-related peptides in different tissues. In the present study the ability of bombesin and its structurally related peptides [litorin, gastrin-releasing peptide (GRP), GRP18-27, neuromedin B, [Leu8]litorin, and bombesin nonapeptide BN(6-14)] to interact with smooth muscle cells isolated from guinea pig stomach was investigated. Each peptide induced a specific contractile response with potencies (D50 in pM) of [Leu8]litorin (0.7) greater than bombesin (1.2) greater than litorin (3) greater than neuromedin B (3.5) = GRP (3.8) = GRP18-27 (3.9) greater than BN(6-14) (70.9). The specific bombesin receptor antagonist psi 13,14-bombesin differed in its potency for inhibiting equipotent concentrations of bombesin, GRP, or neuromedin B, was equipotent for bombesin or GRP (IC50 12.7 and 22.1 nM), and was 11 times less potent for neuromedin B (IC50 234.5 nM), suggesting the presence of subtypes of receptors mediating the action of bombesin-related peptides. To further investigate this possibility, a technique of receptor protection that enables selective preservation of one receptor type was used. GRP or bombesin protected completely the response to GRP or bombesin but abolished the subsequent contractile response to neuromedin B. Neuromedin B, instead, protected only the response to neuromedin B. These results demonstrate that gastric smooth muscle cells possess specific receptors that interact with bombesin-related peptides and that two receptor subtypes mediate the contractile response to these peptides: one subtype is selective for bombesin or GRP, the other for neuromedin B.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.

As an initial characterization of bombesin binding sites in the rat central nervous system, we examined the pharmacological specificity of binding of bombesin (BBS) and several BBS analogs to rat cortex and compared these results to those for rat pancreas. In addition, we used in vitro receptor autoradiography to evaluate binding of 125I-[Tyr4]bombesin and 125I-Bolton-Hunter neuromedin B (NMB) to several regions of the rat brain. The results of the pharmacological study indicated that the pancreas and cortex had different binding affinities for BBS-like peptides. While cortical binding sites had a high affinity for NMB, pancreatic binding sites had almost no affinity for NMB. Results from the autoradiographic study demonstrated that BBS receptor heterogeneity exists in individual nuclei in the rat brain. Some nuclei have a high affinity for NMB, similar to cortical BBS binding sites, other regions have a low affinity similar to pancreatic BBS binding sites. These results provide evidence that subtypes of BBS receptors are present in different tissues and within discrete regions of the rat central nervous system.

Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18-27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of 125I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used 125I-GRP and chemical cross-linking techniques to characterize the mouse pancreatic BN receptor. After binding of 125I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18-27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of 125I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure.

In the present study we examined the effect of carboxyl-terminal modifications of des-Met14-bombesin (Bn) on Bn receptor affinity in murine 3T3 cells, rat and guinea pig pancreatic acini, and the ability to initiate biologic responses by synthesizing 18 des-Met14-Bn(6-13) analogues. With guinea pig acini and 3T3 cells, affinity was affected by the chain length of the alkyl moiety (R) added to [D-Phe6]Bn(6-13)NH2R with relative potencies: propyl greater than ethyl greater than butyl = hexyl greater than heptyl greater than free amide, whereas in rat acini affinity was not increased by the chain length. In each cell system the affinity of the alkylamide was not increased by insertion of a phenyl group in the alkyl side chain, by making the analogue more neuromedin B-like or by addition of a reduced peptide bond. The affinity in each cell system was increased by additions of other electron releasing groups to the COOH-terminal carboxyl group such as [D-Phe6]Bn(6-13)ethyl or methyl ester, or hydrazide. In guinea pig pancreas and 3T3 cells, 12 analogues were antagonists, 1 a full and 5 partial agonists. In rat pancreas, 8 were antagonists, 5 full agonists, and 5 partial agonists. Potent antagonists in each cell system were the methyl and ethyl ester, hydrazide, and ethylamide analogues. In 3T3 cells or guinea pig pancreas, agonist activity of the alkylamide was critically dependent on the chain length, whereas with rat pancreatic Bn receptors any alkylamide longer than the ethylamide had agonist activity. In all three cell systems any alteration that made the alkylamide more neuromedin B-like caused agonist activity. These results demonstrate that the nature of the substitution on the carboxyl terminus of des-Met14-Bn analogues is critically important, not only for determining Bn receptor affinity, but also for determining the ability to initiate a biologic response. In contrast to previous studies, the present results demonstrate that the presence of the COOH-terminal amino acid in position 14 of Bn is not essential for initiating a biologic response. Several des-Met14-Bn analogues were potent partial agonists, whereas others such as the hydrazide or ethyl ester are very potent antagonists.

To determine whether newly described bombesin (BN) receptor antagonists distinguish subtypes of BN receptors, we investigated their abilities to interact with BN receptors on esophageal muscle or pancreatic acinar tissue. For inhibition of binding of 125I-[Tyr4]BN to rat pancreatic tissue, the relative potencies were [D-Phe6]BN-(6-13)ethyl ester (5 nM) greater than Ac-gastrin-releasing peptide (GRP)-(20-26)ethyl ester (17 nM) greater than [D-Phe6,Cpa,14, psi 13-14]BN-(6-14) (40 nM) greater than [Leu14, psi 13-14]BN (0.43 microM) greater than [Tyr4,D-Phe12]BN = [D-Pro4, D-Trp7,9,10]substance P (SP)-4-11 (13 microM) greater than [Leu14, psi 9,10]BN (32 microM) greater than [D-Arg1,D-Trp7,9,Leu11]SP (70 microM). Each antagonist also inhibited binding of 125I-[Tyr4]BN or 125I-Bolton-Hunter-neuromedin B to rat esophageal tissue, and the potency of each antagonist for each tracer was similar. In comparison to rat pancreas, [D-Phe6]BN-(6-13)ethyl ester, Ac-GRP-(20-26)ethyl ester, [D-Phe6,Cpa14, psi 13-14]BN-(6-14), [Leu14, psi 13-14]BN, and [Leu14, psi 9,10]BN had a 10,000-, 2,940-, 1,425-, 122-, and 4-fold, respectively, weaker affinity for BN receptors. In contrast [Tyr4,D-Phe12]BN, [D-Pro4,D-Trp7,9,10]SP-4-11, and [D-Arg1,D-Trp7,9,Leu11]SP had a 4-, 4-, and 9-fold, respectively, higher affinity compared with pancreatic tissue. Comparison of the activity of each peptide at inhibiting the ability of equipotent concentrations of BN or neuromedin B to stimulate contraction of rat esophageal muscle demonstrated that each peptide had the same relative potencies as for inhibiting binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin-related peptides have a large number of physiological functions as well as having an autocrine growth mechanism for the regulation of small cell lung cancer cells. In the present study we have synthesized 21 des-Met amide or alkylamide analogues of bombesin and compared their abilities to function as bombesin receptor antagonists in guinea pig pancreatic acini and Swiss 3T3 cells with those of the previously most potent antagonist described, [Leu13 psi(CH2NH)Leu14]bombesin (analogue I). All des-Met analogues functioned as antagonists. Bn(1-13)NH2 was approximately equipotent to I (Ki = 60-80 nM) whereas Bn(6-13)NH2 was 30-fold less potent (Ki = 1800 nM). Formation of an ethylamide, Bn(6-13)ethylamide, increased the potency 30-fold such that this octapeptide was equipotent to I. The addition of a D-Phe6 moiety to I did not change potency but caused a 30-fold increase in potency of Bn(6-13)NH2 and a 8-fold increase in the potency of Bn(6-13)ethylamide (Ki = 16 nM). Additional studies of both NH2- and COOH-terminal alterations in Bn(6-13)NH2 demonstrated that the most potent antagonist was [D-Phe6]Bn(6-13)propylamide (PA), having IC50's of 1.6 nM and 0.8 nM for bombesin-stimulated amylase release and Swiss 3T3 cell growth, respectively. Detailed studies of the most potent amide analogue, [D-Phe6]Bn(6-13)NH2, and alkylamide analogue, [D-Phe6]Bn(6-13)PA, demonstrated that these analogues functioned as competitive antagonists and that their action was selective for the bombesin receptor. These results demonstrate that, as with CCK- and gastrin-related peptides, the C-terminal amino acid is important for initiating a biologic response but not essential for determining receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

In dispersed acini from guinea pig pancreas two inhibitors of cyclic nucleotide phosphodiesterase, Ro 20-1724 and 3-isobutyl-1-methylxanthine (IBMX), augmented the increase in amylase secretion caused by supramaximal concentrations of cholecystokinin but did not alter the stimulation of enzyme secretion caused by bombesin. The augmentations of the action of cholecystokinin caused by Ro 20-1724 or IBMX could be reproduced by 8-bromo-cAMP. When tested alone or with theophylline, cholecystokinin did not alter cAMP in pancreatic acini; however, with Ro 20-1724 or IBMX, concentrations of cholecystokinin that were supramaximal for stimulating amylase secretion caused a significant increase in cellular cAMP. These findings indicate that Ro 20-1724 and IBMX augment the action of cholecystokinin on enzyme secretion by inhibiting cyclic nucleotide phosphodiesterase and allowing a significant cholecystokinin-induced increase in cellular cAMP. IBMX but not Ro 20-1724 caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion caused by carbachol. IBMX also caused a parallel rightward shift in the dose-response curve for the stimulation of outflux of 45Ca caused by carbachol. These results indicate that IBMX, but not Ro 20-1724, can function as a muscarinic cholinergic antagonist.

When dispersed acini from mouse pancreas are first incubated with bombesin, washed, and then reincubated with fresh incubation solution containing no bombesin, there is significant residual stimulation of amylase release. Induction of residual stimulation is relatively rapid in that significant stimulation occurs as early as after 15 s of first incubation with bombesin. Induction of residual stimulation of amylase release per se is temperature independent, but induction does occur more rapidly when acini are first incubated at 37 degrees C than when they are first incubated at 4 degrees C. Residual stimulation of amylase release persists for at least 75 min in acini that have been first incubated with bombesin at 37 degrees C. The maximal residual stimulation of amylase release obtained with pancreatic acini that have been first incubated with bombesin and then washed is 45% greater than the maximal stimulation obtained when bombesin is added directly to the incubation medium. In terms of their abilities to cause residual stimulation of amylase release, litorin and ranatensin are equal to bombesin in potency and efficacy. Gastrin-releasing peptide is approximately 70% as efficacious as bombesin in causing residual stimulation of amylase release.

Previous attempts to develop analogues of bombesin that function as specific receptor antagonists have been unsuccessful. Alteration of the histidine in luteinizing hormone releasing factor has resulted in analogues that function as competitive antagonists. In the present study we have used a similar strategy and altered the histidine in bombesin. [D-Phe12]bombesin, [D-Phe12,Leu14]bombesin, and [Tyr4,D-Phe12]bombesin did not stimulate amylase release from guinea pig pancreatic acini when present alone, but each analogue inhibited bombesin-stimulated secretion. For each analogue, detectable inhibition occurred at 1 microM and half-maximal inhibition at 4 microM. Each analogue inhibited amylase release by bombesin and other agonists that stimulate secretion by interacting with bombesin receptors. The analogues of bombesin did not alter stimulation by substance P or other agonists that interact with other receptors. The inhibition of the action of bombesin was competitive with Schild plots having slopes of 1.0. Each analogue also inhibited binding of 125I-labeled [Tyr4]bombesin but not 125I-labeled substance P. These results demonstrate that [D-Phe12] analogues of bombesin function as bombesin receptor antagonists and are the only bombesin receptor antagonists that interact only with the bombesin receptor. Because of their specificity, these analogues may prove useful for defining the role of bombesin in various physiological or pathological processes.

When guinea pig pancreatic acini are first incubated with the COOH-terminal octapeptide of cholecystokinin (CCK-8), washed, and then reincubated with 125I-[Tyr4]bombesin (125I-[Tyr4]BN) there is a significant decrease in binding of 125I-[Tyr4]BN compared with that observed with pancreatic acini that have been first incubated with no additions. The CCK-8-induced decrease in binding is maximal after 90 min of first incubation is abolished by reducing the temperature of the first incubation from 37 to 4 degrees C or by adding L364,718 to the first incubation and cannot be reproduced by first incubating acini with A23187, 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cGMP), or 12-O-tetradecanoylphorbol 13-acetate. 125I-[Tyr4]BN interacts with a single class of receptors on pancreatic acini, and first incubating acini with CCK-8 decreases the affinity of BN receptors for BN with no change in the maximal binding capacity. CCK-8 does not alter the rate at which bound 125I-[Tyr4]BN dissociates from pancreatic acini; therefore, CCK-8 must alter the rate at which the radiolabeled BN analogue associates with its receptor. Pancreatic acini possess two classes of CCK receptors: one has a high affinity for CCK-8; the other has a low affinity for CCK-8. The dose-response curve for CCK-8-induced inhibition of binding of 125I-[Tyr4]BN appears to to reflect occupation of low-affinity CCK receptors by CCK-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin (0.62-6.2 nmol) was administered into the cisterna magna of chloralose-anesthetized cats while monitoring tracheal airflow with a pneumotachograph. The peptide produced dose-related increases in respiratory activity which were seen primarily as increases in tidal volume. Intravenous administration of bombesin (6.2 nmol) did not cause any respiratory effects. These results indicate that bombesin acts in the brain to stimulate respiration.

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.

The ability of substance P analogues to inhibit the action of bombesin in the CNS was investigated using receptor binding and biological assays. The putative substance P antagonists inhibited binding to central receptors for both substance P and bombesin-like peptides. Spantide, which was the most potent analogue tested, reversed the bombesin induced hypothermia and grooming. Therefore the putative substance P antagonists may also antagonize the actions of bombesin in the CNS.

Recent studies show that substitutions for the His in position 12 of bombesin (Bn) are important in determining antagonist activity. The present study was designed to investigate the chemical properties of the substitution in position 12 of Bn that determined antagonist activity and affinity. Nine [Leu14]Bn analogues with a single amino acid substitution and two analogues with multiple substitutions in addition to position 12 were synthesized. Replacing His12 with Phe12 resulted in an agonist with 100-fold decrease in potency and as reported previously, replacement with D-Phe12 resulted in an antagonist, but with a 10,000-fold decrease in affinity. Substitution of D-beta-naphthylalanine (D-Nal12), a larger and more hydrophobic group than D-Phe, produced a complete loss of antagonist activity, whereas substitution of D-pyridylalanine (D-Pal12), a group more hydrophilic and similar in size to D-Phe, converted the analogue to a very weak agonist with 300-fold lower affinity than the D-Phe analogue. Antagonist activity depended on the nature of the aromatic moiety, with a D-Trp12 resulting in an inactive analogue, and with a D-Tyr12 resulting in a weak antagonist being 100-fold less potent than the D-Phe12 substitution. The addition of an electron withdrawing group to the D-Phe substitution (D-Cpa12) resulted in a minimal decrease in antagonist activity, whereas the addition of an electron donating group (p-hydroxy in D-Tyr12) resulted in a 30-fold decrease in antagonist activity. The addition of a basic group (D-Arg12 or D-Pal12) resulted in weak agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-[Tyr4]bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-[Tyr4]bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-[Tyr4]bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were as follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-[Tyr4]bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.

Isolated smooth muscle cells from guinea pig stomach were used to study the pharmacological characteristics of a newly synthetized bombesin analog, [Leu13-psi-CH2NH-Leu14]-bombesin (psi 13,14-BN) to function as antagonist of bombesin-induced contractile response. The antagonism caused by this new analog was compared to that obtained with the substance P analog [D-Arg1,D-Pro2,D-Trp7,9,Leu11]Substance P [( APTTL]SP), which has been used until now to characterize bombesin receptors on smooth muscle cells. psi 13,14-BN resulted to be more potent than [APTTL]SP as antagonist of bombesin action on smooth muscle. Comparing the IC50, psi 13,14-BN (IC50 70 nM) was 8 times more potent than [APTTL]SP (IC50 600 nM). In contrast to [APTTL] SP, the action of psi 13,14-BN was shown to be specific toward bombesin receptors in that it does not interfere with receptors for other agents (i.e., cholecystokinin, acetylcholine or substance P). The antagonism induced by both compounds was competitive inasmuch as the slope of the regression lines obtained by Schild plot analysis were not significantly different from the unity. The apparent affinity for the bombesin receptor was 0.8 nM for psi 13,14-BN and 7.8 nM for [APTTL]SP. These results indicate that psi 13,14-BN acts on isolated gastric smooth muscle cells as a competitive bombesin receptor antagonist, with a higher affinity and specificity than the substance P analog used previously.

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.

In the present study the ability of two classes of substance P (SP) antagonists to affect SP- and bombesin (Bn)-stimulated amylase release, as well as binding of 125I-Bolton-Hunter (BH)-SP and 125I-[Tyr4]Bn, was studied to determine their mechanism of action and the relationship between inhibition of the action of SP and the action of Bn. Four SP analogues ([D-Pro2,D-Phe7,D-Trp9]SP, [D-Pro2,D-Trp7,9]SP, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, and [D-Arg1,D-Trp7,9,Leu11]SP) and two SP-(4-11) analogues ([D-Pro4,D-Trp7,9]SP-(4-11) and [D-Pro4,D-Trp7,9,10]SP-(4-11)] did not stimulate amylase release when present alone but inhibited SP- and Bn-stimulated amylase release. For each SP analogue, inhibition was specific for peptides that stimulate release by interacting with SP or Bn receptors, whereas both SP-(4-11) analogues also inhibited cholecystokinin-stimulated amylase release. Each SP analogue's inhibition of Bn-stimulated amylase release was reversible. Each analogue inhibited binding of 125I-BH-SP with the same potency as it inhibited SP-stimulated amylase release and 125I-[Tyr4]Bn binding with the same potency as it inhibited Bn-stimulated amylase. In contrast, SP itself or SP-(4-11) itself did not inhibit Bn-stimulated amylase release or binding of 125I-[Tyr4]Bn. The relative affinities of the analogues for interacting with Bn and SP receptors differed. None of the analogues increased the dissociation of bound 125I-BH-SP or 125I-[Tyr4]Bn.(ABSTRACT TRUNCATED AT 250 WORDS)

The tetradecapeptide bombesin was originally isolated from frog skin. Bombesin-like peptides have since been detected in mammalian gastrointestinal tract, brain and lung. These peptides have potent pharmacological effects on the central nervous system; they cause contraction of intestinal, uterine and urinary tract smooth muscle; and stimulate the release of other peptides including gastrin, cholecystokinin, motilin, pancreatic polypeptide, neurotensin, insulin, enteroglucagon, prolactin and growth hormone. Specific plasma membrane receptors for bombesin have been demonstrated on pancreatic acinar cells, brain membranes and pituitary cells. Studies defining the physiological importance of bombesin have been impeded by the lack of a bombesin receptor antagonist. Here we describe experiments which demonstrate that a peptide originally described as a substance P receptor antagonist, [D-Arg, D-Pro, D-Trp, Leu ]substance P, is also a bombesin receptor antagonist. This peptide competitively inhibits the ability of bombesin to stimulate enzyme secretion from dispersed pancreatic acini, and also inhibits the action of other peptides that interact with the bombesin receptor.

The high inhibitory potency of the previously developed bombesin antagonist [Leu13, psi CH2NHLeu14]bombesin (analogue I) (IC50 values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secretion from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu13, psi CH2NHLeu14]bombesin-(5-14),[Leu13, psi CH2NHLeu14] bombesin-(6-14), and [Leu9, psi CH2NHLeu10]neuromedin C had IC50 values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu13, psi CH2NHLeu14] bombesin did not significantly change the various biological parameters. However, its presence in [Leu13, psi CH2NHLeu14]bombesin-(6-14) and at position 2 of psi-neuromedin C-(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) and des-Gly1-[D-Phe2,Leu9,psi CH2NHLeu10]neuromedin C exhibited IC50 values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH2-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH2- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe6,Leu13,psi CH2NHPhe14]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentrations (IC50 = 0.72 nM and Ki = 3.1 nM for 3T3 cells; IC50 = 7.5 nM and Ki = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) (Ki = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin receptor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a Ki of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibition of bombesin-dependent human small cell lung carcinoma growth.

Incubating dispersed acini from guinea pig pancreas with bombesin and then washing the cells to remove bombesin reduced the subsequent stimulation of amylase secretion caused by bombesin, litorin, or ranatensin by as much as 80%, but did not alter the stimulation of amylase secretion caused by cholecystokinin, carbamylcholine, A23187 or vasoactive intestinal peptide. This bombesin-induced desensitization was reversible, and the onset of the process, as well as its reversal, were time and temperature dependent. Neither desensitization or resensitization were inhibited by abolishing protein synthesis. The concentrations of bombesin required to cause desensitization were in the same range as those required to stimulate amylase secretion. Incubating pancreatic acini with vasoactive intestinal peptide did not reduce the subsequent stimulation of amylase secretion caused by vasoactive intestinal peptide, bombesin, or cholecystokinin. These results indicate that bombesin-induced desensitization of pancreatic acini reflects changes that occur at or close to the bombesin receptor.

We have prepared (125)I-labeled [Tyr(4)]bombesin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of (125)I-labeled [Tyr(4)]-bombesin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 5000 binding sites, and binding of the tracer to these sites could be inhibited by [Tyr(4)]bombesin [concentration for half-maximal effect (Kd), 2 nM], bombesin (Kd, 4 nM), or litorin (Kd, 40 nM) but not by eledoisin, physalemin, somatostatin, carbachol, atropine, secretin, vasocative intestinal peptide, neurotensin, or bovine pancreatic polypeptide. At high concentrations (>0.1 muM), cholecystokinin and caerulein each caused a small (15-20%) reduction in binding of lableled [Tyr(4)]bombesin. With bombesin, litorin, and [Tyr(4)]bombesin, there was a close correlation between the relative potency for inhibition of binding of labeled [Tyr(4)]bombesin and that for stimulation of amylase secretion. For a given peptide, however, a 10-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with [Tyr(4)]bombesin, bombesin, and litorin and that occupation of 25% of these receptors will cause a maximal biological response.

The potency of synthetic bombesin (BN) analogues with D-Phe12 substitutions and substance P analogues was investigated in the rat CNS. (D-Phe12,Leu14)BN, (D-Phe12)BN and (Tyr4,D-Phe12)BN inhibited binding to rat brain slices with IC50 values of approximately 2 microM. Similarly, spantide inhibited binding to rat brain slices with an IC50 value of 1.5 microM. Spantide inhibited specific (125I-Tyr4)BN binding as a result of decreased rate of association, whereas the rate of dissociation was unaffected. Neither the (D-Phe12)BN analogues nor the substance P analogues inhibited specific binding of 125I-VIP to rat brain slices. Central administration of BN (0.5 micrograms) induced grooming and suppressed feeding and resting. (Tyr4, D-Phe12)BN (5 micrograms) antagonized the behavioral effects of BN. Although spantide (2 micrograms) also antagonized many of the BN effects, it had intrinsic effects and hence the behavioral antagonism was not specific. These data suggest that although both (D-Phe12)BN and substance P analogues may function as central BN receptor antagonists, the (D-Phe12)BN analogues may be functionally the more useful class of antagonists.