Biomedical subjects

D H Coy

Publications and source records attributed to D H Coy.

At least 145 records · Page 8Linked to original sources

PubMed1993

Chimeric galanin analogs that function as antagonists in the CNS are full agonists in gastrointestinal smooth muscle.

Galanin has numerous effects on gastrointestinal smooth muscle. However, because of the lack of specific inhibitors, it is not known which are physiological and which are pharmacological. This study investigates the ability of two chimeric galanin analogs, [# 1-galantide = (M-15) = [galanin (1-13)-substance P(5-11)] and #2-M-35[galanin(1-13)bradykinin (2-9)], which were recently reported to function as galanin-receptor antagonists in the CNS, to interact with galanin receptors on rat jejunal muscle strips or dispersed smooth muscle cells from guinea pig stomach. In both systems each chimeric analog had agonist activity and was as efficacious as galanin. Cross-desensitization experiments demonstrated that in the jejunal muscle strips, both chimeric analogs were causing muscle contraction by interacting with the galanin receptor. In dispersed smooth muscle cells, galanin, as well as each chimeric analog, caused muscle relaxation, whereas substance P and bradykinin both caused muscle contraction. Each chimeric analog was equipotent to galanin in inhibiting binding of 125I-galanin, and there was close agreement between their abilities to occupy the galanin receptor and cause relaxation. Each chimeric analog also activated adenylate cyclase and increased cAMP characteristic of relaxants. These studies demonstrate these chimeric analogs will not be useful for defining the physiological role of galanin in altering gastrointestinal motility, because they function as full galanin-receptor agonists instead of as galanin-receptor antagonists.

Animals↗

PubMed1993

Discovery of a novel class of neuromedin B receptor antagonists, substituted somatostatin analogues.

Bombesin-related peptides have widespread activities in the central nervous system and peripheral tissues. Recent studies show two subtypes of receptors; a gastrin-releasing peptide (GRP) receptor subtype and a neuromedin B (NMB) receptor subtype exist. In contrast to the GRP receptor, no antagonists exist for the NMB receptor. In the present study we report that certain somatostatin (SS) octapeptide analogues function as selective NMB receptor antagonists. The most potent analogue, D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2, inhibited binding of 125I-[D-Tyr degree]NMB to NMB receptor-transfected 3T3 cells and C6 cells. This analogue had 100-fold lower affinity for GRP receptors. Structure-function studies were performed by synthesizing 18 structurally related SS octapeptide analogues; each of these analogues, but not native SS-14 or SS-28, also inhibited binding to NMB receptors. The stereochemistry at positions 1, 2, 7, and 8, the hydrophobicity and ring size of the substitution in positions 1, 3, and 4, and the basicity of the group in position 5 were all important in determining NMB receptor affinity. No SS octapeptide analogue increased [3H]inositol phosphates in NMB receptor-transfected cells; however, each analogue inhibited NMB-stimulated increases. The most potent analogue, D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2, caused a parallel rightward shift of the NMB dose-response curve, the Schild plot slope was not significantly different from unity, and the affinity was 230 nM. SS octapeptide analogues also interacted with SS receptors and mu-opioid receptors; however, there was no correlation between the affinities of the analogues for these receptors and their affinities for NMB receptors, demonstrating that these activities can be separated. The results demonstrate for the first time a class of antagonists with > 100-fold selectivity for NMB versus GRP receptors. Because the structural requirements for determining NMB, SS, and mu-opioid receptor activity differ, it is likely that highly selective, specific, high affinity NMB receptor antagonists can now be developed that will be useful in defining the role of NMB in various physiological processes.

3T3 Cells↗

PubMed1993

Cloned somatostatin receptors: identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides.

The recent molecular cloning of the genes encoding three somatostatin (SRIF) receptor subtypes has allowed for the individual expression of these receptors in mammalian cells and characterization of their respective pharmacological profiles. In the present study, we have investigated the affinities of a battery of SRIF analogues to bind to SRIF receptor subtypes SSTR1 (cloned somatostatin complex), SSTR2, and SSTR3, as well as their abilities to inhibit the release of growth hormone from anterior pituitary cells in vitro. We labeled SSTR1 and SSTR3 receptors expressed in Chinese hamster ovary and COS-1 cells, respectively, with the metabolically stable SRIF analogue 125I-CGP 23996. SSTR2 receptors expressed in Chinese hamster ovary cells were labeled with the SSTR2-specific radioligand 125I-MK-678. Inhibition studies were performed using SRIF analogues of differing structures, including hexapeptide analogues similar to MK-678, octapeptide analogues similar to SMS 201-995, pentapeptide analogues similar to c[Ahep-Phe-D-Trp-Lys-Thr(Bzl)] (SA), and linear SRIF analogues. SSTR1 bound SRIF and SRIF-28 with high affinity and the peptide SA and its structural analogues with low affinity. The hexapeptides did not interact with SSTR1 at concentrations as high as 1 microM, and only a few of the octapeptides or linear peptides bound, with very low affinities. In contrast, 125I-MK-678 binding to SSTR2 was potently inhibited by the hexapeptides, octapeptides, and some of the linear compounds, whereas SA and its analogues did not bind to SSTR2. The potencies of the various SRIF agonists to inhibit growth hormone release in vitro was highly correlated with their potencies to inhibit radioligand binding to SSTR2, but not to SSTR1 or SSTR3. SSTR3 bound analogues of each class but with moderate to low affinities, with the exception of several linear peptides and one of the octapeptides. We report for the first time the binding affinities of linear analogues of SRIF, some of which display subnanomolar affinities and are highly selective for SRIF receptor subtypes. Most importantly, these studies identify several peptide analogues that are highly potent, specific, and selective for individual subtypes of SRIF receptors. Such information, coupled with the knowledge of the distribution of these receptor subtypes in normal and pathological tissues, will be critical for more specific experimental and therapeutic interventions.

Amino Acid Sequence↗

PubMed1992

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

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

3T3 Cells↗

PubMed1992

Identification and initial characterization of a putative neuromedin B-type receptor from rat urinary bladder membranes.

Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr4,Leu14]bombesin, and a 50,000 x g total particulate preparation. The binding was specific, reversible, saturable, time- and concentration-dependent. A dissociation curve showed that both bombesin and neuromedin B equally displaced the radioligand in the first 10 min after saturation. From the rate constant of association K + 1 = 7.60 x 10(9) M-1 min-1, and the rate constant of dissociation k-1 = 0.050 min-1, the apparent equilibrium dissociation constant Kd = 6.57 +/- 1.09 pM was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr4,Leu14]bombesin to the membranes revealed that the radioligand bound with high affinity, Kd = 6.38 +/- 0.86 pM, to a single class of sites (Bmax = 2.3 fmol/mg protein). The Hill coefficient of the same binding data was 1.05 +/- 0.21, indicating that the radioligand was binding to a single population of noninteracting binding sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe6]bombesin-(6-13) methyl ester, [D-F5Phe6,D-Ala11]bombesin-(6-11) methyl ester, [D-Phe6]bombesin-(6-13) propylamide, [D-Phe6,Leu13psi(CH2NH)Leu14]bombesin or [D-Cpa6,Phe14(psi13-14)]bombesin-(6-14) had an IC50 greater than 1 microM. The results presented suggest the presence of neuromedin B receptor sites on the rat urinary bladder membranes that can be occupied also by some other peptides, notably bombesin, neurokinin A and neurokinin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Reduced gastric acid inhibitory effect of a pGIP(1-30)NH2 fragment with potent pancreatic amylase inhibitory activity.

Gastric inhibitory polypeptide (GIP) strongly stimulates insulin secretion in the presence of glucose and also stimulates somatostatin release from gastric mucosa. It was reported recently that both stimulatory activities can be dissociated by removing the C-terminal 12 amino acid residues. Since insulin and somatostatin are involved in regulation of exocrine pancreatic and gastric secretion in rats, we compared the inhibitory effects of pGIP and the pGIP(1-30)NH2 fragment on pancreatic amylase and gastric acid secretion. pGIP(1-30)NH2 displayed full activity on inhibition of bombesin (BN)-stimulated amylase release relative to GIP itself, but was about 10-fold less potent in inhibiting gastric acid secretion. These results suggest that the receptors involved in these two events have quite different ligand binding requirements and that more specific analogues of GIP can be designed which should be of value in elucidating the physiological roles of this hormone.

Amylases↗

PubMed1992

Structural requirements for galanin inhibition of pentagastrin-stimulated gastric acid secretion in conscious rats.

The effects of rat galanin, together with a number of its N- and C-terminal fragments, on pentagastrin-stimulated gastric acid secretion were studied in conscious rats equipped with chronic gastric fistulas. Similarly to its porcine counterpart studied previously, at a dose of 3 nmol/kg per h rat galanin was a potent inhibitor of gastric acid secretion. The N-terminal fragments, rat galanin-(1-10) and -(1-15), retained about 60% of the inhibitory potency of the whole galanin sequence whilst the C-terminal fragments, rat galanin-(2-29), -(3-29) and -(9-29), were unable to produce significant inhibition over comparable dose ranges. Surprisingly, however, simply acetylating the alpha-amino group in position 9 of rat galanin-(9-29) restored almost full gastric acid inhibitory activity in a homologous rat model. We speculate that this could be due to a favorable conformational effect on the C-terminal region produced by alpha-acetylation. These results also suggest that structural features within either the N-terminal or C-terminal regions of rat galanin are able to elicit this particular biological response. One possible explanation for this could be the involvement of more than one rat galanin receptor having different ligand recognition requirements.

Animals↗

PubMed1992

Effect of [D-Phe6] bombesin (6-13) methylester, a bombesin receptor antagonist, towards bombesin-induced contractions in the guinea-pig and rat isolated urinary bladder.

1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Actions of somatostatins on gastric smooth muscle cells.

The effects of somatostatin-28, somatostatin-14, and a synthetic somatostatin octapeptide analogue, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Nal-NH2 (cyclo SS-8) were examined on contraction of dispersed gastric smooth muscle cells from guinea pigs. The somatostatins did not cause contraction of gastric smooth muscle cells, nor did they inhibit carbachol-stimulated contraction. However, they reversed vasoactive intestinal peptide (VIP)-induced inhibition (relaxation) of carbachol-stimulated contraction. Somatostatin-28 had a half-maximal effect (EC50) at 1.6 +/- 0.8 nM, cyclo SS-8 at 0.6 +/- 0.3 nM, but somatostatin-14 had no effect even when used in concentrations as high as 1 microM. Incubation of muscle cells with peptidase inhibitors phosphoramidon (1 microM) plus amastatin (10 microM) had no effect on the EC50 of somatostatin-28 or cyclo SS-8 but increased the potency of somatostatin-14 greater than 1,000-fold. When peptides were incubated with muscle cells and the products applied to high-performance liquid chromatography, cyclo SS-8 was not degraded, but somatostatin-14 was rapidly degraded when present alone, and the addition of peptidase inhibitors partially inhibited the degradation. Cyclo SS-8 had its maximal effect at 0.5-1 min and inhibited relaxation induced by VIP, isoproterenol, glucagon, or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Cyclo SS-8 partially inhibited the increase in VIP-stimulated cAMP. Preincubation with pertussis toxin blocked the inhibitory action of cyclo SS-8 on VIP or DBcAMP-induced relaxation. These results indicate that gastric smooth muscle cells rapidly degrade somatostatin-14 and suggest that muscle cell peptidases could have a major effect on the actions of somatostatin-14.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Peptone stimulates gastrin secretion from the stomach by activating bombesin/GRP and cholinergic neurons.

The mechanism by which partly digested protein (peptone) stimulates gastrin secretion was examined in isolated antral tissues with intact intramural innervation. In the isolated vascularly perfused rat stomach, luminal perfusion with 0.5% peptone increased gastrin (62 +/- 14 pg/min; P less than 0.01) and decreased somatostatin (74 +/- 19; P less than 0.01) secretion. The axonal blocker tetrodotoxin (TTX) abolished the gastrin and somatostatin responses indicating that the responses were neurally mediated. Atropine partly inhibited the gastrin response (50%) and converted the somatostatin response to an increase above basal level. The selective bombesin/gastrin-releasing peptide (GRP) antagonist [Leu13-psi(CH2NH)-Leu14]-bombesin partly inhibited the gastrin response (65%) and caused a further decrease in somatostatin secretion. A combination of atropine and the bombesin/GRP antagonist, like TTX, abolished the gastrin and somatostatin responses. The pattern of response to peptone in superfused antral segments was identical to that in the vascularly perfused stomach. In fundic segments that do not secrete gastrin, the somatostatin response to peptone alone and with various antagonists was identical to that in antral segments. The results indicate that peptone stimulates gastrin secretion by activating stimulatory cholinergic and bombesin/GRP neurons. Cholinergic neurons stimulate gastrin directly as well as indirectly by eliminating the inhibitory paracrine influence of somatostatin.

Animals↗

PubMed1992

Analysis of systemic and pulmonary vascular responses to PACAP and VIP: role of adrenal catecholamines.

Systemic and pulmonary vascular responses to pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide with 68% sequence homology to vasoactive intestinal peptide (VIP), were investigated in the anesthetized cat. Intravenous injections of PACAP in doses of 0.1-3.0 nmol/kg produced decreases in arterial pressure (AP) at low doses and biphasic changes (decreases followed by increases) at higher doses, which were accompanied by increases in central venous pressure (CVP) and cardiac output (CO), and decreases and biphasic changes in systemic vascular resistance (SVR). In contrast, VIP in doses of 0.1-3.0 nmol/kg produced only dose-dependent decreases in AP and SVR and produced little change in CVP and CO. PACAP produced increased pulmonary arterial pressure (PAP), left atrial pressure (LAP), and increases in pulmonary vascular resistance (PVR). PACAP increased heart rate (HR) and right ventricular contractile force (RVCF), while VIP had no effect. Increases in AP and SVR in response to PACAP were changed to decreases following the administration of phentolamine or after adrenalectomy. Under constant flow conditions, PACAP and VIP produced dose-dependent decreases in lobar arterial pressure when tone was elevated, with PACAP being threefold more potent than VIP. Meclofenamate and nitro-L-arginine methyl ester (L-NAME) had no effect on pulmonary responses to the peptides. PACAP produced dose-dependent biphasic changes in hindquarters perfusion pressure, whereas VIP produced only decreases that were unchanged by indomethacin, L-NAME, and glibenclamide. Phentolamine and adrenalectomy eliminated the hindquarters pressor response to PACAP and D-Phe2-VIP, a VIP antagonist, reduced responses to VIP but not to PACAP. These data suggest that responses to PACAP and VIP are mediated by distinct receptors and that pressor responses to PACAP are due to the release of catecholamines from the adrenal gland.

Adrenal Glands↗

PubMed1992

Differential effects of PACAP and VIP on the pulmonary and hindquarters vascular beds of the cat.

Responses to pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide derived from ovine hypothalamus with 68% sequence homology with vasoactive intestinal polypeptide (VIP), were investigated in the pulmonary and hindquarters vascular beds of the anesthetized cat under conditions of controlled blood flow. Injection of the peptide into the perfused lung lobe under elevated tone conditions produced dose-dependent decreases in lobar arterial pressure that were accompanied by biphasic changes in systemic arterial pressure characterized by an initial decrease followed by a secondary increase in pressure. When compared with other vasodilator agents in the pulmonary vascular bed, the relative order of potency was isoproterenol greater than PACAP greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP. In the hindquarters vascular bed, intra-arterial injections of PACAP produced biphasic changes in hindquarters perfusion pressure characterized by initial decreases followed by secondary increases, which were accompanied by biphasic changes in systemic arterial pressure. In terms of relative vasodilator activity in the hindlimb, the order of relative potency was isoproterenol greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP greater than PACAP. PACAP was the only agent that caused a secondary vasoconstrictor response in the hindlimb and produced biphasic changes in systemic arterial pressure. D-Phe2-VIP, a VIP receptor antagonist, blocked the hindquarters vasodilation in response to VIP but had no effect on responses to PACAP. The present investigation shows that PACAP produces pulmonary vasodilation, as well as dilation, and vasoconstriction in the systemic (hindlimb) vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A comparison of the clearance of ovine and human corticotrophin-releasing hormone (CRH) in man and sheep: a possible role for CRH-binding protein.

A specific binding protein for human corticotrophin-releasing hormone (hCRH), which does not bind to the ovine hormone (oCRH), has recently been demonstrated in human plasma. No such binding protein has been found in sheep plasma. We have investigated the half-life of human and ovine CRH in man and in sheep. Peptides were measured directly in plasma with two-site immunoradiometric assays, as these assays are unaffected by the presence of inactivated peptide fragments. In man, the half-life of hCRH (30.5 +/- 3.3 min; mean +/- S.E.M.) was significantly (P less than 0.001) less than that of oCRH (42.8 +/- 6.4 min). In sheep, there was no significant difference between the half-life of hCRH (46.5 +/- 7.2 min) and that of oCRH (39.8 +/- 10.1 min); these half-lives were also significantly (P less than 0.001) longer than that of hCRH in man. One possible explanation for the shorter half-life of hCRH in man is that the clearance of hCRH is enhanced by CRH-binding protein, although other binding proteins often have the opposite effect. Peak ACTH and cortisol responses occurred earlier in sheep than in man, although no differences were found in the response times to oCRH or hCRH within either species. The responses were more sustained in sheep than in man, and the previously reported biphasic response was only seen in some of the sheep and not in man. Absolute responses to either peptide were greater in sheep than in man; however, in man an 8.1-fold rise in ACTH was measured in response to oCRH, while hCRH gave a significantly (P = 0.043) smaller 4.4-fold response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1992

Pituitary adenylate cyclase-activating polypeptide relaxes rat gastrointestinal smooth muscle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a new member of the secretin/glucagon peptides family, being most homologous to vasoactive intestinal peptide (VIP). The present study was designed to investigate a possible effect of PACAP on the rat gastrointestinal smooth muscle in vitro. We demonstrated that 1) PACAP reduced basal smooth-muscle contractions in all portions of the gastrointestinal tract, but the effect of VIP was region-specific. The inhibitory effect of PACAP in midcolon was approximately 100 times greater than that of VIP. 2) PACAP significantly inhibited smooth-muscle contractions induced by acetylcholine or carbachol. The inhibitory effect of PACAP was not affected by hexamethonium and was additive to the inhibitory effect of atropine and pirenzepine. 3) PACAP inhibited smooth-muscle contractions induced by substance P, cholecystokinin, and galanin, even after atropine treatment. Although the exact mechanism of the inhibitory action of PACAP remains to be clarified, PACAP appears to exert its effect in the rat at a site other than muscarinic receptors, probably through a direct effect on gastrointestinal smooth muscle in vitro.

Animals↗

PubMed1992

Neuromedin B binds with high affinity, elevates cytosolic calcium and stimulates the growth of small-cell lung cancer cell lines.

Previously, gastrin-releasing peptide (GRP) receptors were identified on small-cell lung cancer (SCLC) cells and GRP functioned as a SCLC autocrine growth factor. Here the effects of neuromedin B (NMB) on SCLC cells were investigated. [125I-Tyr0]NMB bound with high affinity to three of seven SCLC cell lines examined. [125I-Tyr0]NMB bound to SCLC cell line NCI-H209 and NCI-H345 in a time-dependent and reversible manner. [125I-Tyr0]NMB bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 800/cell). Specific [125I-Tyr0]NMB binding was inhibited with high affinity by NMB (IC50 = 1 nM) and moderate affinity by bombesin, GRP and [D-Arg1, D-Pro2, D-Trp7,9, Leu11]substance P ([APTTL]SP) but not GRP1-16 (IC50 = 50, 100, 1,000 and > 10,000 nM, respectively). In Fura 2 AM loaded NCI-H345 cells, NMB elevated cytosolic calcium in a concentration-dependent manner. NMB (10 nM) elevated the cytosolic calcium from 150 to 180 nM and calcium was released from intracellular pools. The increase in cytosolic calcium caused by 10 nM NMB was reversed by 1 microM [APTTL]SP but not 1 microM [D-Phe6]bombesin6-13methylester, a GRP receptor antagonist. Also, NMB stimulated the clonal growth of NCI-H209 and NCI-H345 in a concentration-dependent manner. The increase in the clonal growth caused by NMB was reversed by 1 microM [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of some SCLC cells.

Binding Sites↗

PubMed1992

Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors.

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.

3T3 Cells↗

PubMed1992

Systematic development of bombesin/gastrin-releasing peptide antagonists.

Several families of very potent bombesin (Bn) receptor antagonist analogues have recently been developed and their biological potencies evaluated in a number of in vitro systems including guinea pig and rat pancreatic acini and Swiss 3T3 cells. These studies showed that analogues can exhibit diverse properties ranging from full antagonists, partial agonists, or full agonists depending on the assay system and animal species employed. We have developed two classes of more potent, shorter chain antagonists based on [psi CH2NH(13-14)]Bn(6-14) and desMet14Bn(6-13)NH2 structures. [D-Phe6 psi Leu13-Leu14] Bn(6-14)NH2 was a potent antagonist (Ki 6nM) in Swiss 3T3 cells and guinea pig acini but exhibited 10% partial agonist activity and lower binding affinity (Ki 60 nM) in rat acini. The partial agonism could be eliminated by using p-Cl-Phe or D-Phe at the C-terminus and partially eliminated using D-4-Cl-Phe in position 6. With the antagonist [D-Phe6]Bn(6-13)NH2 (Ki 96 nM), alkyl substituents on the amide group increased affinity 25-fold with the propylamide being the most potent peptide (Ki 4 nM) in 3T3 cells or guinea pig acini. It did, however, have high 40% partial agonist activity in rat acini. Alkyl esters or hydrazide derivatives were, in contrast, pure antagonists in all systems tested with [D-Phe6]Bn(6-13)OMe having the highest affinity in all systems and also excellent in vivo properties. All of the potent antagonists examined had little affinity for neuromedin B--preferring bombesin receptors, which had entirely new ligand structure-activity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1991

Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides.

Bombesin (BB), neuromedin C (NMC) and neuromedin B (NMB) stimulated amylase secretion to similar maximum levels, with EC50 values (concentrations causing 50% of maximum effect) of 0.2, 0.3 and 2 nM respectively. Treatment of pancreatic acini with BB or NMB (10 nM) for 30 min resulted in cross-desensitization of secretory responses to subsequent BB and NMB, but not to acetylcholine, which suggests that NMB and BB activate the same receptor. BB, NMC and NMB stimulated production of similar maximum amounts of inositol mono-, bis- and tris-phosphates, with EC50 values of 3, 5 and 141 nM respectively. The bombesin receptor antagonist [Leu13-psi(CH2NH)Leu14]BB inhibited stimulation of amylase secretion and inositol phosphate formation by BB, NMC and NMB. Binding of 125I-labelled gastrin-releasing peptide (GRP; 200 pM) to rat pancreatic membranes at 22 degrees C was inhibited with relative potencies and IC50 (concn. causing 50% of maximal inhibition; nM) as follows: NMC (0.4) = BB (0.5) greater than NMB (1.8 = GRP (2.6). IC50 values for BB, NMC and NMB inhibition of 125I-GRP binding to intact acini were 5-, 19- and 68-fold higher than their respective values in membranes. The guanine nucleotide analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p) produced rightward shifts of NMC and NMB competition curves by 3.5- and 16-fold respectively, but had little effect on the BB and GRP curves. Elevation of the temperature to 37 degrees C or inclusion of NaCl (40 mM) produced quantitatively similar effects to those of Gpp[NH]p. In the presence of both NaCl and Gpp[NH]p the affinities of peptides for membrane receptors were similar to those for intact cells. Modulation of NMB competition curves by Gpp[NH]p was not attenuated by prior treatment of acini with activated pertussis toxin. These results suggest that BB, NMB and NMC stimulate pancreatic secretion by interaction with a common phosphoinositide-linked receptor. Differences in guanine nucleotide regulation suggest that secretagogue-induced receptor-protein interactions may not be identical for NMB and BB.

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