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D H Coy

Publications and source records attributed to D H Coy.

At least 127 records · Page 7Linked to original sources
PubMed1994

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP.

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 +/- 2 microns and, with carbachol treatment, contracted to 89 +/- 2 microns. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC50 of 3-7 nM. Galanin (1 microM) and VIP (1 microM) increased cellular cAMP from 118 +/- 10 pmol/10(6) cells in control to 212 +/- 14 and 214 +/- 12 pmol/10(6) cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 microM, completely inhibited the relaxant effect of an EC50 concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, L-NNA (NG-nitro-L-arginine), at 100 microM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, L-NNA (100 microM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

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

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

Amino Acid Sequence↗
PubMed1994

A chimeric VIP-PACAP analogue but not VIP pseudopeptides function as VIP receptor antagonists.

The ability to assess the importance of VIP in different physiological processes is limited by the lack of specific potent antagonists. In the present study, we have adopted two different approaches used successfully with other peptides in an attempt to identify new VIP receptor antagonists. One involves the formation of pseudopeptides by insertion of reduced peptide bonds in the NH2-terminus from position 2 to 8 of VIP. The other methodology involves the formation of a COOH-terminal chimeric analogue by combining VIP(6-28) and PACAP(28-38). The ability of each of these peptides to function as an antagonist was compared with reported VIP antagonists. All of the peptides inhibited [125I]VIP binding to VIP receptors on guinea pig pancreatic acini. For the pseudopeptides the affinities were: [psi 3-4]VIP (0.2 microM) = 4 x [psi 4-5]VIP = 8 x [psi 8-9]VIP = 14 x [psi 6-7]VIP, [psi 2-3]VIP = 25 x [psi 5-6]VIP. Each nonpseudopeptide analogue also inhibited VIP binding with relative potencies of VIP(6-28)-PACAP(28-38) (1 microM) = 2.5 x [4-Cl-D-Phe6,Leu17]VIP, VIP(10-28), neurotensin(6-11)-VIP(7-28) = 6 x [Ac-Tyr1,D-Phe2]GRF. All pseudopeptides were agonists with relative potencies: [psi 3-4]VIP > [psi 6-7], [psi 4-5]VIP > [psi 5-6] > [psi 8- 9]VIP > [psi 2-3]VIP. The reported VIP receptor antagonist, neurotensin(6-11)-VIP(7-28), was also an agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cyclic lactam analogues of ovine pituitary adenylate cyclase activating polypeptide (PACAP): discovery of potent type II receptor antagonists.

Binding of [125I]PACAP-38 to rat liver membranes was investigated. It was rapid at 37 degrees C, reversible, and saturable, and it was time, concentration, and temperature dependent. Scatchard plots showed that [125I]PACAP-38 bound to single noninteracting site(s), and [125I]VIP bound to high- and low-affinity binding site(s). The order of potency of displacing [125I]PACAP-38 from rat liver membranes was: PACAP-38 > PACAP-27 > VIP (IC50 = 5, 180, and 350 nM, respectively). Surprisingly, the order of potency of displacing [125I]VIP was also the same (IC50 = 1, 8, and 52 nM, respectively). The order of potency of stimulating adenylate cyclase to release cyclic AMP was: PACAP-27 > VIP > PACAP-38 (EC50 = 0.06, 1, and 6 nM, respectively). Modification of PACAP-27 or PACAP-38 structures either through deletions, substitutions, or cyclization involving amino acid residues, Asp3, Asp8, Lys15, Lys20, or Lys21 indicated that the N-terminal region of the molecule is important for both binding and transduction. Of the various lactam analogues synthesized, cyclo[Asp3,Lys15]PACAP-38 and cyclo[Asp8,Lys15]PACAP-38 appear to be competitive receptor antagonists of the release of cAMP by PACAP-38. The results presented suggest that liver membranes possess distinct PACAP and VIP receptors, and that the PACAP receptor(s) is probably similar, but not identical, to type I receptor(s) characteristic of the brain.

Animals↗
PubMed1994

Chief cells possess somatostatin receptors regulated by secretagogues acting through the calcium or cAMP pathway.

Inhibition both in vivo and in vitro of pepsinogen secretion by somatostatin (SS) and the histological demonstration that fundic D-cells contain long cytoplasmic processes extending to chief cells suggest a possible direct effect of SS on chief cell function. The aim of the present study was to determine whether SS interacts directly with receptors on isolated gastric chief cells and, if so, how SS alters cell function. Binding of 125I-[Tyr11]SS14 to chief cells was saturable, time and temperature dependent, and was inhibited by both SS14 (Ki 1.6 nM) and SS28 (Ki 5.2 nM). SMS-201-995 was 1,300-fold less potent than SS14. Calcium-mobilizing secretagogues reduced binding of 125I-[Tyr11]SS14 with efficacies of cholecystokinin octapeptide (CCK-8) > carbachol > gastrin. Adenosine 3',5'-cyclic monophosphate (cAMP)-activating secretagogues also inhibited binding with efficacies of secretin > vasoactive intestinal polypeptide (VIP). 12-O-tetradecanoylphorbol 13-acetate (TPA) or A-23187 also decreased binding. Analyses demonstrated that CCK-8 and TPA were decreasing the affinity of SS receptors for 125I-[Tyr11]SS14 without affecting their binding capacity. Both SS14 and SS28 at a maximally effective concentration inhibited cAMP production caused by VIP or secretin (20-30%) but did not alter cytosolic calcium ([Ca2+]i), inositol phosphates, or pepsinogen release. We conclude that chief cells possess SS receptors with a high affinity for both SS14 and SS28 but low affinity for SMS-201-995 and thus resemble the SSB receptors described in the rat cerebral cortex. Although occupation of these receptors by SS has no effect on pepsinogen release induced by secretagogues acting through either the calcium or the cAMP pathway, SS receptor occupation is regulated by agents activating phospholipase C, adenylate cyclase, protein kinase C, and [Ca2]i.

Animals↗
PubMed1994

Smooth muscle cells from guinea pig stomach possess high-affinity galanin receptors that mediate relaxation.

Galanin-like immunoactivity occurs in nerves and plexi in muscle layers throughout gastrointestinal tract including the stomach. Galanin can affect gastric emptying and contraction or relaxation of gastric muscle in different species. The aim of this study was to investigate the direct effect of galanin on dispersed gastric smooth muscle cells and to characterize any galanin receptors that mediated any effect. Dispersed gastric smooth muscle cells were prepared from guinea pig stomach by collagenase digestion. Porcine galanin (p-galanin; 1 microM) did not stimulate contraction when present alone; however, p-galanin (1 microM) inhibited carbachol-induced contraction with a half-maximal effect at 7 nM. p-Galanin (1 microM) increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 10 s and caused a maximal increase of 80% over basal. 125I-galanin (porcine) bound to dispersed cells in a time- and temperature-dependent manner. Binding was saturable, reversible, and specific. Binding of 125I-galanin was inhibited almost equally by porcine and rat galanin (Ki = 6-8 nM) but was not inhibited by the galanin-associated peptide [preprogalanin-(108-123)]. The fragment galanin-(1-16) was equally potent to rat galanin; however, the fragment galanin-(9-29) was 56-fold less potent (Ki = 370 nM). Computer analysis demonstrated there were two binding sites for p-galanin on gastric smooth muscle cells, a high-affinity site (Kd = 2.6 nM) with low capacity (Bmax = 175 fmol/mg protein) and a low-affinity site (Kd = 150 nM) with large capacity (Bmax = 3,611 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Potent inhibitory effects of a type four receptor-selective somatostatin analog on rat insulin release.

A total of 5 somatostatin (SS) receptors have been characterized, cloned, and transfected into various cell types which have recently been used to discern peptide ligands displaying high degrees of selectivity for binding to types 2, 3, and 4. These have now allowed us to examine which receptor(s) is involved in SS inhibition of glucose-stimulated rat pancreatic insulin release. The type 4 selective ligand, DC-23-99, which had little affinity for receptor types 1, 2, or 5, was at least equipotent to SS in preventing insulin release. In contrast, the type 3 selective peptide, DC-25-20, was totally devoid of inhibitory effects. Peptides, such as NC-8-12, which have extremely high affinity for type 2 receptors but far less for types 1, 3, 4 and 5, were considerably less potent than SS in this assay. Thus, 2 major inhibitory physiological functions of SS on GH (type 2) and insulin release appear to be mediated by entirely different receptor types.

Amino Acid Sequence↗
PubMed1993

Comparison of responses to pituitary adenylate cyclase activating peptides 38 and 27 in the pulmonary vascular bed of the cat.

Responses to pituitary adenylate cyclase activating polypeptide (PACAP)-38 were investigated and compared with responses to PACAP-27 and vasoactive intestinal polypeptide (VIP) in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Under low resting tone baseline conditions, injections of PACAP-38 had little or no effect on lobar arterial pressure; however, when tone in the pulmonary vascular bed was raised to a high steady level (35-40 mm Hg) with U46619, intralobar injections of PACAP-38 caused dose-related decreases in lobar arterial pressure without altering left atrial pressure. The peptide induced biphasic changes in systemic arterial pressure. PACAP-38 was more potent than VIP in decreasing lobar arterial pressure, and both peptides were significantly less potent than PACAP-27 in dilating the pulmonary vascular bed. The present data show that PACAP-38 has significant vasodilator activity in the pulmonary vascular bed of the cat, and that the 27 amino acid form of the peptide is approximately 3-fold more potent than PACAP-38.

Animals↗
PubMed1993

Galanin: structure-dependent effect on pancreatic amylase secretion and jejunal strip contraction.

Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1-29) = porcine galanin-(1-29) = galanin-(1-15) = rat galanin-(3-29) > rat galanin-(2-29) = porcine galanin-(2-29) > galanin-(1-10). Fragments of rat galanin-(9-29) and N alpha-acetyl-galanin-(9-29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15-29) and rat galanin-(21-29) were unable to produce significant inhibition. Rat and porcine galanin-(1-29), galanin-(1-15) and rat N alpha-acetyl-galanin-(9-29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1-29) and porcine galanin-(1-29) have similar potencies. Galanin-(1-15) and galanin-(1-10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly1 from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3-29) and (9-29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9-29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.

Amino Acid Sequence↗
PubMed1993

Bombesin receptor antagonists differentiate receptor subtypes in rat brain.

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.

Animals↗
PubMed1993

Solubilization and purification of bombesin/gastrin releasing peptide receptors from human cell lines.

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.

Amino Acid Sequence↗
PubMed1993

Effect of bombesin antagonist D-Phe6-BN(6-13)OMe on vagally induced gastrin release from perfused rat stomach.

The aim of the present study was to evaluate the effect of the bombesin antagonist D-Phe6-BN(6-13)OMe (BN-antagonist) on vagally stimulated gastrin release from the isolated rat stomach, which was perfused via the celiac artery with Krebs-Ringer buffer. Vagal stimulation was performed for 10 minutes with 1 ms, 10 V and 10 or 2 Hz, respectively. Gastrin secretion increased significantly during stimulation with 10 and 2 Hz. BN-antagonist was added to the perfusate at the concentration of 10(-6) M, which induced a significant reduction of vagally stimulated gastrin release at 10 Hz (619 +/- 65 vs. 252 +/- 62 pg/10 min, p < 0.05), but not at 2 Hz (564 +/- 117 vs. 493 +/- 113 pg/10 min, p > 0.05). In contrast, atropine (10(-7) M) reduced significantly the gastrin response at 2 Hz (270 +/- 78 pg/10 min, p < 0.01), but not at 10 Hz (446 +/- 87 pg/10 min, p > 0.05). The combination of BN-antagonist and atropine elicited an inhibition of vagally stimulated gastrin release similar to each substance when given alone. Basal gastrin release was not changed by the BN-antagonist. The present data suggest, that in the rat stomach endogenously released bombesin-related peptides contribute to the noncholinergic stimulation of gastrin release at higher stimulation frequencies (10 Hz), however, bombesin-related peptides are not involved, when lower stimulation frequencies (2 Hz) are employed. At both stimulation frequencies additional mechanisms are activated which are noncholinergic and not related to bombesin peptides.

Animals↗
PubMed1993

Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP.

Sensitive and specific two-side enzyme immunoassays (two-site EIAs) for pituitary adenylate cyclase activating polypeptides, PACAP38, and PACAP27, have been established using six monoclonal antibodies against PACAP38, and a rabbit antibody against a C-terminal portion of PACAP27. In extracts of rat hypothalamus, these EIAs detected not only PACAP38 and PACAP27 but also an immunoreactive (ir-) PACAP lacking an epitope of a monoclonal antibody, PA-1C, which recognizes the C-terminal portion of PACAP38. By the use of these EIAs, it was found that one of the human neuroblastoma cell lines, IMR-32, produced ir-PACAP. In reverse-phase (RP-)HPLC, intracellular and extracellular ir-PACAPs were separated into two peaks, of which one was eluted at a position close to that of PACAP38 and the other in rather hydrophobic fractions. Those ir-PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot analysis of the two peaks of the RP-HPLC indicated that they consisted of several components including those with apparent molecular weights of 6.5 k and 10 k for the first peak ir-PACAP, and 14 k and 20 k for the second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processing steps. Although the significance of the modification in the C-terminus of PACAP38 is unknown, IMR-32 may be a cell line useful for studying the regulation of the biosynthesis of PACAP.

Animals↗
PubMed1993

Effect of [(CH2NH)4,5]secretin on pancreatic exocrine secretion in guinea pigs and rats.

[psi 4,5]Secretin was shown to be a secretin receptor antagonist that inhibits secretin-stimulated increase in adenosine 3',5'-cyclic monophosphate in isolated pancreatic acini of the guinea pig. To determine whether it inhibits pancreatic exocrine secretion in vivo, we have studied the effect of [psi 4,5]secretin on the pancreatic secretion stimulated by secretin in anesthetized guinea pigs and rats. In basal state, [psi 4,5]secretin given intravenously for 2 or 3 h in varying doses of 1.6-32.7 nmol.kg-1.h-1 dose dependently increased pancreatic secretion of both fluid and bicarbonate during the 1st h, but it returned gradually to basal level within 2 or 3 h. On the other hand, [psi 4,5]secretin significantly inhibited the pancreatic secretion stimulated by either exogenous or endogenous secretin in a dose-related manner. The inhibitory effect of [psi 4,5]secretin in guinea pigs was greater than that in rats. However, it did not completely block the secretin-stimulated pancreatic secretion, whereas a rabbit antisecretin serum suppressed it completely. We conclude that 1) in the unstimulated state, [psi 4,5]secretin is a partial agonist of pancreatic exocrine secretion of both fluid and bicarbonate; and 2) when pancreatic secretion is stimulated by secretin, unlike an antisecretin serum, it is a partial inhibitor in intact guinea pigs and rats.

Animals↗
PubMed1993

Angiopeptin enhances acetylcholine-induced relaxation and inhibits intimal hyperplasia after vascular injury.

The effects of the somatostatin analogue, angiopeptin (BIM-23014), on neoendothelial function, as evidenced by formation of prostaglandin (PG) I2 and by acetylcholine-induced relaxation (formation of endothelial-derived relaxing factor), were investigated in the rabbit aorta. A balloon catheter injury of the thoracic and abdominal aorta was induced in New Zealand White rabbits. Animals treated with angiopeptin for 2 or 4 wk were compared with untreated rabbits at 2 or 4 wk after the induction of injury, as well as to sham-operated controls. When the rabbits were killed, vascular rings were assessed for arachidonic acid-stimulated PGI2 formation, acetylcholine-induced relaxation, and the degree of intimal hyperplasia. Vascular rings from animals treated with angiopeptin exhibited enhanced acetylcholine-induced relaxation; however, angiopeptin treatment had no effect on arachidonic acid-stimulated PGI2 formation. Intimal hyperplasia in treated animals was reduced by 36%. Treatment with another somatostatin analogue, BIM-23030, did not enhance relaxation or inhibit intimal hyperplasia. These data suggest that treatment with angiopeptin may inhibit intimal hyperplasia in part by its beneficial effect on neoendothelial function.

Acetylcholine↗
PubMed1993

Pituitary adenylate cyclase activating polypeptide: a neuropeptide with potent inotropic and coronary vasodilatory effects in neonatal pig hearts.

Cardiac effects of the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) have not previously been reported. We investigated the influence of PACAP, vasoactive intestinal polypeptide (68% homology with PACAP) and the beta-adrenergic receptor agonist isoproterenol on contractile function and coronary vascular tone in isolated piglet hearts (1 to 5 d of age). Paced (180 beats/min) isovolumically beating hearts underwent retrograde aortic perfusion at constant coronary flow (approximately 3 mL.min-1.g-1) with an erythrocyte-enriched (hematocrit 15 to 20%) solution (37 degrees C). Agonists were injected into the aortic root of hearts, and the positive (+) and negative (-) changes in maximum rate of change of systolic pressure with respect to time (dP/dtmax) and in coronary perfusion pressure (that reflected alterations in vascular tone) were measured. PACAP (n = 8, 0.1 and 0.5 nmol) increased (+) dP/dtmax from 944 +/- 59 to 1519 +/- 206 mm Hg/s and from 867 +/- 40 to 2010 +/- 226 mm Hg/s (p < 0.05); increased (-) dP/dtmax from 1114 +/- 41 to 1439 +/- 95 mm Hg and from 999 +/- 37 to 1668 +/- 145 mm Hg/s (p < 0.05); and decreased perfusion pressure from 61.4 +/- 3.1 to 48.9 +/- 2.3 mm Hg and from 60.5 +/- 2.4 to 43.9 +/- 2.3 mm Hg (p < 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

Characterization of bombesin receptors using a novel, potent, radiolabeled antagonist that distinguishes bombesin receptor subtypes.

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.

3T3 Cells↗