CCK2 receptors mediate inhibitory effects of cholecystokinin on the motor activity of guinea-pig distal colon

Cholecystokinin and related peptides are involved in the control of intestinal motility and cholecystokinin receptor ligands might represent new pharmacological tools for the treatment of symptoms associated with functional bowel disorders. However, the respective roles played by cholecystokinin receptor subtypes and the mechanisms underlying these regulatory actions remain undetermined. This study was designed to examine the influence of cholecystokinin receptor subtypes on the motor activity of guinea-pig distal colon. The effects of drugs acting on CCK1 and CCK2 receptors were assessed in vitro on the contractile activity of longitudinal smooth muscle, both under basal conditions and in the presence of transmural electrical stimulation or KCl-induced contractions. The application of cholecystokinin octapeptide sulphate (cholecystokinin-8S) to colonic preparations induced concentration-dependent contractions which were prevented by devazepide (CCK1 receptor antagonist), enhanced by GV150013 (CCK2 receptor antagonist) or N(omega)-nitro-L-arginine methylester (L-NAME, nitric oxide synthase inhibitor), and unaffected by tetrodotoxin. The application of gastrin-17 to colonic preparations resulted in relaxant responses which were insensitive to devazepide, and prevented by GV150013, L-NAME or tetrodotoxin. L-NAME, N(omega)-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) or GV150013 enhanced electrically evoked contractile responses, whereas devazepide did not. When tested in the presence of L-NAME or NPA the enhancing effect of GV150013 on electrically induced contractions no longer occurred. In the presence of KCl-induced pre-contractions, cholecystokinin-8S or gastrin-17 evoked concentration-dependent relaxations, which were unaffected by devazepide and were counteracted by GV150013, L-NAME, NPA or tetrodotoxin. In conclusion, the present results indicate that, at level of distal colon, CCK1 receptors mediate direct contractile effects on smooth muscle, whereas CCK2 receptors on enteric neurons mediate relaxant responses via nitric oxide release.     

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist

The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2相似文献   

Effect of rokitamycin on upper gastrointestinal contractile activity. Analysis of side-effects on gastrointestinal tract

In order to determine whether rokitamycin (RKM), one of the macrolide antibiotics, has any side effects on the gastrointestinal tract, the effect of intraduodenal administration of RKM (1.0, 3.0 and 9.0 mg/kg) on gastrointestinal contractile activity was studied by means of force transducers implanted chronically on the gastric body, gastric antrum, duodenum and upper jejunum in conscious dogs. Erythromycin (EM 0.3, 1.0 and 3.0 mg/kg) and kitasamycin (LM 1.0, 3.0 and 9.0 mg/kg), both macrolide antibiotics, were used as control drugs. RKM, when given at 3.0 mg/kg and 9.0 mg/kg doses, induced segmentation contractions only in the duodenum where it was administered. The duration of the RKM-induced contractions was 7.5 +/- 2.5 minutes for 3.0 mg/kg and 15.8 +/- 3.0 minutes for 9.0 mg/kg, and the contractile force of the contractions was 43 to 82% of the maximum contractile force of the interdigestive contractions in the duodenum. EM, at 0.3 mg/kg, evoked a series of strong contractions quite different from those induced by RKM but similar to the natural interdigestive contractions, and with large doses, dose-dependent long-lasting interdigestive contractions were induced. On the other hand, LM did not stimulate notable gastrointestinal contractile activity even at a 9.0 mg/kg dose. In order to eliminate the possibilities of the contraction being caused by the effect of RKM on the duodenum through the general circulation upon absorption, 3.0 mg/kg RKM was given intravenously. It was found that intravenous injection of RKM did not evoke any contractions attributable to the direct action of RKM in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholecystokinin CCK2 receptors mediate the peptide's inhibitory actions on the contractile activity of human distal colon via the nitric oxide pathway

BACKGROUND AND PURPOSE: Cholecystokinin is known to exert stimulant actions on intestinal motility via activation of type 1 cholecystokinin receptors (CCK(1)). However, the role played by cholecystokinin 2 (CCK(2)) receptors in the regulation of gut motility remains undetermined. This study was designed to examine the influence of CCK(2) receptors on the contractile activity of human distal colon. EXPERIMENTAL APPROACH: The effects of compounds acting on CCK(2) receptors were assessed in vitro on motor activity of longitudinal smooth muscle, under basal conditions as well as in the presence of KCl-induced contractions or transmural electrical stimulation. KEY RESULTS: Cholecystokinin octapeptide sulphate induced concentration-dependent contractions which were enhanced by GV150013 (CCK(2) receptor antagonist; +57% at 0.01 microM). These effects were unaffected by tetrodotoxin. The enhancing actions of GV150013 on contractions evoked by cholecystokinin octapeptide sulphate were unaffected by N(omega)-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor), while they were prevented by N(omega)-nitro-L-arginine methylester (L-NAME, non-selective nitric oxide synthase inhibitor). In the presence of KCl-induced contractions, cholecystokinin octapeptide sulphate elicited concentration-dependent relaxations (-36%), which were unaffected by NPA, but were counteracted by GV150013 or L-NAME. The application of electrical stimuli evoked phasic contractions which were enhanced by GV150013 (+41 % at 0.01 microM). CONCLUSIONS AND IMPLICATIONS: CCK(2) receptors mediate inhibitory actions of cholecystokinin on motor activity of human distal colon. It is suggested that CCK(2) receptors exert their modulating actions through a nitric oxide pathway, independent of the activity of the neuronal nitric oxide synthase isoform.     

Interactions between AT1 and AT2 receptors in uterine arteries from pregnant ewes.

This study was performed to investigate the roles of angiotensin receptors (AT1 and AT2) in the contractility of uterine arteries during normal pregnancy and after angiotensin II levels have been elevated. Pregnant ewes were given intravenous infusions of saline for 24 h (control) or angiotensin II (30 ng kg(-1) min(-1)) for 2 or 24 h. The contractile responses of uterine arterial rings to angiotensin II (4 microM) and antagonists were then examined in vitro. Most uterine arteries were relatively insensitive to the vasoconstrictor effects of angiotensin II. In rings from control ewes an angiotensin AT2 antagonist enhanced (P < 0.05) the contractile responses to angiotensin II, suggesting that angiotensin AT2 receptors inhibited the angiotensin AT1 receptor mediated contractions. Uterine arterial rings from ewes given intravenous infusions of angiotensin II displayed greater (P < 0.05) contractile responses to angiotensin II in vitro compared to rings from control ewes. This was in part due to down regulation of angiotensin AT2 receptors. Surprisingly, while performing these experiments a small number of ewes had uterine arteries which were "hyperreactive" to angiotensin II (contractile responses 6-fold greater). These ewes also had abnormal renin angiotensin systems and had some features which are characteristic of those seen in preeclampsia. The "hyperreactivity" of these arteries could only in part be explained by down regulation of angiotensin AT2 receptors. It is concluded that in normal pregnancy angiotensin AT2 receptors play a role in maintaining an adequate uterine blood flow for the fetus. When angiotensin II levels are elevated for a prolonged period this protective effect is lost partly because angiotensin AT1 receptors are down regulated.     

The involvement of the serotonergic transmission system in neonatal and adult rat ileum contractility varies with age

The relevance of age on serotonergic involvement in the control of alimentary contractility has not been pharmacologically described. Experiments were performed to investigate the effects of acetylcholine, atropine, 5-hydroxytryptamine (5-HT) and its related drugs on intestinal segments taken from the neonatal and adult ileum. 5-HT induced concentration-dependent contractions of ileum irrespective of age; however, these contractions were diminished by pretreatment with atropine only in neonatal tissues. In tissues taken from both the neonatal and adult ileum, methysergide (5-HT(1/2/5-7) receptor antagonist), ritanserin (5-HT(2) receptor antagonist), and RS23597-190/SB204070 (5-HT(4) receptor antagonists) all differentially reduced 5-HT-induced contractions at a concentration <100 μmol/l. At higher concentrations, the contractions were comparable to those in control tissues. Granisetron and ondansetron (5-HT(3) receptor antagonists) significantly reduced contractions induced by 5-HT at concentrations >30 μmol/l in both neonatal and adult ileum. Combined treatments with ritanserin, granisetron, plus RS23597-190 reduced or abolished contraction responses induced in neonatal ileum by 5-HT. SB269970A (5-HT(7) receptor antagonist) and WAY100635 (5-HT(1A) receptor antagonist) failed to influence contractile responses induced by 5-HT or 5-HT receptor agonists. Pretreatments with WAY100635 and SB267790A also had no influence on the contractile responses induced by 5-HT(1A/7) receptor agonist, 5-CT, and 5-HT(1A) receptor agonist, 8-OH-DPAT, which itself failed to induce a measurable response. It is concluded that the 5-HT-induced contractions in segments taken from both the neonatal and adult rat ileum were mediated via 5-HT(2) receptors, 5-HT(3) receptors and 5-HT(4) receptors. However, the effect of atropine on the neonatal rat intestine indicates that the mechanism of serotonergic involvement in ileal contractility is influenced by age.     

CONTRACTILE ACTION OF GALANIN ANALOGUES ON RAT ISOLATED GASTRIC FUNDUS STRIPS IS MODIFIED BY TACHYPHYLAXIS TO SUBSTANCE P

This study was undertaken to characterize the interaction of porcine galanin (Gal) and some of its analogues with their receptors on rat gastric fundus muscle strips.Gal, galantide (M15) and Gal(1–14)-[Abu⁸]SCY-I evoked concentration-dependent contractions of gastric smooth muscle strips. Reproducible effects were observed in concentrations of 1–300, 3–1000 and 100–3000n , respectively. Specific EC₅₀for the contractile effect equalled 13, 70 and 187n .Hill's coefficient for Gal is 1.03 indicating an interaction of one Gal molecule with one receptor, fulfilling the criteria of classical receptor theory. For M15 and Gal(1–14)-[Abu⁸]SCY-I Hill's coefficients are different from 1, namely 0.73 and 1.56, pointing out that the principle of interaction of one drug molecule with one receptor may not apply. The contraction induced by 300n of Gal was not significantly modified by tachyphylaxis to substance P (SP). On the contrary the introduction of tachyphylaxis to SP decreased the contractile effects of M15 and Gal(1–14)-[Abu⁸]SCY-I by about 57.7±3% and 39.6±5%, respectively. The findings suggest that contractile actions of M15 and Gal(1–14)-[Abu⁸]SCY-I are probably not only due to their agonist activities at Gal receptors but may result from a subsequent stimulation of receptors for SP or release of endogenous SP.     

Tachykinin NK(2) receptors and enhancement of cholinergic transmission in the inflamed rat colon: an in vivo motility study

In the gastrointestinal tract, tachykinin NK(2) receptors are localized both on smooth muscle and nerve fibres. NK(2) receptor antagonists reduce exaggerated intestinal motility in various diarrhoea models but the site of action contributing to this effect is unknown. In this study we investigated the effects of atropine (1.4 micromol kg(-1), i.v.), hexamethonium (13.5 micromol kg(-1), i.v.), and nepadutant (0.1 micromol kg(-1), i.v.), a selective tachykinin NK(2) receptor antagonist, on distension (0.5 and 1 ml)-, or irritation (acetic acid, 0.5 ml of 7.5% v v(-1))-induced motility in the rat distal colon in vivo. The effects of atropine, hexamethonium or N(omega)-nitro-L-argininemethylester (L-NAME, 1.85 micromol kg(-1), i.v.) on [betaAla(8)]NKA(4-10) (10 nmol kg(-1), i.v.)-induced colonic contractions were also investigated. When the colonic balloon was filled with a subthreshold volume (0.5 ml), the intraluminal instillation of acetic acid triggered a high-amplitude phasic colonic motility which was partially reduced by nepadutant and suppressed by either hexamethonium or atropine. Filling of the balloon with 1 ml evoked reflex (hexamethonium-sensitive), atropine-sensitive phasic colonic motility: nepadutant had no significant effect on the distension-evoked motility. Neither hexamethonium nor atropine significantly reduced [betaAla(8)]NKA(4-10)-induced colonic contractions, whereas nepadutant suppressed them. Following L-NAME pretreatment, [betaAla(8)]NKA(4-10)-induced colonic contractions were inhibited by both atropine and hexamethonium. In hexamethonium-pretreated animals, an atropine-sensitive component of [betaAla(8)]NKA(4-10)-induced colonic contractions was also evident. These results indicate that the application of irritants onto the colonic mucosa induces the release of endogenous tachykinins which enhance excitatory cholinergic mechanisms through the stimulation of NK(2) receptors.     

Investigation of the effects of P2 purinoceptor ligands on the micturition reflex in female urethane-anaesthetized rats

1 The effects of purinoceptor ligands for P2X1 and/or P2X3 receptors (alpha,beta-meATP, IP(5)I, TNP-ATP, MRS 2179, PPADS, Phenol red and RO116-6446/008; i.v., n=4-5) and for P2Y1 receptors (PPADS, MRS 2179 and MRS 2269; i.v., n=3-5) were investigated on the distension-evoked 'micturition reflex' in the urethane-anaesthetized female rat. 2 Alpha,beta-meATP (180 nmol kg(-1) min(-1)), IP5I (10, 30 and 100 nmol kg(-1)), TNP-ATP (1 micromol kg(-1)), MRS 2179 (1 micromol kg(-1)) and PPADS (17 micromol kg(-1)) each caused maintained bladder contractions to occur during the infusion of saline into the bladder. PPADS (17 micromol kg(-1) min(-1)) had a similar effect when infused intravesicularly. Regular bladder contractions were not observed until the infusion of saline was halted. For IP5I, TNP-ATP, MRS 2179 and PPADS, the magnitude of postinfusion isovolumetric contractions was significantly reduced and, for IP5I, this action was also associated with a significant reduction in urethral relaxation. Additionally, TNP-ATP caused a significant increase in the pressure and volume thresholds required to initiate a reflex. 3 Phenol red (a P2X1/P2X3 antagonist; 0.1 and 1 micromol kg(-1)) caused a significant increase in the pressure and volume thresholds required to initiate a reflex and, at the higher dose, also caused a reduction in postinfusion isovolumetric contractions. 4 RO116-6446/008 (a P2X1-selective antagonist; 1 and 10 micromol kg(-1)) only caused a reduction in postinfusion isovolumetric contractions. 5 It is concluded that P2X1 and P2X3 receptors play a fundamental role in the micturition reflex in urethane-anesthetized female rats. P2X3 receptor blockade raised the pressure and volume thresholds for the reflex, whereas P2X1 receptor blockade diminished motor activity associated with voiding. P2Y1 receptors may be involved in inhibition of rat detrusor tone.     

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 nM for the CCK1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg(-1) was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine.     

Green tea catechins evoke a phasic contraction in rat aorta via H2O2-mediated multiple-signalling pathways

1. The contractile effects of tea polyphenols (TP) and its four principle catechins, namely (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG), on rat aorta contractility were investigated using the isometric tension recording technique. 2. At concentrations of 5-100 mg/L, TP evoked phasic contraction of rat aorta in a concentration-dependent but endothelium-independent manner. Of the four catechins tested, EGCG and EGC (3-300 micromol/L), but not EC and ECG, mimicked the contractile response to TP, suggesting that the epigallol moiety in the B ring may be associated with the contractile effect. 3. Contractions in response to EGCG and EGC were not affected by several endogenous vasoconstrictor receptor antagonists, but could be abolished by 10 micro mol/L BAPTA-AM, a membrane-permeable Ca2+ chelator, or attenuated by removal of extracellular Ca2+, suggesting the involvement of both intracellular and extracellular Ca2+ in evoking the contraction. 4. Pretreatment with non-selective Ca2+ channel antagonists mefenamic acid (10 micro mol/L), tetrandrine (30 micro mol/L) and SKF 96365 (30 micromol/L), but not nifedipine (1 micromol/L), the selective inhibitor of voltage-dependent Ca2+ channels, inhibited the contractile responses to EGC and EGCG, indicating the involvement of Ca2+ influx via non-voltage dependent Ca2+ channels. 5. Several intracellular Ca2+ channel modulators, including procaine (5 mmol/L), dantrolene (30 micromol/L) and 2-amino ethoxydiphenyl borate (50 micromol/L; an inositol 1,4,5-trisphosphate receptor inhibitor), also inhibited EGCG- and EGC-induced contractions, thus suggesting a role of intracellular Ca2+ release in these contractions. 6. Both EGCG- and EGC-induced contractions were depressed, to different degrees, by inhibitors of several receptor-coupled enzymes, including phospholipase C, protein kinase C, phospholipase A2 and tyrosine kinase. Furthermore, both EGCG- and EGC-induced contractions were completely abolished by catalase, but not by superoxide dismutase or mannitol/dimethyl sulphoxide. 7. Taken together, these data show, for the first time, that TP and its related catechins that contain an epigallol structure in the B ring, as in EGCG and EGC, exert direct contractile effects on rat aortic smooth muscle via a H2O2-mediated pathway.     

Pharmacological study of IQM-97,423, a potent and selective CCK1 receptor antagonist with protective effect in experimental acute pancreatitis

The pharmacological profile of the new CCK1 receptor antagonist IQM-97,423, (4aS,5R)-2-benzyl-5-(tert-butylaminocarbonyl-tryptophyl)amino-1,3-dioxoperhydropyrido-[1,2-c]pyrimidine, was examined in in vitro and in vivo studies and compared with typical CCK1 antagonists such as devazepide and lorglumide. IQM-97,423 showed a high affinity at [3H]-pCCK8-labeled rat pancreatic CCK1 receptors, and was virtually devoid of affinity at brain CCK2 receptors. IQM-97,423 antagonized CCK8S-stimulated alpha-amylase release from rat pancreatic acini with a potency similar to devazepide and much higher than lorglumide. In the guinea pig isolated longitudinal muscle-myenteric plexus preparation, IQM-97,423 produced a full antagonism of the contractile response elicited by CCK8S and a weaker effect on the contraction elicited by CCK4, suggesting a selective antagonism at CCK1 receptors. The protective effect of IQM-97,423 and devazepide was tested in two models of acute pancreatitis in rats, induced by injection of cerulein or by combined bile and pancreatic duct obstruction. The new compound fully prevented the cerulein-induced increase in plasma pancreatic enzymes and in pancreas weight with a potency similar to devazepide. In common bile-pancreatic duct ligature-induced acute pancreatitis, IQM-97,423 partially prevented, like devazepide, the increase in plasma pancreatic enzyme activity and in pancreas weight. Consequently, the pyridopyrimidine derivative IQM-97,423 is a potent and highly selective CCK1 receptor antagonist with preventive effects in two experimental models of acute pancreatitis and a potential therapeutic interest.     

Tachykinin receptors mediate atropine-resistant rat duodenal reflex contractions in vivo

The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 mol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2–0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 mol/kg i.v.) or hexamethonium (28 mol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 mol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4–5 mmHg by further distension of the balloon (0.4–0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 mol/h), to provide a suitable baseline (amplitude of contractions was 7–8 mmHg) for studying the effect of tachykinin receptor antagonists.I.v. administration of the selective tachykinin NK₂ receptor antagonists, MEN 10,627 (10–100 nmol/kg) and SR 48968 (100–300 nmol/kg) or of the selective NK₁ antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 µmol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR 48965 (300 nmol/kg), the enantiomer of SR 48968 devoid of NK₂ receptor blocking activity, was without effect.I.v. administration of the selective NK_i receptor agonists [Sar⁹] substance P sulfone and septide or of the NK₂ receptor selective agonist, Ala⁸] neurokinin A(4–10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar⁹] substance P sulfone and septide without affecting those produced by [Ala⁸] neurokinin A(4–10). On the other hand, MEN 10,627 (30–100 nmol/kg i.v.) and SR 48968 (100–300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK₂ and NK₁ receptor agonists.We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK₁ and NK₂ receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK₁ receptor agonists, [Sar⁹] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK₂ receptors.     

Tocolytic effect of parecoxib, a new parenteral cyclo-oxygenase-2-specific inhibitor, on the spontaneous and prostaglandin-induced contractions of rat isolated myometrium

1. The present study was undertaken to elucidate the effects of parecoxib, a novel cyclo-oxygenase (COX)-2 inhibitor, on spontaneous and prostaglandin-induced contractions of uterine smooth muscle. 2. Non-pregnant adult Wistar rats were decapitated and dissected to isolate myometrial strips. The tissue was mounted in 5 mL organ baths filled with Krebs' solution that was maintained at 37 degrees C and bubbled continuously with a mixture of 95% O(2)-5% CO(2) to give pH 7.4. Contractions were recorded through transducers for isometric tension recording. The dose-dependent effects of parecoxib on contractility were quantified by changes in the mean amplitude, frequency and area under the contractile curve (AUC; percentage of control conditions) of the isometric tension recordings, averaged over 5 min intervals. Statistical analyses were performed using ANOVA. 3. Application of parecoxib (50-900 micromol/L) caused dose-dependent decreases in mean amplitude, mean frequency and mean AUC of both spontaneous and prostaglandin-induced contractions. Mean percentage inhibition of the AUC of spontaneous contractions was found to be 29, 56, 74 and 84% in the presence of 50, 150, 300 and 600 micromol/L parecoxib, resepctively (n = 8). In the case of prostaglandin (PG) F(2alpha)-induced contractions, 100, 300, 600 and 900 micromol/L parecoxib resulted in a 27, 43, 61 and 73% inhibition, respectively (n = 9). Moreover, pretreatment with parecoxib (600 micromol/L) reduced the responsiveness and maximum contractility to PGF(2alpha) compared with non-treated strips. 4. The data from the present study indicate, for the first time, that parecoxib inhibits spontaneous and prostaglandin-induced contractions of rat myometrium in vitro. These results raise the possibility that parecoxib may be of therapeutic use in the management of preterm labour and dysmenorrhoea.     

Regional and functional differences of 5-hydroxytryptamine-receptor subtypes in guinea pig stomach

Functions and the presence of 5-hydroxytryptamine (5-HT) receptors in the fundus, corpus and antrum of the guinea pig stomach were examined by measuring contractile force and acetylcholine (ACh) release. Stimulation of the 5-HT1 receptor caused tetrodotoxin (TTX)-insensitive relaxations in the preparations from 3 regions. Stimulation of the 5-HT2 receptor caused TTX-insensitive contractions in the preparations of fundus and antrum. Stimulation of 5-HT3 receptors caused contractions that were sensitive to TTX and atropine and enhanced the outflow of [3H]ACh from preparations of only antrum. Stimulation of 5-HT4 receptors caused contractions of antral strips and decreased relaxations of corporal strips and enhanced the outflow of [3H]ACh from the preparations of both corpus and antrum. In the guinea pig stomach, the fundus possesses relaxant 5-HT1 receptor < contractile 5-HT2 receptors and caused the contractile response to 5-HT. The corpus possesses relaxant 5-HT1 receptors and relaxant receptors other than 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptors > contractile 5-HT4 receptor, and therefore 5-HT caused relaxations. The antrum possesses relaxant 5-HT1 receptor < contractile 5-HT2, 5-HT3 and 5-HT4 receptors, and thus 5-HT caused contractions.     

Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor

1 The full-length, canine cholecystokinin 1 (CCK1) receptor was cloned from gallbladder tissue using RT-PCR with a combination of primers designed to interact with conserved regions of the human and rat CCK1 receptor, which also shared homology with the canine genomic sequence. 2 Analysis of the sequence of the canine CCK1 receptor revealed a 1287 base pair product, which encoded a 429 amino-acid protein. This protein was 89% identical to the human and 85% identical to the rat CCK1 receptor. 3 The canine CCK1 receptor was expressed in CHO-K cells for pharmacological characterization. In competition studies, using [(125)I]BH-CCK-8S as radioligand, the affinity values estimated for CCK receptor-selective compounds were not significantly different between the canine and human CCK1 receptors (pK(I)+/-s.e.m. at canine CCK1 receptor; L-364,718=8.82+/-0.08, L-365,260=6.61+/-0.05, YF476=7.91+/-0.15, YM022=8.28+/-0.06 and dexloxiglumide=7.53+/-0.11). Furthermore, the selectivity of these compounds between canine CCK1 and CCK2 receptors was consistent with the selectivity between the human CCK1 and CCK2 receptors. 4 Two additional forms of the canine CCK1 receptor were identified during the cloning procedure. These had three (variant #1) and six (variant #2) amino-acid differences from the wild-type canine CCK1 receptor. Variant #1 bound [(125)I]BH-CCK-8S and displayed an identical pharmacological profile to the wild-type receptor using the ligands described above. No significant binding was measured with variant #2. 5 In conclusion, we have cloned and pharmacologically characterized the canine CCK1 receptor. The data obtained will facilitate the interpretation of numerous pharmacological experiments that have been performed using canine tissue to elucidate the actions of CCK and gastrin.     

L364,718 antagonizes the cholecystokinin-induced suppression of locomotor activity

To determine the role of CCK-A receptors in the cholecystokinin (CCK)-induced suppression of locomotor activity in the rat, the ability of the selective CCK-A receptor antagonist L364,718 to block these responses was investigated. Cholecystokinin octapeptide (CCK8) (10, 100 micrograms/kg IP) and caerulein (1, 5, 10 micrograms/kg IP) produced marked reductions in locomotor activity whereas cholecystokinin tetrapeptide (CCK4) (100 micrograms/kg IP) was without effect. The reductions in activity produced by CCK8 (10 micrograms/kg) and caerulein (10 micrograms/kg) were antagonized by L364,718 (100 micrograms/kg IP). In an open field test CCK8 (10 micrograms/kg IP) reduced locomotor activity and total number of rears and increased pause duration. These effects of CCK8 on open-field behaviour were also antagonized by L364,718 (100 micrograms/kg IP). It is concluded that L364,718 is a potent antagonist of the actions of CCK8 and caerulein on locomotor activity, suggesting that the effects of these peptides are mediated by a CCK-A receptor.     

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.     

Long-lasting cholecystokinin(2) receptor blockade after a single subcutaneous injection of YF476 or YM022

Histamine-forming ECL cells in the rat stomach operate under the control of gastrin. They represent a convenient target for studying cholecystokinin-B/gastrin (CCK(2)) receptor antagonists in vivo. We examined the effectiveness and duration of action of two CCK(2) antagonists, YM022 and YF476, with respect to their effect on ECL-cell histidine decarboxylase (HDC) activity in the rat. Oral administration of subcutaneous deposition of YF476 or YM022 reduced the HDC activity. The maximum/near-maximum dose for both drugs and for both modes of administration was 300 micromol kg(-1) (effects measured 24 h after dose). At this dose and time the serum concentration of YF476 was 20 - 40 nmol l(-1). The dose 300 micromol kg(-1) was used in all subsequent studies. A single subcutaneous injection of YF476 inhibited the HDC activity for 8 weeks. The circulating concentration of YF476 remained high for the same period of time (>/=15 nmol l(-1)). Subcutaneous YM022 suppressed the HDC activity for 4 weeks. A single oral dose of YF476 or YM022 inhibited the HDC activity for 2 - 3 days. Chronic gastric fistula rats were used to study the effect of subcutaneous YF476 on gastrin-stimulated acid secretion. A single injection of YF476 prevented gastrin from causing an acid response for at least 4 weeks (the longest time studied). We conclude that a single subcutaneous injection of 300 micromol kg(-1) YF476 causes blockade of CCK(2) receptors in the stomach of the rat for 8 weeks thus providing a convenient method for studies of the consequences of long-term CCK(2) receptor inhibition.     

The contractile action of leukotriene B4 in the guinea-pig lung involves a vascular component

Leukotriene B4 (LTB4) is a potent leukocyte chemoattractant, acting on specific receptors, BLT receptors. The aim of this study was to examine the mechanism of action of LTB4 in the guinea-pig lung, using strips of lung parenchyma (GPLP), spirals of trachea (GPT) and bronchus (GPB) and rings of pulmonary artery (GPPA). Mechanical responses were studied in organ baths, and mediator release was assessed using enzyme immuno assay. LTB4 induced similar contractions of GPLP and GPPA, whereas LTB4 had only small contractile effects in GPT and GPB. In addition, the contractile response to LTB4 was reproduced in the human pulmonary artery. In the GPLP, the unselective BLT receptor antagonist ONO-4057 abolished the contractions induced by LTB4, whereas the selective BLT1 receptor antagonist U-75302 only partly inhibited the LTB4-induced contractions. In the GPPA, both antagonists abolished the response to LTB4. The effect of LTB4 in GPPA and GPLP was indirect and mediated by the release of thromboxane A2 and histamine, as supported by selective pharmacologic interventions and measurements of thromboxane B2 and histamine in the organ baths. In conclusion, the results indicate a new biological function of LTB4, namely to constrict isolated pulmonary arteries. Moreover, the findings suggest that the LTB4-induced contractions of GPPA were mediated by a BLT1 receptor, whereas BLT2 receptor activation accounted for a major part of the contraction of GPLP, making the latter preparation a suitable assay for BLT2 receptors.British Journal of Pharmacology (2004) 141, 449-456. doi:10.1038/sj.bjp.0705641