Oesophageal Muscularis Mucosae Research Articles

Post-synaptic 5-HT 4 receptor modulation of tachykinergic excitation of rat oesophageal tunica muscularis mucosae

Interaction between the 5-HT 4 receptor and cholinergic-dependent and -independent contraction of the rat oesophageal muscularis mucosae was determined. Substance P- (in the presence of atropine) and carbachol-precontracted tissue was relaxed by tryptamines and the substituted benzamides with the following rank order of potency: 5-HT>5-methoxytryptamine>cisapride>( R)-zacopride>lintopride>metoclopramide, consistent with 5-HT 4 receptor activation. The response to 5-HT was not antagonized by tetrodotoxin, methysergide or ondansetron, but was shifted to the right by GR113808 ([1-[2-[methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl-1- H-indole-3-carboxylate) in substance P- and carbachol-precontracted tissue, confirming 5-HT 4-mediated relaxation. This study shows for the first time that although 5-HT 4 receptors are involved in the modulation of cholinergic neurotransmission they can also act independently of this system modulating tachykinergic responsiveness. © 1997 Elsevier Science B.V. All rights reserved.

Pharmacologic characteristics of rabbit esophageal muscularis mucosae in vitro.

The aim of this study was to investigate in vitro the pharmacologic properties of rabbit esophageal muscularis mucosae. Stimulation of its motor innervation elicited only atropine-sensitive excitatory responses. This tissue was not refractory to histamine but appeared to be rapidly desensitized to its effects. Responses to adrenoreceptor agonists were produced by excitatory alpha1 and beta1 adrenoreceptors, inhibitory beta3 receptors but not alpha2 receptors. The esophageal muscularis mucosae was only weakly stimulated by neurokinins and bradykinin, and these responses were mediated via NK2, NK3, B1, and B2 receptors, respectively. Adenosine diphosphate and adenosine triphosphate produced approximately 40% maximal contractions through the activation of P2 receptors, whereas adenosine monophosphate and adenosine were without effect. Responses to prostaglandins E2 and F2alpha were < or =10% of the tissues' maximum response to acetylcholine. These data demonstrate that rabbit esophageal muscularis mucosae has a simple excitatory innervation and is only weakly stimulated by a variety of pharmacologic agents. For these reasons it is distinct from muscularis mucosae found elsewhere in the rabbit gastrointestinal tract.

P-675 - The role of receptor-operated Ca2+ influx in acetylcholIne-induced contraction of the esophageal muscularis mucosae

P-675 - The role of receptor-operated Ca2+ influx in acetylcholIne-induced contraction of the esophageal muscularis mucosae

Influence of adrenodemedullation on beta 2- and beta 3-adrenoceptors mediating relaxation of oesophageal smooth muscle of spontaneously hypertensive rats.

1. In oesophageal smooth muscle strips from spontaneously hypertensive rats (SHR) of 8-10 and 22-24 weeks of age, respectively, beta-adrenoceptor-mediated relaxation was investigated, by use of the beta-agonists, (-)-isoprenaline and fenoterol (both in the absence and presence of the beta 2-selective antagonist ICI 118,551) and the selective beta 3-agonist, BRL 37,344. 2. In preparations from 8-10 week SHR, (-)-isoprenaline- and fenoterol-induced concentration-response curves (CRCs) were hardly antagonized by ICI 118,551 at concentrations up to 1 microM, indicating only a minor contribution of beta 2-adrenoceptors. pA2-values for ICI 118,551 of 5.30 ((-)-isoprenaline as agonist) and 5.46 (fenoterol as agonist), estimated from the shifts at the highest (10-100 microM) antagonist concentrations, are consistent with affinity at a beta 3-adrenoceptor, similar to that in Wistar rat oesophageal smooth muscle. 3. In 8-10 week SHR, adrenodemedullated at 4 weeks of age (SHR-ADM4) the potency of fenoterol was markedly increased and CRCs were shallow. In addition, ICI 118,551 (0.1 microM) now produced a clear rightward shift accompanied by a steepening of the CRC. A marked further shift was observed only at 100 microM of the antagonist. The data are compatible with the involvement of both beta 2- and beta 3-adrenoceptors. 4. In 22-24 week animals, the same differences between SHR and SHR-ADM4 were observed with fenoterol as in 8-10 week animals, though beta-adrenoceptor responsiveness was slightly decreased. The potency of ICI 118,551 at beta 3-adrenoceptors (pA2 = 5.11) was significantly different from the pA2 value of 5.46 obtained with the younger animals. 5. Responses to the beta 3-adrenoceptor agonist, BRL 37,344, were similar in Wistar rat and SHR preparations. In 8-10 week SHR, a small decrease in the maximal response was observed, which in animals of 22-24 weeks of age was accompanied by a small decrease in the pEC50 value as well. 6. The results clearly indicate that beta 2-adrenoceptors in SHR oesophageal muscularis mucosae are desensitized, whereas beta 3-adrenoceptor-mediated responses are unaffected and similar to the responses observed in the Wistar rat oesophagus. The functional presence of beta 2-adrenoceptor-responses in SHR-ADM4 suggests a major role for adrenal-derived adrenaline in the desensitization of the beta 2-adrenoceptor-population.

Functional interactions between muscarinic M2 receptors and 5-hydroxytryptamine (5-HT)4 receptors and beta 3-adrenoceptors in isolated oesophageal muscularis mucosae of the rat.

1. Relaxations of isolated oesophageal muscularis mucosae of rat are mediated by 5-hydroxytryptamine (5-HT), acting at 5-HT4 receptors, and isoprenaline, principally acting via beta 3-adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M2 receptors, also present in this tissue, functionally oppose 5-HT and beta-adrenoceptor-relaxant effects in this preparation. 2. Contractions of rat oesophageal muscularis mucosae were induced, in a concentration-dependent manner, by the muscarinic receptor agonist, oxotremorine M (pEC50 = 6.7 +/- 0.1). The contractile responses to oxotremorine M were surmountably antagonized by the following compounds, (pKB values in parentheses): atropine (9.1 +/- 0.2), 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide, 8.7 +/- 0.1), p-F-HHSiD (para-fluoro-hexa-hydro-siladifenidol, 7.5 +/- 0.1), zamifenacin (8.6 +/- 0.3), himbacine (7.2 +/- 0.2), pirenzepine (6.8 +/- 0.3) and methoctramine (6.2 +/- 0.2). These data are consistent with a role for muscarinic M3 receptors mediating contractions to oxotremorine M. The contractile response was associated with a low receptor reserve, since the responses were shifted to the right and virtually abolished by the alkylating agent, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl) piperidine, 40 nM; 60 min equilibration). 3. In tissues precontracted with U46619 (0.7 microM; approx. EC90), isoprenaline (pEC50 = 8.0 +/- 0.1) and 5-HT (pEC50 = 7.5 +/- 0.2) induced concentration-dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine M (isoprenaline, pEC50 = 7.4 +/- 0.2). In contrast, the potency of 5-HT (pEC50 = 7.5 +/- 0.2), in tissues that were precontracted with 1 microM (EC90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M2 receptor antagonist, methoctramine (1 microM), there was no effect on the relaxant potencies to either 5-HT or isoprenaline. Collectively, these data suggest that muscarinic M2 receptors do not, under these conditions, modulate relaxant potencies to either 5-HT or isoprenaline. 4. In a second protocol, tissues were pre-contracted with U46619 (0.7 microM) and relaxed with either 5-HT (0.1 microM) or isoprenaline (0.1 microM). In these tissues (in which the muscarinic M3 receptor population was extensively depleted by alkylation), oxotremorine M caused concentration-dependent re-contractions (i.e. reversal of relaxations). In tissues relaxed with 5-HT, the potency of oxtremorine M was 5.9 +/- 0.2, while in tissues relaxed with isoprenaline, the potency (pEC50) = 5.6 +/- 0.3. These re-contractions were antagonized, in a surmountable fashion, by methoctramine (1 microM; pKB = 7.6 +/- 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 microM; pKB = 7.5 +/- 0.2). Under these conditions, therefore, the pKB values are consistent with activation of muscarinic M2 receptors, and inconsistent with activation of M3 receptors. 5. It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M3 receptors mediate direct contractions and are associated with a low receptor reserve. When this population is depleted, and the tissues relaxed via activation of receptors that augment adenylyl cyclase activity, a functional role for muscarinic M2 receptors is revealed.

Potential anti-diabetic applications of a new molecule with affinity for β3-Adrenoceptors

β 3-adrenoceptors have been described through both molecular and pharmacological studies. In this context, it has been characterized the β 3-adrenoceptors agonist activity of a new compound (Trecadrine ®), and evaluated whether it exhibits additional activity at β 1 and β 2- adrenoceptor subtypes. In addition, it has been tested its potential anti-diabetic properties in a model of alloxan-induced diabetes in rats. Our data show that Trecadrine ® induces oesophageal muscularis mucosae relaxation, indicating a putative action on β 3-adrenoceptors. In the absence of β 3-adrenoceptors antagonists, assays with β 1 and β 2-adrenergic antagonists reveal a quite remarkable selectivity for β 3-adrenoceptors. Furthermore, it has been suggested that this molecule has hypoglycaemic and lipomobilizing effects, although hypoglycaemia was not related to an increase in insulin secretion in diabetic rats. It is concluded that Trecadrine ® mainly acts through β 3-adrenoceptors, and it may constitute a potential drug in the treatment of diabetes.

Reduced expression of basic fibroblast growth factor and its receptor mRNAs and proteins in portal hypertensive esophageal mucosa: A mechanism responsible for muscularis mucosae thinning and variceal rupture

Basic fibroblast growth factor (bFGF) enhances cell migration, proliferation, and tissue integrity. This especially pertinent to the smooth muscle cells in which it stimulates cell proliferation and promotes their growth. The aim of this study was to determine whether expression of bFGF and its receptors (FGFR-1 and -2) is altered in portal hypertensive esophageal mucosa, especially in the muscularis mucosal layer, which constitutes a physical barrier to variceal rupture. Portal hypertension (PHT) was produced by staged portal vein ligation. In 30 PHT rats and 30 sham-operated controls 2 weeks after operation, specimens of lower esophagus were obtained for (1) quantitative histologic assessment including thickness of epithelium and muscularis mucosae; (2) immunostaining with specific antibodies against bFGF and its receptors 1 and 2 (intensity of bFGF, FGFR-1 and FGFR-2 immunostaining in esophageal structures was measured with a video image system); and (3) expression of bFGF and FGFR-1 and -2 mRNAs was assessed with reverse transcription-polymerase chain reaction. The esophageal muscularis mucosae and epithelium overlaying large submucosal veins in PHT rats significatly thinner than those in controls (muscularis mucosae, 28.3 +/- 1.4 versus 52.2 +/- 8.0 micrometer, respectively, p<0.05); epithelium, 39.0+/- 7.1 versus 49.3 +/- 1.9 micrometer, respectively, p<0.05). The immunostaining intensity of bFGF and FGFR-2 was significantly reduced in PHT rats (42.1 +/- 2.3 and 71.3 +/- 6.5 units, respectively) versus controls (49.5 +/- 5.6 and 78.6 +/- 5.7 units, respectively, p< 0.05). Expressions of bFGF and FGFR-2 mRNAs in PHT esophageal mucosa were significantly reduced versus controls by 30.8% and 30.3%, respectively (p < 0.01, p 0.05). (1) Esophageal mucosa of PHT rats has thinner muscularis mucosae and reduced bFGF and FGFR-2 mRNAs and proteins. (2) Because bFGF stimulates smooth muscle cell proliferation and their growth, our findings can explain thinning of esophageal muscularis mucosae in PHT rats, thus indicating a possible mechanism for rupture of varices in the esophagus.

Portal hypertension activates the nitric oxide synthase genes in the esophageal mucosa of rats

BACKGROUND & AIMS: Nitric oxide is associated with hyperdynamic circulation and development of collaterals in portal hypertension. The aim of this study was to investigate whether NO synthase is activated in the portal-hypertensive esophagus. METHODS: In esophageal specimens after portal ligation or sham operation, the expression of constitutive and inducible NO synthase messenger RNA was assessed by reverse transcription-polymerase chain reactions. NO synthase protein at 14 postoperative days was visualized with immunofluorescence staining with specific antibodies against constitutive and inducible NO synthase. RESULTS: The esophageal muscularis mucosae and epithelium overlying large submucosal veins in portal-hypertensive rats were significantly thinner than in controls (muscularis mucosae, 24.3% thinner, P < 0.01; epithelium, 23.0% thinner, P < 0.05). Expression of NO synthase proteins in endothelia of submucosal veins was significantly higher in portal-hypertensive rats than in controls (constitutive NO synthase, 17.6%; inducible NO synthase, 18.0% increased over controls, respectively; P < 0.01). Expression of both constitutive and inducible NO synthase messenger RNA in portal-hypertensive rats was significantly increased (constitutive NO synthase, 10-fold; inducible NO synthase, 20- fold at 14 days vs. controls; P < 0.01). CONCLUSIONS: Portal hypertension triggers overexpression of NO synthase messenger RNA and protein in the rat esophageal mucosa. This phenomenon, combined with the thinness of muscularis mucosae and epithelium, may facilitate development and rupture of esophageal varices. (Gastroenterology 1996 Feb;110(2):549-57)

Characterization of muscarinic receptor and β-adrenoceptor interactions in guinea-pig oesophageal muscularis mucosae

Smooth muscles of a number of species contain both muscarinic M 2 and M 3 receptors in differing proportions and while muscarinic M 3 receptors mediate contraction, the role of muscarinic M 2 receptors is unclear. Muscarinic M 2 receptor-mediated inhibition of adenylyl cyclase activity has been demonstrated in smooth muscle and since β-adrenoceptor relaxation of this tissue is mediated via stimulation of adenylyl cyclase, an interaction between muscarinic M 2 receptors and β-adrenoceptors in smooth muscle has been postulated. Such an interaction has been demonstated in guinea-pig ileum and trachea using two different approaches. The present study investigates whether interactions between muscarinic M 2 receptors and β-adrenoceptors also occur in guinea-pig oesophageal muscularis mucosae. Using the technique of selective muscarinic M 3 receptor alkylation, we were unable to demonstrate muscarinic M 2 receptor-mediated re-contractions in oesophageal smooth muscle, as described previously in ileum. In addition, while increased functional antagonism of relaxant responses to isoprenaline could be demonstrated in tissues pre-contracted with oxotremorine M compared to histamine, muscarinic M 2 receptor activation did not contribute to this effect, as described previously in trachea. These data suggest a lack of interaction between muscarinic M 2 receptors and β-adrenoceptors in guinea-pig oesophageal smooth muscle, but suggest an interaction between muscarinic M 3 receptors and β-adrenoceptors.

Characterization of the interaction of zamifenacin at muscarinic receptors in vitro

The interaction of zamifenacin ((3 R)-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)piperidine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following p K i values; rat cerebral cortex (M 1) 7.90 ± 0.08, myocardium (M 2) 7.93 ± 0.13, submaxillary gland (M 3) 8.52 ± 0.01 and rabbit lung (M 4) 7.78 ± 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M 1) 7.93 ± 0.09, guinea-pig left atria (M 2) 6.60 ± 0.04, guinea-pig ileum (M 3) 9.31 ± 0.06, guinea-pig oesophageal muscularis mucosae (M 3) 8.84 ± 0.04, guinea-pig trachea (M 3) 8.16 ± 0.04, and guinea-pig urinary bladder (M 3) 7.57 ± 0.15. Therefore, zamifenacin is selective for muscarinic M 3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M 2 receptors in atria. The degree of muscarinic M 3 M 2 receptor selectivity depends upon the muscarinic M 3 receptor preparation studied.

SPECIAL REPORT Noradrenaline‐induced relaxation of rat oesophageal muscularis mucosae: mediation solely by innervated β3‐adrenoceptors

We investigated the effects of cocaine and corticosterone on the noradrenaline-induced relaxation of rat oesophageal smooth muscle in the absence and presence of the selective beta2-antagonist, ICI 111,551. It was found that the concentration-response curve (CRC) of noradrenaline was not shifted by ICI 118,551 at 1 microM, whereas a clear shift to the right was observed at 100 microM of the antagonist. In the presence of corticosterone (10 microM), CRC's were clearly shifted to the left; with cocaine (10 microM) additionally present, a further leftward shift was observed, indicating the involvement of both neuronal and extraneuronal uptake sites. It was concluded that the relaxation of rat oesophageal muscularis mucosae by noradrenaline is solely mediated by beta3-adrenoceptors which are sympathetically innervated.

Muscarinic suppression of ATP-sensitive K+ channel in rabbit esophageal smooth muscle

Smooth muscle cells from the rabbit esophageal muscularis mucosae were studied for the presence of ATP-sensitive K+ channel (KATP) and its inhibition by carbachol. Lemakalim (10 microM), a synthetic K+ channel opener, increased whole cell currents by -174 +/- 15 pA with 0.1 mM intracellular ATP concentration ([ATP]i) and -70 +/- 11 pA with 5 mM [ATP]i. Glibenclamide (10 microM) completely abolished the lemakalim-induced currents. These currents were therefore denoted as KATP. Carbachol (10 microM) suppressed KATP by 74 +/- 4% with 10 mM intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) concentration and 100% when EGTA was omitted from the pipette solution. Carbachol suppression was attenuated to 23 +/- 16% by the M3 receptor antagonist, p-flurohexahydrosiladifenidol (0.1 microM). KATP was also suppressed by phorbol 12-myristate 13-acetate (PMA; 100 nM) by 63 +/- 9%. The effects of both PMA and carbachol were significantly reduced by inhibitors of protein kinase C and tyrosine kinase. These results suggest that carbachol suppression of KATP is via M3 receptor subtype and the signaling pathway involves Ca2+, protein kinase C, and tyrosine kinase.

Antagonistic properties of McNeil‐A‐343 at 5‐HT4 and 5‐HT3 receptors

1. This study describes the in vitro interaction of the muscarinic ligand McNeil-A-343 with two 5-hydroxytryptamine (5-HT) receptor subtypes, the 5-HT4 and 5-HT3 receptors, using functional as well as radioligand binding studies. 2. In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil-A-343 was a competitive antagonist (pA2 6.2) of the 5-HT4 receptor which mediates the relaxation induced by 5-HT. The compound per se relaxed the oesophagus at high concentration only (> or = 10 microM), an effect unchanged by desensitization of the 5-HT4 receptor with 10 microM 5-methoxytryptamine. In the same preparation in the absence of tone, McNeil-A-343 displaced the carbachol concentration-response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3. In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil-A-343 caused a concentration-dependent depolarization that was unaffected by 100 nM ondansetron. The concentration-fast depolarization curve to 5-HT, mediated by the 5-HT3 receptor, was displaced to the right by McNeil-A-343, which showed an apparent affinity (pA2) of 4.8 for the 5-HT3 subtype. 4. In binding studies, McNeil-A-343 recognized a single population of 5-HT4 receptors in pig caudate nucleus, with a pKI of 5.9. The binding affinity of McNeil-A-343 for 5-HT3 receptors in NG 108-15 cells was approximately four times lower (pKI 5.3). Binding affinities (pKI) for muscarinic receptor subtypes in rat tissues were 5.3 (M1, cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively. 5. McNeil-A-343 is an antagonist at 5-HT4 and 5-HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5-HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors.

ATP-sensitive K+ (KATP) channels in rabbit esophageal muscularis mucosae: Inhibition by muscarinic M3 receptor

ATP-sensitive K+ (KATP) channels in rabbit esophageal muscularis mucosae: Inhibition by muscarinic M3 receptor

(R) and (S) RS 56532: Mixed 5-HT 3 and 5-HT 4 receptor ligands with opposing enantiomeric selectivity

The pharmacological properties of the (R) and (S) enantiomers of RS 56532 have been studied in vitro and in vivo. In radioligand binding studies at 5-HT 4 receptors in guinea-pig striatum, (S) RS 56532 exhibited a higher affinity than (R) RS 56532 (− log i = 7.6 and 6.5, respectively). (S) RS 56532 acted as a potent agonist at 5-HT 4 receptors mediating relaxation of rat oesophageal muscularis mucosae (−log EC 50 = 7.9) while (R) RS 56532 acted as a weaker agonist at this receptor (− log EC 50< 6.0). These data suggest that at 5-HT 4 receptors, the enantiomeric selectivity of RS 56532 was (S) > (R). In binding studies at 5-HT 3 receptors in rat cortex, (R) RS 56532, conversely, exhibited a higher affinity than (R) RS 56532 (− log K i = 9.1 and 8.0, respectively). At 5-HT 3 receptors in guinea-pig isolated ileum, (R) RS 56532 exhibited an affinity (− log K B) of 7.9, whereas (S) RS 56532 (1 nM–1 μM) was inactive. No agonism was observed at ileal 5-HT 3 receptors with either enantiomers. These data suggest that at 5-HT 3 receptors in rat and guinea-pig, both enantiomers acted as antagonists, with (R) > (S) RS 56532. At the non-5-HT 3, high affinity ‘(R) zacopride’ site, (R) RS 56532 exhibited a higher affinity than (S) RS 56532 (− log K i = 6.1 and 4.9). This site was insensitive to potent 5-HT 3 antagonists such as (R) YM 060 or ondansetron. However, it was recognized with relatively high affinity (− log K i = 7.5) by the (R), but not (S)d enantiomer, of RS 42358 (− log K i = 4.7). Since (S) RS 42358 is a high affinity 5-HT 3 receptor antagonist, these data further highlight the dissimilarity between the 5-HT 3 receptor and the ‘(R) zacopride’ site. The ‘(R) zacopride’ site also appeared to be pharmacologically distinct from the 5-HT 4 receptor, since 5-HT 4 ligands such as renzapride, SDZ 205,557 or RS 23597-190 exhibited low affinities. The enantiomeric selectivity of (R) and (S) RS 56532 in vivo was consistent with findings in vitro. At 5-HT 4 receptors mediating tachycardia in the pig, 5-HT induced a dose-dependent tachycardia (ED 50 = 3μg kg −1, i.v.; maximum response = 90–100 beats min −1). (S) RS 56532 increased heart rate by 88 min −1 with a potency of (ED 50) of 3 μg kg −1, i.v. In contrast, a tachycardia effect (23 beats min −1) of (R) RS 56532 was seen only at 1 mg kg −1, i.v. (R) RS 56532 was more potent than (S) RS 56532 (ID 50 = 3 and 78 μg kg −1, i.v. respectively) at inhibiting the von Bezold Jarisch reflex, a response mediated by 5-HT 3 receptor activation. Similarly, (R) RS 56532, at 0.1 mg kg −1 p.o., inhibited cisplatin induced emesis in the ferret, from 19.8 to 5.8 emetic episodes. In contrast, (S) RS 56532 was inactive at this oral dose. The emetic response to neoplastic agents such as cisplatin is also mediated by 5-HT 3 activation. In summary, RS 56532 in vitro and in vivo, exhibits opposing enantiomeric selectivity at 5-HT 3 and 5-HT 4 receptors, i.e. 5-HT 3—(R) > (S); 5-HT 4—(S) > (R). The affinity of the (R) enantiomer at 5-HT 3 receptors and the potency of the (S) enantiomer at 5-HT 4 receptors render them useful pharmacological tools to further define the binding domains of these two 5-HT receptor subtypes. Furthermore, these data show that 1,8-naphthalimides, such as (S) RS 56532, represent a novel class of potent 5-HT 4 receptor agonists.

The β‐adrenoceptors mediating relaxation of rat oesophageal muscularis mucosae are predominantly of the β3‐, but also of the β2‐subtype

1. beta-Adrenoceptor-mediated relaxation of rat oesophageal smooth muscle was investigated by studying the effects of beta 1- and beta 2-selective antagonists on the relaxation induced by (-)-isoprenaline, the beta 2-selective agonists fenoterol and clenbuterol and the beta 3-agonist, BRL 37344. 2. The highly beta 1-selective antagonist CGP 20721A did not antagonize (-)-isoprenaline- or BRL 37344-induced relaxations in concentrations up to 10 microM. Only at 100 microM of CGP 20712A were clear rightward shifts of the agonist concentration-response curves (CRCs) observed, with pA2 values of 4.70 and 4.97 against (-)-isoprenaline and BRL 37344, respectively. 3. ICI 118,551, a potent and selective beta 2-antagonist, at 100 nM caused moderate rightward shifts of the CRCs of (-)-isoprenaline, fenoterol and clenbuterol; with fenoterol and clenbuterol, this was accompanied by a clear steepening of the curve. Only at the highest concentration (100 microM ICI 118,551) did the shifts to the right further increase substantially. Resulting Schild-plots were clearly biphasic. BRL 37344-induced relaxations were only antagonized at 100 microM ICI 118,551, yielding a pA2 value of 5.48. 4. These results clearly demonstrate that the BRL 37344-induced relaxation of rat oesophageal muscularis mucosae is mediated solely through beta 3-adrenoceptors, whereas (-)-isoprenaline-, fenoterol- and clenbuterol-induced relaxations were shown to involve both beta 2- and, predominantly, beta 3-adrenoceptors.

RS 23597-190: a potent and selective 5-HT4 receptor antagonist.

1. The pharmacological properties of RS 23597-190 (3-(piperdine-1-yl)-propyl-4-amino-5-chloro-2-methoxy benzoate hydrochloride) have been studied in vitro and in vivo. 2. RS 23597-190 competitively antagonized 5-HT4 receptor-mediated relaxations of rat, carbachol precontracted oesophageal muscularis mucosae, (pA2 = 7.8 +/- 0.1; Schild slope = 1.2 +/- 0.2). Affinity estimates (-log KB) at 5-HT4 receptors using either renzapride or SC-53116 as agonists yielded a -log KB value of 8.0 +/- 0.01. In contrast, RS 23597-190 failed to antagonize contractile responses to 5-HT of guinea-pig ileal 5-HT3 receptors, even at concentrations up to 10 microM. 3. Increases in short-circuit current, induced by 5-HT, were studied in guinea-pig ileal mucosal sheets. Concentration-response curves to 5-HT were biphasic, with the high potency phase to 5-HT inhibited by RS 23597-190 and mimicked by 5-methoxytryptamine. The -log KB value for RS 23597-190 at the high potency phase was 7.3 confirming that 5-HT4 receptors mediated the high potency phase. 4. In rat isolated vagus nerve, 5-HT elicited a slow, maintained depolarization at low concentrations and a rapid, transient depolarization at higher concentrations. The high potency, slow depolarizing phase to 5-HT was abolished selectively in the presence of 1 microM RS 23597-190 and the low potency phase was abolished selectively in the presence of 1 microM ondansetron. These data confirm that 5-HT4 and 5-HT3 receptors mediated slow and fast depolarization responses, respectively. 5. At 5-HT3 binding sites in membranes from NG 108-15 cells, labelled by [3H]-quipazine, RS 23597-190 exhibited an apparent affinity (- log Ki) of 5.7 +/- 0.1. At 5-HT3 receptors in membranes from rat cerebral cortex, labelled by [3H]-RS 42358-197, the apparent affinity (- log Ki) of RS 23597-190 was also 5.7 +/- 0.1. In both studies, Hill coefficients were not significantly different from unity. At 5-HT1A, 5-HT2,muscarinic M1, M2, M3, M4 and dopamine D1 and D2 receptors, RS 23597-190 exhibited low apparent affinities, with all - log Ki values less than 5.5.6. Intravenous infusion of RS 23597-190 in the conscious, restrained rat antagonized the von Bezold Jarisch reflex induced by 2-methyl 5-HT, with an ID50 of 300 microg kg-1 min-1, i.v. In the anaesthetized,bilaterally vagotomized micropig, RS 23597-190 (6 mg kg-1, i.v.) antagonized 5-HT-induced tachycardia with a half-life of 77 (63-99) min. Transient arrhythmic effects were noted after administration of the compound.7. In conclusion, RS 23597-190 acts as a high affinity, selective competitive antagonist at 5-HT4 receptors. Thus, the compound appears to be a useful tool for 5-HT4 receptor identification in vitro. In vivo, the compound is rapidly metabolized in pigs such that 5-HT4 blockade is not maintained. However,in the rat, when given by infusion, RS 23597-190 antagonizes 5-HT3 mediated responses, at doses consistent with a low affinity 5-HT3 receptor. These data suggest that, under appropriate experimental conditions, RS 23597-190 may also be used in vivo to characterize further 5-HT4 receptor function.

K+ currents in rabbit esophageal muscularis mucosae.

Single cells were obtained from esophageal muscularis mucosae of the rabbit using enzymatic dispersion. Their electrophysiological properties were studied with both conventional whole cell and nystatin-perforated patch techniques. The latter technique was used to prevent "washout" of intracellular constituents and to maintain endogenous buffering of Ca2+. The average resting potential of these cells was -54 +/- 3.2 mV in the conventional recording and -51 +/- 4.4 mV in perforated patch recordings. In the current-clamp mode, regenerative responses were consistently observed in perforated patch recordings, but not when conventional whole cell gigaseal methods were used. Conventional whole cell voltage-clamp methods revealed outward currents on depolarization from a holding potential of -70 mV. These currents were inhibited by extracellular tetraethylammonium (TEA, 5-10 mM) and CoCl2 (4 mM), indicating that the predominant outward current is a Ca(2+)-activated K+ current. In the presence of TEA, inward Ca2+ currents were unmasked. In contrast, when the nystatin-perforated patch technique was used, depolarizations resulted in a net inward current followed by an outward current. The outward current was inhibited by CoCl2 (2 mM) and TEA (5 mM) to the same extent as in conventional recordings. A second component of K+ current was observed in both types of recordings when extracellular Ca2+ influx was abolished and also in the presence of TEA. This slowly activating persistent K+ current resembled a delayed rectifier K+ current. These studies show that the rabbit tunica muscularis mucosal cells possess voltage-activated Ca2+ and K+ channels as well as the capability to elicit action potentials.

Ca2+ and Ca(2+)-activated Cl- currents in rabbit oesophageal smooth muscle.

1. An inward current carried by Ca2+ was recorded from single smooth muscle cells of rabbit oesophageal muscularis mucosae using a whole-cell gigaseal technique with physiological (2 mM) external calcium concentration ([Ca2+]o) in the presence of intracellular Cs+ ([Cs+]i 130 mM). Only one type of Ca2+ current could be identified. The threshold for its activation was approximately -30 mV and maximum inward current (approximately 300 pA) was recorded at 0 mV. 2. This inward current was blocked by Co2+ (4 mM), Cd2+ (0.5 mM) and nifedipine (1 microM) and was enhanced by Bay K 8644 (5 microM). We therefore classify it as a L-type Ca2+ current and denote it ICa. 3. Steady-state inactivation data were well-fitted by a Boltzmann distribution, indicating that inactivation of the Ca2+ current is strongly modulated by membrane potential. However, the inactivation of ICa slowed significantly and became less complete when BaCl2 replaced CaCl2 in the Tyrode solution suggesting that the inactivation of ICa may also be dependent on [Ca2+]i. The steady-state activation and inactivation curves for ICa overlap between -40 and 0 mV indicating that there may be a Ca2+ window current in this range of potentials. 4. When EGTA was omitted from the pipette-filling solution, depolarizations positive to -10 mV resulted in a transient as opposed to a maintained inward Ca2+ current which was followed by a relatively large outward current. Under these conditions, slowly decaying inward tail currents were also recorded upon repolarization to the holding potential, -60 mV. However, when EGTA was omitted from the pipette, marked 'run-down' of the Ca2+ current occurred within 10 min after starting the whole-cell recording. 5. This run-down of ICa was reduced significantly when the nystatin perforated patch technique was used. Under these conditions stable ICa records could be obtained for over 1 h. Outward currents and slow decaying inward tail currents similar to those recorded with no EGTA in the pipette were also obtained consistently using the nystatin recording technique. 6. In nystatin perforated patch recordings, CoCl2 (2 mM) completely abolished the Ca2+ current, the outward currents and the slow inward tails. These findings suggest that the outward currents and slow inward tails are activated by a transmembrane influx of Ca2+. 7. Ion replacement and pharmacological tests provided evidence that both the outward currents and the slow inward tails are due to Ca(2+)-activated Cl- current (ICl(Ca)).(ABSTRACT TRUNCATED AT 400 WORDS)

Anomalous responses to histamine stimulation of guinea-pig oesophageal muscularis mucosae.

The incomplete tachyphylaxis of the contractile response to the H1-stimulants observed on guinea-pig oesophageal muscularis mucosae seems to be H2- and H3-antagonist as well as atropine- and tetrodotoxin-resistant. Lidocaine and eserine partially prevented this process probably by a mechanism independent of their main activity. The dualistic antagonism exerted by mepyramine and methysergide on reproducible histamine responses could be explained by a kinetic condition of "hemi-equilibrium state" together with changes of drug-receptor interaction and by non-specific properties of methysergide. On the whole, the present data indicate that the role of histamine in this tissue has still to be defined.