In vitro effects of bethanechol on equine gastrointestinal contractility and functional characterization of involved muscarinic receptor subtypes

The goal of this study was to investigate the effect of bethanechol (BeCh) on contractility patterns of smooth muscle preparations of equine duodenum descendens, jejunum, caecum and pelvic flexure in vitro. Concentration-response relationships were developed for BeCh using in vitro assays with and without preincubation of muscarinic (M) receptor antagonists for M2 and M3 receptors. BeCh induced a significant, concentration-dependent increase in contractile response in equine intestine in specimens with circular orientation. The maximal effect was largest for jejunal specimens with no difference in EC50 within the different locations investigated. The M2 antagonist, AF-DX 116, caused a rightward shift of the concentration-response curve and the M3 antagonist, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide), almost completely inhibited the effect of BeCh over the entire concentration-response curve. These data provide evidence that, although the effect of BeCh is predominantly mediated by M3 receptors, M2 muscarinic receptors also play a role in BeCh-induced contraction in specimens of equine intestine. The involvement of other muscarinic receptor subtypes cannot be excluded. Further studies are necessary to understand the effect of BeCh in vivo including diseased animals.     

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

OBJECTIVE: To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. SAMPLE POPULATION: Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. PROCEDURES: Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A(max)), and area under the curve (AUC) were evaluated. RESULTS: Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M(3) antagonists (more commonly for basal tone than for A(max) and AUC). The M(2) receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M(3) receptor antagonists were generally more potent than the M(2) receptor antagonists. In a protection experiment, an M(3) receptor antagonist was less potent than when used in combination with an M(2) receptor antagonist. Receptor antagonists for M(1) and M(4) did not affect contractility variables. CONCLUSIONS AND CLINICAL RELEVANCE: Bethanechol acting on muscarinic receptor sub-types M(2) and M(3) may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.     

Effects of muscarinic receptor antagonists on acetylcholine-induced contractions of jejunal smooth muscle in horses

This study investigated the effects of a muscarinic type 1 (M(1)), 2 (M(2)), and 3 (M(3)) antagonists (4-DAMP, pirenzepine, and methoctramine, respectively) on acetylcholine (Ach)-induced contractions of longitudinal jejunal muscle strips of horses. Strips were irrigated with Krebs-Henseleit solution gassed with 95% O(2) and 5% CO(2), and the developed tension in response to Ach was recorded before and after incubation with increasing concentrations of 4-DAMP (10(-8)-10(-6) M), pirenzepine (10(-6)-10(-4) M), and methoctramine (10(-5)-10(-3) M). When competitive antagonism was characterized, the affinity constant (pA(2)) was calculated by Schild plots. A parallel rightward shift in the concentration-response curves was observed after 4-DAMP and pirenzepine. Methoctramine presented a dual effect on the concentration-response curves: lower concentrations induced a parallel rightward shift without altering the maximum intensity of contraction (E(max)), while the highest concentration increased slope of the concentration-response curve and increased E(max). The pA(2) for 4-DAMP and pirenzepine was 9.18 and 7.13, respectively. Acetylcholine-induced contractions of longitudinal jejunal smooth muscle are mediated mainly via M(3) receptors. The complex role of M(2) receptors in jejunal smooth muscle contractions was evident because methoctramine potentiated the contractile response to higher doses of Ach.     

In vitro effects of cisapride, metoclopramide and bethanechol on smooth muscle preparations from abomasal antrum and duodenum of dairy cows

The objective of this study was to investigate the effects of cisapride (CIS), metoclopramide (MET) and bethanechol (BET) on contractility parameters from smooth muscle preparations of the abomasal antrum and proximal duodenum of cows. Smooth muscle preparations were harvested shortly post-mortem from 42 healthy dairy cows, and concentration-response curves were performed by cumulative application of the drugs. Cisapride and MET did not have any significant effect on the contractility parameters studied, while BET induced a significant, concentration-dependent increase in basal tone (BT), mean amplitude (Amean), and area under the curve (AUC) in smooth muscle preparations from the abomasal antrum, but not from the duodenum. The effect of BET on BT was more pronounced in specimens with longitudinal orientation while the maximal obtainable effect (Vm) in Amean was more pronounced in circular-oriented preparations. Atropine (1 x 10-5 m) significantly inhibited the effect of BET, whereas pre-incubation with hexamethonium or tetrodotoxin (TTX) had no effect, suggesting that the effect was mediated by cholinergic receptors on the smooth muscle. The results may be relevant to diseases or disorders associated with gastric emptying and gastric hypomotility. Further investigations are warranted to investigate the potential ability of BET to enhance abomasal emptying of adult dairy cows.     

Adrenergic, cholinergic, and nonadrenergic-noncholinergic intrinsic innervation of the jejunum in horses.

OBJECTIVE: To determine the major neurotransmitters that regulate contractile activity in the jejunum of horses. SAMPLE POPULATION: Jejunal specimens from 65 horses without gastrointestinal tract lesions. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscular layer, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 30 and 70 V intensities and at various frequencies on muscle strips maintained at low or high muscle tone. To detect residual nonadrenergic-noncholinergic neurotransmission, the response of muscle to EFS in the presence of adrenergic and cholinergic blockade was compared with the response in the presence of tetrodotoxin. RESULTS: Atropine (ATR) decreased the contractile response of muscle strips to EFS under most conditions. However, ATR increased the contractile response of high-tone circular muscle. Adrenergic blockade generally increased the muscle responses to 30 V EFS and in high-tone longitudinal muscle but decreased contractile responses in high-tone circular muscle. Tetrodotoxin significantly altered the responses to EFS, compared with adrenergic and cholinergic receptor blockade. CONCLUSIONS: Acetylcholine and norepinephrine appear to be important neurotransmitters regulating smooth muscle contractility in the equine jejunum. They induce contraction and relaxation, respectively, in most muscle preparations, although they may cause opposite effects under certain conditions. In addition, nonadrenergic-noncholinergic excitatory and inhibitory influences were detected. CLINICAL RELEVANCE: Acetylcholine or norepinephrine release within the myenteric plexus of horses may alter gastrointestinal motility.     

In vitro evaluation of the contractile response to endothelin-1 of the circular and longitudinal myometrial layers of the uterine horn of nongravid mares

OBJECTIVE: To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. SAMPLE POPULATION: Uteruses from 10 nongravid mares in anestrus. PROCEDURE: Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10(-9), 10(-7), and 10(-5)M) of BQ-123, IRL-1038, or BQ-123 and IRL-1038 before concentration-response curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10(-9) to 10(-6)M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose. RESULTS: ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue. CONCLUSIONS AND CLINICAL RELEVANCE: ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility.     

Modification of spontaneous contractility of smooth muscle preparations from the bovine abomasal antrum by serotonin receptor agonists

The effects of serotonin (5-hydroxytryptamine, 5-HT), a 5-HT2-receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-M-5-HT) and RS-67506, a 5-HT4 receptor partial agonist, on spontaneous contractility of bovine abomasal smooth muscle preparations were investigated in vitro. Preparations from the abomasal antrum of freshly slaughtered healthy dairy cows were cut parallel to the longitudinal fibres, suspended in isolated organ baths, and concentration-response curves were performed by cumulative application of the 5-HT receptor agonists. Blockade of 5-HT2-induced response was tested with atropine and hexamethonium. Serotonin evoked a significant increase in the area under curve (AUC), whilst the 5-HT2 receptor agonist alpha-M-5-HT significantly increased the AUC and resting tone (RT). RS-67506 induced a significant increase in AUC and RT and a significant decrease in the maximum force. The effect of alpha-M-5-HT was mediated by a muscarinic cholinergic pathway, as the effect of alpha-M-5-HT was inhibited in the presence of atropine but not hexamethonium. It is concluded that 5-HT2 and 5-HT4 receptors are present in the bovine abomasal antrum. Muscarinic receptors are involved in the increase in RT seen after 5-HT2 receptor stimulation.     

Effect of endotoxins on contractility of smooth muscle preparations from the bovine abomasal antrum

The effects of endotoxin (ET) on spontaneous contractility and of carbachol- and alpha-methyl-5-hydroxytryptamine-(alpha-M-5-HT; 5-HT2 receptor agonist) induced contractions of smooth muscle preparations from the bovine abomasal antrum were investigated in vitro. Preparations from the abomasal antrum of freshly slaughtered healthy dairy cows were cut parallel to the longitudinal and circular fibres, suspended in isolated organ baths, and contractility was recorded and analyzed, using digitalized data. The traits maximum amplitude, time till maximum amplitude, frequency, basal tone, and area under curve were calculated. The contractile effect of Carbachol (CH) was concentration dependent. Repeated administration of CH (3.75 x 10(-6) M), each time interrupted by flushing of the organ baths, did not reveal any significant effect on contractility traits of CH-induced contractions. Endotoxin (10 micrograms/ml; lipopolysaccharide from E. coli, O26:B6) significantly reduced some of the spontaneously occurring contractility traits and of carbachol-(3.75 x 10(-6) M) and alpha-M-5-HT-induced (2.14 x 10(-5) M) contractions. The effects of higher and lower concentrations of ET occurred less consistently. The inhibitory effect of endotoxin was more pronounced after 6 hours as compared to 2 hours of incubation. The results of the present study (i) support the hypothesis of a possible role of endotoxin in reducing motility of the abomasum during the development of spontaneous abomasal displacement in dairy cows, and (ii) may serve as the basis for the development of an in vitro model of abomasal displacement with endotoxemia for future studies on the effect of motility modulating drugs.     

Comparative responses of bronchial rings to mediators of airway hyperreactivity in healthy horses and those affected with summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To compare responses of bronchial rings obtained from healthy horses and horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) to selected mediators of airway hyperreactivity in vitro. SAMPLE POPULATION: Bronchial rings from 6 healthy horses and 6 horses affected with SPAOPD. PROCEDURE: Bronchial rings obtained from each group of horses were mounted in organ baths and attached to force transducers interfaced with a polygraph. After applying 2g of tension, each ring was allowed to equilibrate for 45 minutes in Tyrode's solution at 37 C. Cumulative concentration-response relationships to graded concentrations of selected mediators (10(-8) to 10(-4) M) were determined and analyzed for significance at each concentration. RESULTS: Acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 induced concentration-dependent contractile responses in bronchial rings. Prostaglandin F2alpha induced weak and inconsistent contractile responses. The other 2 agents, norepinephrine and substance P, did not induce concentration-dependent responses. Considering the overall group-drug effect, acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 were effective in inducing consistent concentration-dependent contractile responses in both groups. Only 5-hydroxytryptamine and histamine induced significant responses in contractility between groups. The response of bronchial rings from horses with SPAOPD to 5-hydroxytryptamine was significantly greater than those from control horses, whereas the response to histamine was significantly lower. Significant responses were evident at concentrations ranging from 10(-6) to 10(-4) M for both drugs. CONCLUSIONS AND CLINICAL RELEVANCE: Because the airways of horses with SPAOPD had increased responsiveness to 5-hydroxytryptamine in vitro, treatment modalities using 5-hydroxytryptamine antagonists should be investigated to address this phenomenon.     

Acetylcholine-induced contractions in the porcine internal mammary artery: possible role of muscarinic receptors

The effect of acetylcholine on the isolated, non-precontracted, porcine internal mammary artery (IMA) was investigated. Acetylcholine induced concentration-dependent contractions of non-precontracted IMA rings with denuded endothelium (pEC50 = 5.80 +/- 0.04) and was without effect on arterial segments with intact endothelium. The muscarinic receptor antagonists atropine, pirenzepine, methoctramine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) antagonized the response to acetylcholine. The constrained pA2 values were 10.14, 7.74, 7.34 and 10.5, respectively. It is concluded that acetylcholine induces concentration-dependent contractions of porcine internal mammary artery rings on basal tone and that this contractile effect is probably due to direct cholinergic stimulation of smooth muscle cells, maybe including activation of muscarinic M1 receptors.     

Spontaneous in vitro contractile activity of specimens from the abomasal wall of healthy cows and comparison among dairy breeds

OBJECTIVE: To characterize and compare in vitro contractility patterns of sections of abomasal wall harvested from cattle of 3 dairy breeds. SAMPLE POPULATION: Longitudinal and circular smooth muscle preparations harvested from the antrum and body of the abomasum of 30 recently slaughtered Holstein-Friesian, Brown Swiss, and Simmental X Red Holstein cows. PROCEDURE: Spontaneous isometric contractions of specimens in tissue baths of modified Krebs solution were recorded during a 4-hour period. Maximal amplitude, frequency of contractions, and change of basal tension were used to characterize contractility. Statistical analyses were used to test for differences among time periods, among breeds, between specimen locations, and between fiber orientations. RESULTS: Myoactivity patterns of abomasal smooth muscle preparations are highly variable and differ on the basis of location and fiber orientation. Frequency of contractions differed significantly among time periods for longitudinally oriented specimens with decreasing frequencies of contractions over time. Maximal amplitude of the longitudinally oriented specimens from the antrum increased significantly, whereas maximal amplitude of the circularly oriented specimens from the antrum decreased significantly. Values did not differ significantly among breeds. CONCLUSIONS AND CLINICAL RELEVANCE: Patterns of spontaneous contractility of abomasal wall specimens are not homogeneous. During a 4-hour recording period, maximal amplitude and frequency of contractions of specimens varied significantly with respect to orientation and location; however, spontaneous contractile myoactivity did not differ significantly among breeds. Therefore, breed predisposition for displaced abomasum is not correlated with spontaneous activity of smooth muscle specimens.     

Effect of sodium butyric acid, sodium valerianic acid, and osmolarity on contractility of specimens of intestinal wall obtained from the cecum and spiral colon of healthy cows

OBJECTIVE: To compare the effect of various concentrations of sodium butyric acid and sodium valerianic acid, as well as various osmolarities, on contractility of ex-vivo intestinal wall specimens obtained from the cecum and spiral colon of each of several healthy cows. SAMPLE POPULATION: Full-thickness preparations of intestinal wall, dissected parallel to the longitudinal smooth muscle layers, harvested from freshly slaughtered healthy cows. PROCEDURE: Specimens of intestinal wall were incubated for 5 minutes with various concentrations of sodium butyric acid and sodium valerianic acid as well as various osmolar concentrations of NaCl, using a crossover design. Isometric contractions were induced 7 times with carbachol (CH; 5 X 10(-6) mol/L). Contractility was defined as the maximum amplitude of contraction and the amplitude of contraction 2 minutes after addition of CH. RESULTS: Repeated addition of CH did not result in a significant effect on contractility of specimens from the cecum and spiral colon. Contractility after addition of CH was not significantly affected by prior incubation with various concentrations of sodium butyric acid or sodium valerianic acid or after an increase of osmolarity. Maximum amplitude of contraction was significantly higher in specimens from the spiral colon, compared with specimens from the cecum. CONCLUSIONS: Increases in concentrations of sodium butyric acid or sodium valerianic acid and increases in osmolarity did not inhibit contractility of intestinal wall specimens from the cecum and spiral colon of a group of healthy cows.     

The effect of Pasteurella haemolytica cytotoxin on bovine polymorphonuclear leukocytes can be attenuated by beta-adrenoceptor antagonists

It was investigated whether beta-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 micrograms cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The beta-adrenoceptor antagonists alprenolol (10(-5) M) and propranolol (5 X 10(-7) - 5 X 10(-6) M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their beta-adrenoceptor blocking potency because other beta-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the beta-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether beta-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.     

Estrous cycle-dependent differences in responsiveness to prostaglandins and contractile agents in sheep (Ovis aries) cervical smooth muscle

We investigated the influence of the phase of the estrous cycle on mechanical responses elicited in sheep cervix by potassium chloride (KCl), acetylcholine chloride (ACh), prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1). The cervix of adult ewes (n = 48) were classified according to the presence or absence of corpora lutea (luteal or follicular phase, respectively). Muscle strips of the circular and longitudinal layers were prepared in an organ bath and coupled to an isometric force transducer. Concentration-response curves were obtained noncumulatively. KCl and ACh produced concentration-dependent contractions in all preparations in both phases of the estrous cycle. However, maximum effect, EC50 and slope values of KCl and ACh were not significantly different between muscle layers, as well as between the phases of the estrous cycle. The prostanoid, PGF2 alpha, produced a significant reduction in the amplitude of spontaneous contractions for all preparations. The depressant effect of PGF2 alpha on spontaneous contractions of circular smooth muscle was significantly greater during the follicular than the luteal phase, whilst the depressant effect of PGF2 alpha on the longitudinal layer did not differ between phases of the estrous cycle. PGE1 significantly reduced the amplitude of spontaneous contractions on circular but not on longitudinal preparations. In conclusion, we have characterized with in vitro preparations of circular and longitudinal muscle layers of ewes during the follicular and luteal phases of the estrous cycle, the parameters of the K- and ACh-induced contractions on cervix and the efficacy of PGF2 alpha and PGE1 on inhibition spontaneous contractile activity.     

An in vitro study on spontaneous cervical contractility in the cow during oestrus and diestrus

Cervical smooth muscle specimens were collected from slaughtered cows in oestrus and diestrus. Longitudinal and circular specimens were mounted in organ baths and after equilibration time and 2 g pre-load, their physiologic isometric contractility was recorded for 2.5 h. Area under the curve (AUC), maximal amplitude (Amax), mean amplitude (Amen) and minimal amplitude (Amin) were studied. Differences between cycle phases, between muscle layers and over the recorded time periods were statistically evaluated using linear mixed-effect models. In the cow, physiologic contractility of the cervix decreased significantly over time for all variables evaluated (time as covariate on a continuous scale). Furthermore, for Amax, there was a tendency for an influence of muscle layer (longitudinal > circular). Contractility was not influenced by cycle phases.     

In vitro evaluation of gut contractile response to histamine in rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

The contractile response of intestinal strips in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) to the administration of histamine was assessed by means of the organ bath technique. Intestinal strips were isolated from 16 clinically healthy fish and mounted in organ baths. Histamine was compared with the full agonist serotonin, to evaluate their contractile efficacy and potency. Serotonin elicited a concentration-related contraction in all examined intestinal strips, whereas histamine induced the contraction only in 14 exemplars. Of these, seven exhibited a concentration-related response. A sigmoidal curve was fitted from data (R(2) = 0.55) and its best fit values were compared with those of serotonin. Interestingly, histamine exhibited the same efficacy (E(max)) as serotonin (F-test, p > 0.05), but showed lower potency (by an order of magnitude) (F-test, p < 0.01). Moreover, the effect of the H1 antagonist, pyrilamine, has been tested to exclude aspecific contraction due to other agonists eventually released in situ following histamine administration. Pyrilamine showed a marked concentration-related antagonist action on the contractility induced by histamine with complete contractility antagonism at 10(-4)M. The authors suggest that the responses to histamine measured in the present study reflect a less sensitive response to an exogenous source of histamine, possibly due to bacterial metabolism.     

Diazepam potentiates the positive inotropic effects of histamine and forskolin in guinea-pig papillary muscles

There have been diverse reports on the effects of diazepam on cardiac contractility. The purpose of this study was to examine whether diazepam modifies the inotropic response elicited by histamine on an isolated guinea-pig papillary muscle. The responses of electrically driven papillary muscle to histamine and cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Histamine and forskolin, which directly stimulate adenylate cyclase, significantly increased the contractile force in the papillary muscle in a concentration-dependent manner. A histaminergic H₂-receptor antagonist, cimetidine, but not a H₁-receptor antagonist, diphenhydramine, at 10 μ M produced a rightward shift in the concentration-response curve for histamine. Diazepam (10 μ M ) shifted the concentration-response curve for histamine and forskolin to the left by 1.8 and 1.6 times, respectively. Neither a central type (fulmazenil) nor a peripheral type (PK11195) of benzodiazepine receptor antagonist modified the effect of diazepam on the histaminergic-evoked contraction. Phosphodiesterase blockade by 3-isobutyl-1-methylxanthine shifted the concentration-dependent curve for histamine to the left. A combination of 3-isobutyl-1-methylxanthine also produced a leftward shift of the curve. However, there was no significant difference between the 3-isobutyl-1-methylxanthine only group and the combination group. These results indicate that diazepam potentiates the positive inotropic effect produced by histamine, probably mediated via an increase in cyclic AMP levels induced by histamine.     

5-Hydroxytryptamine mediated contractions in isolated preparations of equine ileum and pelvic flexure: pharmacological characterization of a new 5-HT(4) agonist

The effects of 5-hydroxytryptamine (5-HT), HTF 919, a new 5-HT(4) agonist, and the antagonists SB 203-186 (5-HT(4)) and tropisetron (5-HT(3)) on intestinal motility were tested in vitro on isolated preparations of horse ileum and pelvic flexure. Concentration-response curves were created by cumulative application of the agonists with or without preincubation of the antagonists. The 5-HT preparation induced a concentration-dependent contraction in equine ileum and pelvic flexure. The results indicate that 5-HT receptors are present in all parts of equine intestine investigated in this study. Tropisetron was found to act as a noncompetitive antagonist in all locations of the equine intestine. SB 203-106 was confirmed as an antagonist to 5-HT in the equine ileum circular muscle, in pelvic flexure circular and longitudinal muscle. Nevertheless, a discernible increase of smooth muscle contractions caused by HTF 919 could only be observed in pelvic flexure. In accordance with an earlier study in the guinea pig, in the equine gut HTF 919 acted as a partial agonist for the 5-HT(4) receptor with an affinity constant in the nanomolar range. It is concluded that 5-HT receptors, and especially their subtypes, may represent a promising target for the treatment and prevention of gastrointestinal (GI) motility disorders in horses.     

Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.     

Contractile effects of 5-hydroxytryptamine (5-HT) in the equine jejunum circular muscle: functional and immunohistochemical identification of a 5-HT1A-like receptor

REASONS FOR PERFORMING STUDY: Prokinetic drugs used to treat gastrointestinal ileus in man have equivocal results in horses. In man, prokinetic drugs have 5-hydroxytryptamine4(5-HT4) receptors as their target, but little is known about the 5-HT-receptor subtypes in the equine small intestine. OBJECTIVE: Functional and immunohistochemical identification of the serotonin receptor subtype(s) responsible for the 5-HT induced contractile response in the equine circular jejunum. METHODS: Isometric organ-bath recordings were carried out to assess spontaneous and drug-evoked contractile activity of equine circular jejunum. Histological investigations by immunofluorescence analyses were performed to check for presence and localisation of this functionally identified 5-HT receptor subtype. RESULTS: Tonic contractions were induced by 5-HT in horse jejunal circular muscle. Tetrodotoxin, atropine and NG-nitro L-arginine did not modify this response. A set of 5-HT receptor subtype selective antagonists excluded interaction with 5-HT1B, 1D, 2A, 3, 4 and 7 receptors. The selective 5-HT1A receptor antagonists WAY 100635 and NAN 190 caused a clear rightward shift of the concentration-response curve to 5-HT. The contractile effect of 5-CT, that can interact with 5-HT1A, 1B, 1D, 5 and 7 receptors was also antagonised by WAY 100635, identifying the targeted 5-HT receptor as a 5-HT1A-like receptor. Immunohistology performed with rabbit polyclonal anti-5-HT1A receptor antibodies confirmed the presence of muscular 5-HT1A receptors in the muscularis mucosae, and both longitudinal and circular smooth muscle layers of the equine jejunum. CONCLUSIONS: Contractile responses in equine jejunal circular smooth muscle induced by 5-HT involves 5-HT1A-like receptors.