Segment-dependent Expression of Muscarinic Acetylcholine Receptors and G-protein Coupling in the Equine Respiratory Tract

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-³H]scopolamine chloride ([³H]NMS) binding to all three tissues was saturable and of high affinity, with K _D values ranging between 1.6±0.7 and 1.9±0.3 nmol/L. [³H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720±59 nmol/L) than in bronchi (438±48 nmol/L) or lung (22 ± 3 nmol/L). Competitive binding studies using [³H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M₁), methoctramine and himbacine (M₂), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M₃), tropicamide (M₄) and mamba toxin (MT-3) (M₄) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24–28%: M₂>M₃>M₁), whereas in bronchus and trachea M₂ subtypes (87–90%) predominated over M₃ (14–22%), and M₁ subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31–36%) or presence of guanosine 5^′-triphosphate (GTP) (∼100%). Western blotting for G-protein α-subunits showed a much more robust expression of G_αi1/2 in the trachea (with highest receptor density) than in the lung or bronchi, whereas G_αs-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.     

Muscarinic receptor subtypes in equine tracheal smooth muscle

Selective muscarinic receptor antagonists were used to identify muscarinic receptor subtypes in equine trachealis strips. The M₁ receptor antagonist pirenzepine (10^–7 mol/L to 3 × 10^–5 mol/L) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10^–9 mol/L to 3 × 10^–7 mol/L³) dose dependently inhibited the contractile responses to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). Schild plots yielded a pA₂ value for pirenzepine vs ACh of 6.75 ± 0.09, which is consistent with the affinity for M₂ or M₃ receptors, and a pA₂ value for 4-DAMP vs ACh of 8.47 ± 0.09, which is in agreement with the affinity for M₃ receptors. The M₂ receptor antagonist gallamine (10^–5 mol/L and 10^–4 mol/L) did not affect the response of trachealis to exogenous ACh and low-frequency EFS (0.1–2 Hz) but decreased the responses to high-frequency EFS (4–16 Hz). These results suggest that the muscarinic receptors mediating contractions induced by ACh in equine tracheal smooth muscle are of the M₃ subtype. The lack of an increase in the response to EFS following gallamine suggests that functional prejunctional inhibitory M₂ receptors are not present on the cholinergic nerves innervating equine tracheal smooth muscle.     

Pharmacological characterization of muscarinic receptors in the contractions of isolated bronchi in the horse

We investigated the effects of nonselective muscarinic antagonist (atropine) and of selective muscarinic subtype 1 (M₁), 2 (M₂), 3 (M₃) antagonists (VU0255035, methoctramine, pFHHSiD, respectively) on the contractions evoked by electrical field stimulation (EFS) or by exogenous ACh in isolated horse bronchial muscle. Atropine completely inhibited neurogenic contractions in a concentration‐dependent fashion, whereas selective muscarinic antagonists induced relevant modifications only at the highest concentration tested. Experiments with selective muscarinic antagonists in combination showed that only the simultaneous blockade of M₁/M₃ or M₂/M₃ receptors was able to induce a nearly complete suppression of contractions. The contractions induced by exogenous ACh were competitively antagonized only by atropine (pA₂ = 9.01 ± 0.05). M₃ selective antagonist, up to 10⁻⁶ m , caused a moderate concentration‐dependent rightward shift of ACh curve (pA₂ = 7.96 ± 0.10). These data show that M₃ muscarinic receptors possess a central role in mediating cholinergic contraction of horse bronchi, while M₁ and M₂ receptors seem to have a cooperative role. Selective muscarinic antagonists seem unlikely to be useful against bronchoconstriction associated with airway diseases in horses. Conversely, compounds with selectivity for both M₁ and M₃ receptors could be as effective as traditional anticholinergics and induce fewer cardiac side effects.     

Muscarinic receptor subtypes, β-adrenoceptors and cAMP in the tracheal smooth muscle of conventional and double-muscled calves

The total muscarinic (M₁ + M₂ + M₃) and -adrenergic receptors in the tracheal smooth muscle of conventional and double-muscled calves were identified and characterized with the non-specific antagonists [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]dihydroalprenolol ([³H]DHA) respectively.Although the quantity of -adrenoceptors in double-muscled calves was 25% lower (p<0.05) than in conventional calves (B _max=327±89 fmol/mg protein), adenylate cyclase assays indicated that the basal adenylate cyclase activity and the (–)-isopropylnoradrenaline (ISO)- and sodium fluoride (NaF)-stimulated values were not significantly different between these calves. However, the density of muscarinic receptors in double-muscled calves was 40% higher (p<0.01) than in conventional calves (B _max=2955±625 fmol/mg protein). Subtypes of muscarinic receptors were studied with [³H]telenzepine (M₁-receptors), [³H]AF-DX 384 (M₂-receptors) and [³H]4DAMP (M₁ and M₃-receptors). It was found that in both double-muscled and conventional calves about 40% of the receptors were of the M₃-subtype, the remaining 60% being M₂-receptors.From these results, it is suggested that inflammation of the respiratory tract in double-muscled calves may be complicated by an imbalance between the cholinergic bronchoconstrictor and the -adrenergic bronchodilator components of the autonomic nervous system.     

Tachykinin receptors in the equine pelvic flexure

Tachykinins, of which substance P (SP) is the prototype, are neuropeptides which are widely distributed in the nervous systems. In the equine gut, SP is present in enteric nerves and is a powerful constrictor of enteric muscle; in other species, SP is also known to have potent vasodilatory and pro-inflammatory effects. The specific effects of SP are determined by the subtype of receptor present in the target tissue. There are 3 known subtypes of tachykinin receptors, distinguished by their relative affinities for SP and other tachykinins. The distribution of SP binding sites in the equine pelvic flexure was determined using 125I-Bolton Hunter SP (I-BHSP) autoradiography. Most I-BHSP binding sites were determined to be saturable and specific, therefore presumably representing tachykinin receptors. The greatest degree of I-BHSP binding occurred over very small vessels, and over the muscularis mucosae; I-BHSP binding was also intense over the circular muscle of the muscularis externa and mucosa, and present, although less intense, over the longitudinal muscle of the muscularis externa. Competition of I-BHSP with specific receptor agonists for binding sites in the equine pelvic flexure were used to determine the subtypes of tachykinin receptors present. The neurokinin-1 receptor subtype predominated in the equine pelvic flexure, followed by the neurokinin-3 receptor subtype.     

β-Adrenoceptors in Equine Trachea and Heart

The density and subtype pattern of -adrenoceptors in equine tracheal epithelium, tracheal smooth muscle and heart from 6–9 horses were investigated by radioligand binding studies using the non-selective -adrenoceptor antagonist 125I-cyanopindolol (ICYP). The specific binding of ICYP was 341±162 fmol/mg protein (mean±SD) for epithelium, 42±13 fmol/mg for smooth muscle and 124±39 and 101±19 fmol/mg for the cardiac atrium and ventricle, respectively. The Kd value of ICYP was 6.7–10.2 pmol/L.In competition studies, different concentrations of either the 2-selective drug ICI 118551 or the 1-selective CGP 20712A competed with 25 pmol/L ICYP for the binding sites. The competition curves for tracheal smooth muscle and epithelium were monophasic with an approximate Kd value for ICI 118551 of 1 nmol/L and for CGP 20712A of 10 000 nmol/L. This corresponds to known Kd values for these substances binding to 2-adrenoceptors. 2-Adrenoceptors were also found in the heart, most pronounced in the atrium, where the density was 29%±6% (mean±SD) of the total receptor density. CGP 20712A and ICI 118551 bound to the dominating binding site of 1-adrenoceptors in the heart with Kd values of approximately 1 nmol/L and 100 nmol/L, respectively.     

Equine recurrent airway obstruction does not alter airway muscarinic acetylcholine receptor expression and subtype distribution

In recurrent airway obstruction (RAO) or heaves, bronchospasm has been attributed to enhanced cholinergic activity. However, the expression and function of muscarinic acetylcholine receptors (mAChR) and their signaling components are not yet known. Thus, we examined the expression, subtype distribution and postreceptor signaling pathways of mAChR in the peripheral lung, bronchial and tracheal epithelia with the underlying smooth muscle from nine horses with RAO and 11 healthy control horses. In RAO horses, no significant segment-dependent alteration in mAChR density and subtype distribution (assessed by [N-methyl-3H]-scopolamine binding; ([3H]-NMS)), was found, except a trend in receptor down-regulation in some peripheral parts of the lung. The total number of high mAChR binding sites (assessed by carbachol-displacement experiments in the presence or absence of guanosine 5'-triphosphate) was not changed in RAO, suggesting that the functional coupling of mAChR to the corresponding G-proteins is intact. The M2-mediated inhibition of adenylate cyclase (AC) as well as the M3-receptor-G(q/11)-phospholipase C (PLC) activity was not different between RAO and control airway tissues. In conclusion, in equine RAO airways, mAChR expression and function were not altered, and thus appear not to account for the enhanced cholinergic activity in RAO.     

Characterization of muscarinic receptor subtype mediating contraction and relaxation in equine coronary artery in vitro

In coronary arterial rings isolated from horses, 10-^-8-10^-6 mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with I,-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of Ach 10-¹⁴-10-⁸ mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine ≥ parafluoro-hexahydrosiladiphenidol (pFHHSiD) > methoctramine. ACh-induced relaxation in the rings with endothelium was inhibited by LNAG or MB, and by each selective antagonist in the following order of potency: 4-DAMP < pFHHSiD ≥ pirenzepine ≥ methoctramine. These results suggest that the ACh-induced contraction and relaxation in equine coronary arteries are mediated mainly by an M₃-receptor located on the smooth muscle cells and endothelial cells, respectively, and that the stimulation of the M₃-receptor on the endothelial cells liberates nitric oxide.     

Functional characterization of the muscarinic receptors involved in endothelium-dependent relaxation in isolated canine uterine artery

Acetylcholine interacts with endothelial muscarinic receptors releasing nitric oxide and causing vasodilatation. To identify the receptor subtype responsible for acetylcholine-induced relaxation in canine uterine artery, the usual organ bath method for in vitro investigation on isolated blood vessels was applied. Using a range of muscarinic receptor antagonists such as atropine (nonselective), pirenzepine (M₁-selective), methoctramine (M₂-selective) and p -fluoro-hexahydro-sila-difenidol ( p -FHHSiD) (M₁/M₃) and determining pA2 value of those antagonists through Shild analysis, we aimed at establishing a precise receptor mechanism underlying acetylcholine-induced relaxation in isolated canine uterine artery. The relaxation of uterine arterial rings in response to acetylcholine in the presence or absence of selective muscarinic receptors antagonists was calculated using concentration response curves. Acetylcholine induced concentration-dependent and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC₅₀ = 6.90 ± 0.02). Muscarinic receptors antagonists atropine, pirenzepine, methoctramine and p -FHHSiD competitively antagonized the response to acetylcholine and obtained pA₂ values were 9.91 ± 0.06, 6.60 ± 0.04, 6.21 ± 0.08 and 8.05 ± 0.1, respectively. This study showed that acetylcholine induced endothelium-dependent relaxation of canine uterine artery by stimulation of muscarinic receptors localized on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the acetylcholine-induced relaxation of canine uterine artery are predominantly of M₃ subtype.     

Characterization of alpha-adrenoceptor subtypes in smooth muscle of equine ileum

OBJECTIVE: To determine the concentration and binding characteristics of alpha-adrenoceptor subtypes in smooth muscle cell membranes of equine ileum. SAMPLE POPULATION: Segments of longitudinal and circular smooth muscle from the ileum of 8 male and 8 female adult horses. PROCEDURE: Distribution of alpha-adrenoceptor subtypes was assessed by use of radioligand binding assays incorporating [3H]-prazosin and [3H]-rauwolscine, highly selective alpha1- and alpha2-adrenoceptor antagonists, respectively. Characterization of adrenoceptor subtypes was performed by use of binding inhibition assays. RESULTS: On the basis of binding affinity for specific radioligands, low- and high-affinity alpha1- and alpha2-adrenoceptors were detected. Concentration of low-affinity alpha2-adrenoceptors was significantly greater in male horses, compared with females. Competition studies confirmed the specificity of the radioligands used in the binding assays. Alpha1-adrenoceptors of both subtypes in male and female horses had a higher affinity for prazosin than phentolamine, whereas yohimbine did not compete with the radioligand for binding. For alpha2-adrenoceptors regardless of subtype, potency of inhibition elicited by each drug varied between sexes. In males, yohimbine was a more potent inhibitor than phentolamine, which was more potent than prazosin. In females, yohimbine was more potent than prazosin, which was more potent than phentolamine. CONCLUSIONS AND CLINICAL RELEVANCE: High- and low-affinity alpha1- and alpha2-adrenoceptors were detected in smooth muscle of equine ileum. Because alpha-adrenoceptor subtypes, particularly alpha2-adrenoceptors, are involved in the regulation of gastrointestinal tract function, characterization of these receptors may represent the basis for development of new therapeutic strategies for the control of gastrointestinal disturbances in horses.     

The influence of ageing on muscarinic receptors, β-adrenoceptors and adenylate cyclase activity in the bovine lung

Muscarinic and -adrenoceptors were identified in airway epithelium, smooth muscle and lung parenchyma from Holstein-Friesian calves and cows and were characterized with [³H]quinuclidinyl benzilate and [³H]dihydroalprenolol, respectively. The muscarinic receptor density in the smooth muscle of cows (B _max=4803±245 fmol/mg protein) was 33% greater (p<0.01) than in calves. Low receptor numbers were detected in the epithelium and parenchyma. In both calves and cows, the density of epithelial -adrenoceptors was twice as high as in smooth muscle and parenchyma. The quantity of -adrenoceptors in the tracheal epithelium (B _max=994±83 fmol/mg protein) and smooth muscle (B _max=492±41 fmol/mg protein) in cows was respectively 37% (p<0.001) and 35% (p<0.01) lower than in calves. Adenylate cyclase (AC) assays indicated that the basal and the (–)-isopropylnoradrenaline- (ISO-) stimulated cAMP production were not significantly different between the calves and cows. After stimulation with NaF, significantly higher cAMP production was found in all tissues from cows. Significant correlations were found between absolute AC responses to NaF and -adrenoceptor density in epithelium (r=–0.75,p<0.001) and smooth muscle (r=–0.63,p<0.01). It seems that, in older animals, the production of cAMP is independent of the number of receptors, indicating the presence of fully active compensatory mechanisms.Abbreviations AC adenylate cyclase - cAMP intra-cellular adenosine monophosphate - G-protein GTP binding protein - GTP guanosine 5-triphosphate - ISO isopropylnoradrenaline - M muscarine - QNB quinuclidinyl benzilate     

Evaluation of opioid receptors in synovial membranes of horses

OBJECTIVE: To evaluate mu-opioid receptors in synovial membranes of horses and determine whether these receptors are up-regulated in nerve endings during inflammation. SAMPLE POPULATION: Synovial tissue obtained from 39 client-owned horses during arthroscopy and 14 research horses during necropsy; brain and synovial tissues were obtained during necropsy from 1 horse, and control tissues were obtained from a mouse. PROCEDURE: Horses were classified into 7 groups on the basis of histologically determined degree of inflammation. Binding of primary rabbit antibody developed against mu-opioid receptors in equine synovial tissue was studied, using western blot analysis. Synovial membranes were tested for mu-opioid receptors by immunohistochemical staining, using a diaminobenzidine-cobalt chloride chromogen. Homogenates of synovial membranes were evaluated by use of radioligand binding. RESULTS: Examination of western blots of equine thalamus revealed that rabbit antibody developed against mu-opioid receptors yielded a band (molecular weight, 55 kd) that corresponded with that of other opioid receptors. Use of immunohistochemical staining of synovial tissue revealed considerable staining in the proliferative lining layer and in regions surrounding vascular structures. Specific radioligand binding of tissue homogenates was found in all groups. We did not detect significant differences in binding between horses with inflammation and horses without inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: Results of immunohistochemical analysis and radioligand binding of tissue homogenates suggest that there are opioid receptors in synovial membranes of horses. Our results support the practice of intra-articular administration of opioids to relieve pain after arthroscopic surgery in horses.     

Expression and function of 5-HT7 receptors in smooth muscle preparations from equine duodenum, ileum, and pelvic flexure

In horses, gastrointestinal (GI) disorders occur frequently and cause a considerable demand for efficient medication. 5-Hydroxytryptamine receptors (5-HT) have been reported to be involved in GI tract motility and thus, are potential targets for treating functional bowel disorders. Our studies extend current knowledge on the 5-HT₇ receptor in equine duodenum, ileum and pelvic flexure by studying its expression throughout the intestine and its role in modulating contractility in vitro by immunofluorescence and organ bath experiments, respectively.5-HT₇ immunoreactivity was demonstrated in both smooth muscle layers, particularly in the circular one, and within the myenteric plexus. Interstitial cells of Cajal (ICC), identified by c-Kit labeling, show a staining pattern similar to that of 5-HT₇ immunoreactivity.The selective 5-HT₇ receptor antagonist SB-269970 increased the amplitude of contractions in spontaneous contracting specimens of the ileum and in electrical field-stimulated specimens of the pelvic flexure concentration-dependently.Our in vitro experiments suggest an involvement of the 5-HT₇ receptor subtype in contractility of equine intestine. While the 5-HT₇ receptor has been established to be constitutively active and inhibits smooth muscle contractility, our experiments demonstrate an increase in contractility by the 5-HT₇ receptor ligand SB-269970, suggesting it exerting inverse agonist properties.     

A comparison between clenbuterol, salbutamol and terbutaline in relation to receptor binding and in vitro relaxation of equine tracheal muscle

Beta₂-adrenoceptor agonists are used as bronchodilators in both humans and horses. Of these drugs, clenbuterol is the one most frequently used when treating chronic obstructive pulmonary disease in the horse, while salbutamol and terbutaline are used in the treatment of human asthma. Little is known of the properties of the latter two drugs in equine medicine.
  We have compared salbutamol and terbutaline with clenbuterol in relation to their ability to relax muscle strips from equine tracheal muscle, pre- contracted with 40 n m carbachol, in tissue chambers. The affinities of these drugs to the β₂-adrenoceptors in homogenates of the same muscle tissue were also examined. These experiments were performed with radioligand binding studies using the very potent β-adrenoceptor antagonist ¹²⁵I-cyanopindolol.
  The three drugs were almost equipotent in relaxing the muscle strips. The EC₅₀-values for salbutamol, terbutaline and clenbuterol were 5.6, 13.8 and 2.1 n m , respectively, and all three drugs relaxed the preparations completely. In the competitive binding study, however, the K_d-value of clenbuterol was much lower (24 n m ) than that of salbutamol and terbutaline (1100 n m and 3900 n m , respectively). The amount of receptors bound at the EC₅₀-value of clenbuterol was 8% compared to less than 1% for salbutamol and terbutaline. This indicates a lower intrinsic efficacy of clenbuterol than of the other two drugs. The β-adrenoceptor density was 45 ± 14.3 fmol/mg protein (mean ± SD) and the K_d-value of ¹²⁵I-cyanopindolol was 11.4 ± 3.3 p m .     

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.     

Cholinergic activity of intestinal musclein vitro taken from horses with and without equine grass sickness

Equine grass sickness (EGS) is a pan-dysautonomia of horses that involves central and peripheral neuronal degeneration and ultimately depletion. This is the first reported functional study on the motility of equine intestine taken immediatelypost mortem from horses with EGS. Strips of smooth muscle from the small intestine of healthy and EGS-affected horses were suspended in an organ bath and their motility was measured isometrically. The activity of the cholinergic system was studied. Physostigmine enhanced the motility of all muscle strips. Tissues taken from horses suffering from acute grass sickness (AGS) had the longest latency before a measurable response could be obtained (p<0.05). The ileum appeared to be damaged by EGS to a greater extent than the duodenum. For the duodenal strips the enhanced rate of spontaneous contractions was significant (p<0.05) for both normal tissue and that affected by grass sickness but this was not the case for the ileal strips. Muscarinic receptor sensitivity investigation using bethanecol suggested a hypersensitivity of receptors with AGS material,Abbreviations AGS acute grass sickness - CGS chronic grass sickness - ED₅₀ median effective dose - EGS equine grass sickness - VIP vasoactive intestinal peptide     

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.     

The vasomotor effects of 5-hydroxytryptamine on equine basilar arteries In vitro

The vasomotor effects of 5-hydroxytryptamine (5-HT) on isolated equine basilar arteries were studied. 5-HT induced contractions of equine basilar arteries in a concentration-dependent manner, with a pEC₅₀ value (with 95% confidence limits) of 7.35 (7.08–7.62). Similar results were obtained with endothelium-denuded basilar arteries. Contractions were not competitively inhibited by the 5-HT₂ receptor antagonist ketanserin at low concentrations of 5-HT. Conversely, at high concentrations of 5-HT, contractions were inhibited by ketanserin in a concentration-dependent manner, with a pA ₂ value of 8.91 (8.62–9.20). The 5-HT₁ and 5-HT₂ receptor antagonist methiothepin shifted the concentration-response curve of 5-HT downwards and to the right in a concentration-dependent manner. In the presence of 10^-6 mol/L ketanserin, however, methiothepin antagonized 5-HT-induced contractions competitively with a pA ₂ value of 7.95 (7.59–8.31). The 5-HT₃ receptor antagonist MDL 72222 had no effect on 5-HT-induced contractions. The findings of this study indicate that 5-HT₁ and 5-HT₂ receptors are located in equine basilar arterial smooth muscle cells, and that stimulation of these receptors results in contraction.Abbreviations CR concentration ratio - EC₅₀ concentration producing 50% of the maximal response - 5-HT 5-hydroxytryptamine - MDL 72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - pA ₂ negative logarithm of the molar concentration of antagonist that produces a 2-fold rightward shift of the concentration-response curve - pEC₅₀ negative logarithm of EC₅₀ - PGF₂ prostaglandin F₂     

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.     

Contractile responses of isolated equine digital arteries under hypoxic or hyperoxic conditions in vitro: role of reactive oxygen species and Rho kinase

The underlying pathophysiological triggers for equine acute laminitis are unknown, although digital vasoconstriction, ischaemia, hypoxia and reperfusion injury may be involved. The contractile responses of isolated equine digital arteries (EDAs), harvested from the hindlimbs of normal horses postmortem at an abattoir, were studied acutely (up to 3 h) under hyperoxic (95% oxygen, 5% CO₂) and hypoxic (95% nitrogen, 5% CO₂) conditions in organ baths. Phenylephrine (PHE; 10^?6 m ), 5‐hydroxytryptamine (5‐HT; 10^?7 m ) and high potassium (K⁺; 118 mm ) caused contraction in EDAs which was significantly (P < 0.0001) enhanced under hypoxic conditions. In contrast, contraction stimulated by 9,11‐dideoxy‐9α,11α‐epoxymethanoprostaglandin F_2α (U44069; 3 × 10^?8 m ) was not significantly enhanced by hypoxia (P = 0.75). Hypoxia‐enhanced contraction in response to K⁺ was greater (P < 0.03) in vessels with a functional endothelium than in vessels in which the endothelium was removed by rubbing. Fasudil (10^?6 to 10^?5 m ), a Rho kinase inhibitor, and apocynin (10^?3 to 3 × 10^?3 m ), an NADPH oxidase inhibitor, significantly (P ≤ 0.05) inhibited hypoxia‐enhanced contraction in response to PHE and 5‐HT. In conclusion, hypoxia‐enhanced contraction occurred in EDAs. This appears to be partially mediated by reactive oxygen species produced by NAPDH oxidase, which activate Rho kinase to increase calcium sensitisation and enhance smooth muscle contraction.