Endothelin receptor mediating contraction of isolated bovine coronary artery

We studied endothelin (ET) receptors and their subtypes on isolated bovine coronary arteries. Endothelin receptors that mediated contraction of isolated bovine coronary artery were characterized by the use of antagonists and agonists. Contractions induced by the nonselective agonist ET-1 (10-10-10-7 M) were not affected by the removal of the endothelium (pEC50: 8.52, maximal contraction: 105% of that induced by 60 mM KCl). BQ-123 (3 x 10-7 M) antagonized contractions of endothelium-denuded coronary rings induced by low concentrations of ET-1 (10-10 or 10-9 M), but potentiated the contractions induced by higher concentrations of ET-1 (3 x 10-8 and 10-7 M). BQ-788 (10-6 M) potentiated contractions induced by ET-1 (3 x 10-10 and 10-7 M). In the presence of BQ-788 (10-6 M), BQ-123 (3 x 10-8-3 x 10-6 M) concentration - dependently inhibited contractions induced by ET-1 (3 x 10-10 and 10-7 M) (pA2: 6.61). Sarafotoxin S6b (10-9-3 x 10-7 M) evoked contractions in the denuded coronary artery (pEC50: 8.49, maximal contraction: 139% of 60 mM KCl). The BQ-123 caused a concentration-dependent rightward shift of contractions induced by sarafotoxin S6b (pA2: 7.89). The present study indicates that ET-1 and sarafotoxin S6b contract the isolated bovine coronary artery by stimulating ETA receptors on smooth muscle cells, and that ETB receptors might suppress the ET-1-induced contractions.     

Characterization of bradykinin-induced endothelium-independent contraction in equine basilar artery

We investigated the effect of bradykinin (BK) on isolated equine basilar arterial rings with and without endothelium. BK induced concentration-dependent contraction of resting arterial rings and no relaxation when the rings were precontracted by prostaglandin F_2α. The maximal response and pD₂ value were 161.2 ± 28.1% (to 60 m m KCl-induced contraction) and 8.24 ± 0.25 respectively. The cumulative concentration–response curve for BK was not shifted to the right by des-Arg⁹-[Leu⁸]-BK (a B₁-receptor antagonist), HOE140 (a B₂-receptor antagonist) or NPC567 (another B₂-receptor antagonist). In four of six basilar arteries, NPC567 induced concentration-dependent contraction. Indomethacin (a cyclooxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), quinacrine (a phospholipase A₂ inhibitor), tetrodotoxin (a selective blocker of Na⁺ channels), guanethidine (a nor-adrenergic neuron blocking drug), phentolamine (an α-adrenoceptor antagonist), Nω-nitro- l -arginine ( l -NNA, a nitric oxide (NO) synthase inhibitor) and endothelial denudation did not affect the BK-induced contraction. l -NNA and indomethacin induced contraction and relaxation under resting vascular tone respectively. These results suggest that endothelial cells are not involved in BK-induced contraction and that the contraction is not mediated via activation of known B₁ and B₂ receptors. Arachidonic acid metabolites and neurotransmitters like norepinephrine and NO might not play a role in BK-induced contraction in equine basilar artery.     

Epithelium- and mucosa-dependent relaxation and contraction of normal equine trachealis muscle in vitro

Strips of trachealis muscle were dissected from the mid-cervical portion of the trachea from horses that were free of respiratory tract disease. The epithelium and mucosa were removed from one group of tissues and were left intact in a second group of tissues. Each tissue was suspended in a bath filled with Krebs-bicarbonate solution that was aerated with 5% CO2 in oxygen and maintained at 37 degrees C. Isometric tension was continuously recorded. The contractile response to square-wave electrical stimulations increased as frequency (3, 5, 10, 15, 20, 25, and 30 Hz), voltage (10, 15, 18, and 25 V), and pulse duration (0.2, 0.5, 1.0, 1.5, and 2.0 ms) increased in tissues with the epithelium and mucosa intact. A stimulus of 18 V, 20 Hz, and 0.5 ms induced maximal contraction. Atropine (10(-6) M) abolished the response to 18 V and 0.5 ms at all frequencies. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) was added to the baths in 0.5-logarithmic increments. Tissues that were contracted in response to acetylcholine (10(-5) M) had a concentration-dependent decrease in active isometric tension when isoproterenol was added to the baths in 0.5-logarithmic increments (10(-9) to 10(-4) M). The contraction and relaxation curves were qualitatively similar, but quantitatively different in tissues with and without the epithelium and mucosa. Removing the epithelium and mucosa increased the contractile response to acetylcholine at bath concentrations of 3.1 x 10(-7) M and 10(-6) M. The presence of epithelium and mucosa enhanced the magnitude of isoproterenol-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Participation of H1-receptors in histamine-induced contraction and relaxation of horse coronary artery in vitro.

The mechanisms of histamine-induced contraction and relaxation were investigated in rings isolated from a middle part of the left descending coronary arteries of horses. Intact and endothelium-denuded preparations were compared. Rings of horse coronary arteries contracted in response to histamine in a concentration dependent manner, but some of them relaxed with lower concentrations and contracted with higher concentrations. Removal of the endothelium abolished the relaxation and potentiated the contraction. The pD2 values were 4.70 +/- 0.08 in the rings with intact endothelium and 4.95 +/- 0.08 in endothelium-denuded rings. Histamine-induced contractions in intact and denuded preparations were not affected by an H2-antagonist, cimetidine, but were inhibited by an H1-antagonist, diphenhydramine in non-competitive manner in the rings with endothelium and in competitive manner in denuded rings. After precontraction with PGF2 alpha or norepinephrine, histamine relaxed preparations with intact endothelium (pD2 value, 7.80 +/- 0.11), although histamine-induced relaxations were not observed in denuded preparations. The relaxation was competitively inhibited by diphenhydramine. Relaxing response was significantly attenuated by methylene blue, quinacrine, L-nitro-arginine, gossypol and AA861 but not by indomethacin. These results suggest that the histamine-induced contraction and relaxation in horse coronary arteries are mediated mainly by H1-receptors in the smooth muscle and endothelium, respectively, and H1-receptor activation of endothelial cells may liberate vasodilator substances.     

Isoproterenol- and salbutamol-induced relaxation of acetylcholine- and histamine-induced contraction of equine trachealis muscle in vitro

Strips of trachealis muscle were dissected from the midcervical portion of the trachea of horses that were free of respiratory tract disease, and the overlying epithelium and mucosa were removed. Muscle strips were suspended in tissue baths that were filled with Krebs-bicarbonate solution, aerated with 5% CO2 in oxygen and maintained at 37 C. Isometric tension was continuously recorded. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) or histamine (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. When the tissues were contracted with acetylcholine (3.1 x 10(-6) M) or histamine (10(-4) M), the decrease in active isometric tension was concentration dependent when isoproterenol (10(-9) to 10(-4) M) or salbutamol (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. There was no difference between the response to isoproterenol and salbutamol when tissues from the same horses were compared whether the tissues were contracted in response to acetylcholine (3.1 x 10(-6) M) or histamine (10(-4) M). Relaxation was antagonized by 10(-6) M propranolol. The degree of relaxation obtained in these muscle strips was considerably less than that reported from other species' tracheal muscle strips that had the epithelium and mucosa intact. We concluded that equine tracheal smooth muscle contains beta-adrenoceptors that can be stimulated by either a mixed beta-1, beta-2 agonist or a selective beta-2 agonist.     

Equine coronary artery responds to 5-hydroxytryptamine with relaxation in vitro

Isolated equine coronary arteries responded to 5-hydroxytryptamine (5-HT) with relaxations in both endothelium-dependent and endothelium-independent mechanisms. Experiments were designed to characterize the 5-HT receptor sub type mediating these relaxations. Both 5-HT and alpha-methyl-5-HT (α-Me-5- HT; 5-HT₂ agonist) produced concentration-dependent relaxations in equine coronary arteries precontracted with a thromboxane A₂ derivative (0N011113). The degree of the maximal relaxation induced by α t-Me-5-HT was about one-half of that induced by 5-HT. In the coronary arteries without endothelium, α -Me-5-HT produced no relaxation, but 5-HT caused relaxation, which was inhibited by a 5-HT₁ antagonist (methysergide, mianserin and methiothepin), but was inhibited neither by ketanserin (5-HT₂ antagonist) nor by MDL72222 (5-HT₃ antagonist). In the coronary arteries with endothelium, however, the relaxation induced by α -Me-5-HT was inhibited by ketanserin, L-nitro-arginine (NO synthase inhibitor) and methylene blue (soluble guanylate cyclase inhibitor). These results suggest that the relaxation induced by 5-HT in equine coronary arteries depends mainly on the stimulation of both 5-HTi receptor subtype on smooth muscle cells directly, and 5-HT₂ receptor subtype on endothelial cells indirectly by liberating endothe-lium-derived NO.     

Stress relaxation of the equine forelimb in vitro

Studies with in vitro preparations of the foreleg of the horse have shown that stress relaxation, loss of tensile force generating capacity of the tendinous structures, routinely occurs and must be considered in the interpretation of experiments with such preparations.     

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.     

Capsaicin-induced relaxation in rabbit coronary artery.

In the present study mechanism of inhibitory effects of capsaicin on the contractility of rabbit coronary artery were studied by measurement of isometric tension and intracellular Ca2+ concentration. Capsaicin (1 microM to 30 microM) relaxed the coronary artery pre-contracted with prostaglandin (PG) F2alpha (1 microM) in a concentration-dependent manner. The PGF2alpha-induced increase in intracellular Ca2+ concentration was also inhibited. The effects of capsaicin were readily reversed by washing capsaicin from the bath. Capsaicin-induced relaxation was not attenuated by pretreatment with capsazepine (1 microM), a blocker of vanilloid receptor or ruthenium red (1 microM), a blocker of non-selective cation channel. Previous exposure to a high concentration of capsaicin (100 microM) or repeated application of capsaicin did not eliminate the relaxation response to subsequent application of capsaicin. Increasing the external K+ concentration to 80 mM significantly attenuated the capsaicin-induced relaxation with simultaneous change in intracellular Ca2+ concentration. Pretreatment with iberiotoxin (100 nM), a blocker of Ca2+-activated K+ channel, only partially inhibited the capsaicin-induced relaxation. However, application of 4-aminopyridine (4-AP, 1 mM), a blocker of delayed rectifier K+ current significantly inhibited the capsaicin-induced relaxation with concomitant attenuation of the effect on intracellular Ca2+ concentration. These results indicate that capsaicin may have a direct relaxing effect on the smooth muscle contractility, and relaxation may be due to activation of the 4-AP-sensitive, delayed rectifier K+ channels in the rabbit coronary artery.     

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 .     

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.     

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 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.     

Characterization of equine inflammasomes and their regulation

Veterinary Research Communications - Inflammasome, a cytosolic multi-protein complex, assembly is a response to sensing intracellular pathogenic and endogenic danger signals followed by caspase-1...     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Pathogenicity of equine herpesvirus 1 subtype 2 for foals and adult pony mares

Three pony mares and 4 pony foals were inoculated with a subtype 2 strain of equine herpesvirus 1. Foals had periods of fever 12 h and 2.5 days after inoculation and leukopenia, involving both neutrophils and lymphocytes, followed by leukocytosis. Mares had transient fever and leukopenia 24 hours after inoculation that were less severe than in foals. An increase in circulating virus-neutralizing antibody was seen in 2 of 3 inoculated mares, but not in foals. Attempts to isolate virus from blood were unsuccessful. These studies show that equine herpesvirus 1 subtype 2 is a mild pathogen for ponies and infection may result in inapparent clinical disease.     

Evaluation of beta3-adrenoceptor-mediated relaxation in intact and endotoxin-treated equine digital veins

OBJECTIVE: To investigate the functional expression of beta3-adrenoceptors (beta3-ARs) in equine digital veins (EDVs) and to examine whether beta3-AR relaxation was altered in EDVs incubated with endotoxin. SAMPLE POPULATION: Forelimbs obtained from 30 horses. PROCEDURE: Forelimbs were obtained from horses in an abattoir. Equine digital veins were carefully removed from distal portions of the forelimbs. Rings of dissected EDVs were mounted in 5-mL organ baths to record isometric tension in the presence of various beta3-AR agonists (SR 58611A, ZD 2079, and ZM 215001). RESULTS: In intact EDVs, isoprenaline, SR 58611A, ZD 2079, and ZM 215001 induced concentration-dependent relaxation. Isoprenaline and SR 58611A-induced relaxations were reduced or unaffected by nadolol, respectively. In intact EDVs, SR 58611A-induced relaxation was significantly reduced in the presence of 2 microM ZM 215001 (used as a beta3-AR antagonist). In endothelium-denuded EDVs or intact EDVs in the presence of a nitric oxide synthase inhibitor, isoprenaline and SR 58611A-induced relaxations were significantly decreased. The endothelium-independent relaxation to SR 58611A was significantly inhibited in the presence of ZM 215001. In endotoxin-treated EDV, isoprenaline- and SR 58611A-induced relaxations were significantly reduced. In these conditions, cycloheximide (a protein synthesis inhibitor) and ibuprofen (a cyclooxygenase inhibitor) restored the relaxant response to SR 58611A. CONCLUSIONS AND CLINICAL RELEVANCE: Beta3-adrenoceptors are functionally expressed in EDVs. Incubation in the presence of endotoxin, used as an in vitro model of laminitis, induced an alteration of beta-AR-mediated relaxations in EDVs, which could be the consequence of cyclooxygenase induction and subsequent prostanoid production.     

Characterisation of reconstituted equine cartilage formed in vitro

Lesions in cartilage of equine weightbearing joints commonly result in lameness. Cell-based resurfacing techniques are currently being developed for human and veterinary applications. Biopsies of stifle joint cartilage (1 g) were harvested aseptically and chondrocytes were isolated by sequential enzyme digestion. The cells were grown in vitro on filter inserts. Analysis of cultures 8 weeks later showed that the cells had accumulated extracellular matrix and formed a continuous layer of cartilagenous tissue as determined histologically. The cells maintained their phenotype as they synthesised type II collagen and proteoglycans similar in size to those synthesised by chondrocytes in native cartilage, but this reconstituted tissue had more sulphated glycosaminoglycan and lower collagen content than native cartilage. This experiment tests the feasibility of growing equine cartilagenous tissue in vitro. This tissue may be useful in the management of chondral injuries in the horse in a scenario where the patient donates cells, the cells are propagated under laboratory conditions and the resulting tissue becomes the therapeutic agent.