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.     

Endostatin inhibits bradykinin-induced cardiac contraction

.Endogenous fragments of extracellular matrix are known to possess various biological effects. Levels of endostatin, a fragment of collagen type XVIII, increase in certain cardiac diseases, such as cardiac hypertrophy and myocardial infarction. However, the influence of endostatin on cardiac contraction has not been clarified. In the present study, we investigated the effects of endostatin on bradykinin-induced atrial contraction. Isometric contractile force of mouse isolated left atria induced by electrical current pulse was measured. Voltage-dependent calcium current of guinea pig ventricular myocytes was measured by a whole-cell patch-clamp technique. Endostatin (100–1,000 ng/ml) alone treatment had no influence on left atrial contraction. On the other hand, pretreatment with endostatin (300 ng/ml) significantly inhibited bradykinin (1 µM)-induced contraction and voltage-dependent calcium current. These data suggest that endostatin may decrease bradykinin-induced cardiac contraction perhaps through the inhibition of voltage-dependent calcium channel.     

Endothelin mediated contraction of equine laminar veins

REASONS FOR PERFORMING STUDY: Endothelin-1 (ET-1) may be a key mediator in the pathogenesis of laminitis, but endothelin-mediated responses in the venous microcirculation of the equine foot have yet to be fully characterised. OBJECTIVES: To characterise the response of equine laminar veins to ET-1 and evaluate the ET-1 receptor subtypes that mediate this response. METHODS: Small veins (150-500 microns) draining the equine digital laminae from healthy horses and ponies subjected to euthanasia at an abattoir were investigated using wire myography. Concentration response curves were constructed for ET-1 in the presence of ETA (BQ123) and ETB (BQ788) receptor antagonists, and L-NAME, a nitric oxide synthase blocker. The selective ETB receptor agonist BQ3020 was investigated alone and following incubation with L-NAME, with or without BQ788. RESULTS: Endothelin-1 contraction of laminar veins was significantly inhibited by BQ123 but not by BQ788. In the presence of L-NAME, sensitivity of laminar veins to ET-1 was enhanced 4-fold, and further addition of BQ788 did not alter this increased sensitivity. BQ3020 induced no venoconstriction; however, in the presence of L-NAME, it caused contraction of veins with approximately 30% of the efficacy of ET-1. The action of BQ3020 in the presence of L-NAME was abolished by BQ788. CONCLUSIONS: Both ETA and ETB receptors are involved in the net tonic response to ET-1 in normal laminar veins. A population of ETB receptors may be present on the vascular endothelium and on smooth muscle of laminar veins, and the action of ET-1 at these 2 sites is likely to be approximately equal and opposite. POTENTIAL RELEVANCE: Our results clarify the function of the ET-1 receptor subtypes in laminar veins from healthy horses. Further study of ET-1 receptors in laminitic horses is therefore warranted.     

Characterization of equine vocalization

Bioacoustics is the study of sound in animals and includes, but is not limited to, animal communication with associated behavior, sound production anatomy and neurophysiology, auditory capacities and auditory mechanisms, and animal welfare. The present research investigates the vocalizations of horses during stressful situations. Stress can be positive or negative. Distress is anything that affects the animal in a negative way, such as in mare and foal separation. Eustress is anything that affects the animal in a positive way, such as morning feeding time in a horse barn. The purpose of the current research is to find spectral differences in the recorded vocalizations of stalled horses that indicate both distress and eustress using the Hidden Markov Model (HMM). Greenwood Function Cepstral Coefficient values suggest that there are spectral differences between vocalizations in a distress and eustress situation. These consistent results indicate that further research to obtain and evaluate vocalizations of horses may provide a productive tool in understanding equine welfare.     

Vasomotor effects of histamine on bovine and equine basilar arteriesin vitro

The vasomotor effects of histamine on isolated bovine and equine basilar arteries were examined. Histamine induced contractions in both these preparations. The maximal response to and pEC₅₀ value for histamine of the equine artery were larger than those of bovine tissue. Similar results were obtained with endothelium-denuded basilar arteries. Diphenhydramine (H₁-receptor antagonist) inhibited histamine-induced contractions of the basilar arteries from both species in a concentration-dependent manner and its pA ₂ values (with 95% confidence limits) were 7.61 (7.39–7.83) and 8.15 (8.01–8.29) for the bovine and equine preparations, respectively. Cimetidine (H₂-receptor antagonist) slightly potentiated histamine-induced contractions of bovine, but not equine, basilar arteries. 2-Thiazolylethylamine (H₁-receptor agonist) induced contractions in both preparations, whereas impromidine (H₂-receptor agonist) induced weak relaxation of the bovine, but not the equine, tissue. These findings indicate that bovine basilar arterial smooth muscle cells possess H₁- and H₂-receptors. Stimulation of the former results in contraction, whereas stimulation of the latter results in weak relaxation. Equine basilar arterial smooth muscle cells possess H₁-receptors, stimulation of which results in contraction.Abbreviations CR concentration ratio - EC₅₀ concentration producing 50% maximal response - pA ₂ negative logarithm of the molar concentration of antagonist that produces a twofold rightward shift of a concentration-response curve - pEC₅₀ negative logarithm of EC₅₀ - PGF₂ prostaglandin F₂ - PGI₂ prostaglandin I₂     

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     

Characterization of arginase expression by equine neutrophils

Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood neutrophils using quantitative PCR. Arginase expression was also studied by Western blot and enzyme activity assay. The effect of nor-NOHA (1 mM), a specific arginase inhibitor, was assessed on arginase activity in vitro and ex vivo on neutrophil's inflammatory gene expression and viability. Results showed that equine neutrophils constitutively express arginase isoform 2, ODC and OAT. Neutrophil ex vivo stimulation did not induce arginase I or influence arginase II mRNA expression. Ex vivo inhibition of arginase activity by nor-NOHA had no effect on neutrophils inflammatory gene expression induced by LPS/fMLP (5 h) but significantly reversed the cell loss observed after this stimulation.     

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...     

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)     

Characterization of vasodilatory adenosine receptors in equine digital veins

Isolated equine digital veins (EDVs) which had been denuded of their endothelium were used to study adenosine receptors causing vasodilation. When the blood vessel wall tension was raised with the thromboxanemimetic, U44069 (30 n m ), the order of vasodilator potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2- p -(2-carboxyethyl)phenyl amino-5'-N-ethylcarboxamido-adenosine (CGS 21680) > 5'-N-methylcarboxamido-adenosine (MECA) > > N⁶-cyclohexyladenosine (CHA) > N⁶-cyclopentyladenosine (CPA) > N⁶–2-(4-Aminophenyl)ethyladenosine (APNEA) > adenosine. Removal of the endothelium had no significant effect on the responses to NECA. The adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A₁-selective) and xanthine amine cogener (XAC; non-selective antagonist) inhibited responses to NECA and CHA in a competitive manner and XAC proved to be 8–25 times more potent than DPCPX against both agonists. These data support the presence of A₂ adenosine receptors in EDVs, located on the vascular smooth muscle cells, which are most likely to be of the A_2A-adenosine receptor subtype. A direct comparison between the potency and efficacy of NECA and adenosine as vasodilators of EDV and equine digital arteries was made and both agonists proved to be significantly more potent and efficacious as vasodilators of EDVs. These data suggest that adenosine may be an important local mediator regulating blood flow through the digital circulation and that its generation under hypoxic conditions would lead to selective venodilation.     

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.     

Characterization of the equine platelet aggregation response.

Equine platelet aggregation responses to bovine collagen, adenosine diphosphate (ADP), serotonin, epinephrine, and arachidonate in a platelet aggregometer were recorded. Equine platelets exhibited irreversible aggregation when incubated with ADP at a final concentration of 10 microM and bovine collagen. A secondary aggregation wave was recorded from platelets from certain horses at final ADP concentrations of 1 to 5 microM. Serotonin and arachidonate induced a weak reversible aggregation response, but a response was not observed following epinephrine addition. Equine platelet aggregation was influenced by concentration of anticoagulant (sodium citrate). Platelet aggregation responses at 37 C were indistinguishable from those recorded at 39 C. Platelet aggregation responses also were altered if the aggregation tests were not performed within 4 hours of blood sample acquisition. An assessment of platelet aggregation from multiple blood samples from the same horse indicated that the procedures described provide a reliable method to assess equine platelet aggregation in vitro.     

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.     

Finite element analysis of wall stress in the equine pulmonary artery

Reasons for performing study: Arterial calcification is found frequently in the pulmonary artery of racehorses, but the aetiology is unknown. Calcification might be associated with increased wall stress due to arterial geometry (shape) and exercise‐induced hypertension. Hypothesis: High wall stress levels are found in the regions associated with calcified lesion formation, exacerbated as transluminal pressure increases to levels associated with exercise. Methods: The pulmonary arteries of 5 horses, unaffected by calcification, were dissected and pressurised to resting and exercising physiological transluminal pressures and scanned with MRI. Arterial geometries were reconstructed to form 3D computer models and finite element analyses performed. Wall stress levels were measured in 4 regions of interest: the arterial trunk and bifurcation, the wall ipsilateral and contralateral to the bifurcation. Measurements were made for arterial transluminal pressures of 25, 50 and 100 mmHg. Results: High wall stress levels were consistently found at the pulmonary artery bifurcation and wall ipsilateral to the bifurcation, where calcified lesions typically form. Lower wall stress levels were found along the trunk and the wall contralateral to the bifurcation where lesions are less frequently found. Wall stress levels increased 5‐fold over a 4‐fold increase in pressure. The wall stress levels ranged 10 kPa in the wall of the branch contralateral to the bifurcation at 25 mmHg to 400 kPa in the bifurcation at 100 mmHg. Conclusions: Wall stress from arterial geometry and increased pulmonary artery transluminal pressure are factors that may be associated with calcification of the equine pulmonary artery. Potential relevance: Arterial calcification may increase the risk of arterial wall failure in racing horses.     

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 adipose-derived equine and canine mesenchymal stem cells after incubation in agarose-hydrogel

Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive application. Isolated ASCs were expanded and 2.5 × 10⁶ cells were combined with agarose-hydrogel to build a 0.4% hydrogel-cell solution which was stored at two temperatures (room temperature (RT) vs. 37°C). Cell viability was investigated (live-dead assay) at different time points (0, 1, 6 and 24 h) in order to determine i) the effect of different temperatures on the cell survival as well as ii) the maximum possible time span before implantation. CFU-assay and WST-1 assay were performed after 24 h incubation in agarose-hydrogel and the cells were induced into adipogenic and osteogenic differentiation to analyze the effects of the incubation on the cell behaviour. No negative effect of the agarose-hydrogel incubation was determined on the different species’ cell behaviour at either RT or 37°C with any of the assays used. We can recommend agarose-hydrogel as a cell carrier for cell implantation with a storage period of up to 24 h at room temperature or at 37°C prior to implantation.     

Affinity of isoxsuprine for adrenoreceptors in equine digital artery and implications for vasodilatory action

We used isolated equine digital arteries to study the vasodilatory mechanism of isoxsuprine, and fowl caecum preparations to investigate the affinity of the drug for beta-adrenoceptors. Isoxsuprine is a potent vasodilator of arterial smooth muscle that has been precontracted by an alpha-adrenoceptor agonist such as noradrenaline (log EC50 = -6.33 [-5.98; -6.68]). The present study indicates that its effect is due to alpha-adrenoceptor blockade since: (1) after a long lasting exposure to cumulative doses of isoxsuprine the vasoconstricting action of noradrenaline cannot be restored; (2) isoxsuprine does not promote relaxation on preparations precontracted by PGF2alpha; (3) isoxsuprine shifts the dose-response curve of noradrenaline to the right; and (4) its affinity (pK(B) = 6.90 [6.60; 7.20]) in this experiment is comparable to that in noradrenaline-precontracted preparations and is 14 times lower than that of the selective alpha1-adrenergic antagonist prazosin [pK(B) = 8.04 (7.40; 8.68]). The affinity of isoxsuprine for beta-adrenoceptors was 100 times lower than that of isoprenaline when tested on fowl caecum. This preparation has a large beta-adrenoceptor and negligible alpha-adrenoceptor population concerned with the control of smooth muscle motility. Our data suggest that the alpha-mediated effect of isoxsuprine on horse arterial smooth muscle is due to higher affinity of the drug for alpha- than beta-adrenoceptors rather than low concentration or functionality of beta-sites at this site. According to these data, pure beta2-agonists seem to be more profitable tools to determine vasodilation of the arterial bed in horses legs.