Comparison of 2 endothelin-receptor antagonists on in vitro responses of equine palmar digital arterial and venous rings to endothelin-1

The goals of this study were to determine the concentration-response (C-R) relationship of endothelin-1 (ET-1), compare 2 ET-receptor antagonists and determine the antagonist concentrations that block the vasomotor effects of ET-1, and compare the effectiveness of ET-1 and previously studied vasoconstrictors in equine palmar digital arterial and venous rings in vitro. Vessel rings from 8 nonlaminitic horses were placed in Tyrode's solution, 1 side fixed to the floor of an organ bath and the other side fixed to a force-displacement transducer. Two separate studies were conducted: (I) incubation with a single ET-receptor antagonist (PD142893 or PD145065 at a concentration of 10(-7), 10(-6), or 10(-5) M), followed by determination of an ET-1 C-R curve (using concentrations of 10(-10) to 10(-6) M) for medial vessel rings; and (II) comparison of ET-1 with norepinephrine and histamine (10(-10) to 10(-6) M) and comparison of contractile responses of medial and lateral vessel rings. In study I, ET-1 administration caused pronounced and sustained concentration-dependent contraction of vessel rings; these contractile responses were decreased by 10(-5) M PD142893 and were completely blocked by 10(-5) M PD145065. Venous rings had greater apparent maximum contraction in response to ET-1 than arterial rings. In study II, the relative sensitivity of norepinephrine was found to be equivalent to that of ET-1, whereas that of histamine was lower. No significant differences were observed between responses of medial versus lateral vessel rings. Thus, ET-1 is a potent vasoconstrictor of equine palmar digital arteries and veins, and the ET-receptor antagonist PD145065 is more effective than PD142893 in inhibiting these contractile effects in vitro.     

Responses of Guinea-Pig Lung Parenchymal Strips to Tracheobronchial Lavage Fluid from Horses affected with Summer Pasture-Associated Obstructive Pulmonary Disease

The response of parenchymal strips from guinea-pig lungs to tracheobronchial lavage fluid (TBLF) collected from 8 normal horses and from 8 affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) was determined. TBLF was collected during the summer (July) and winter (February) seasons. The serum/TBLF urea nitrogen ratio was used to standardize the mediator concentration in the TBLF. Four strips were used from each guinea-pig. The first strip did not receive any antagonist and served as the control. The second, third and fourth strips received antagonists of PGE2, LTD4 and PAF, respectively at 10–6 mol/L for 30 min. The tissues were then precontracted with a dose of histamine (10–5 mol/L) and their responses to 1 ml of TBLF were determined. The study showed that TBLF obtained in the summer from unaffected horses produced a significantly greater relaxation than that from the affected horses, whereas TBLF obtained in the winter from unaffected or affected horses did not cause a significantly different degree of relaxation. Among the antagonist-treated strips, only those exposed to the PGE2 blocker showed a significant reduction in the relaxation caused by TBLF obtained in the summer from SPAOPD horses. This suggests that PGE2 is an important mediator present in the summer in the TBLF from horses affected with SPAOPD.     

Plasma and bronchoalveolar fluid concentrations of nitric oxide and localization of nitric oxide synthesis in the lungs of horses with summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To determine concentrations of nitric oxide (NO) in plasma and bronchoalveolar lavage fluid (BALF) and localize nitric oxide synthesis in the lungs of horses with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 7 adult horses with SPAOPD and 6 clinically normal adult horses. PROCEDURE: Severity of SPAOPD was determined by use of clinical scores, change in intrapleural pressure (APpl) during tidal breathing, cytologic analysis of BALF, and histologic evaluation of lung specimens obtained during necropsy. Nitric oxide concentrations in plasma, BALF and epithelial lining fluid (ELF) were determined by use of a chemiluminescent method. Inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) were localized in formalin-fixed lung specimens by use of immunohistochemical staining, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity was localized in cryopreserved specimens by use of histochemical staining. RESULTS: Plasma concentration of NO in affected horses was slightly but not significantly greater than concentration in nonaffected horses. Nitric oxide concentrations in BALF or ELF did not differ between groups. Immunoreactivity of iNOS in bronchial epithelial cells of 3 of 5 lung lobes was greater in horses with SPAOPD, compared with nonaffected horses. However, staining for NT and NADPHd activity did not differ between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of iNOS was greater in bronchial epithelial cells of horses with SPAOPD, compared with nonaffected horses, suggesting that NO may play a role in amplifying the inflammatory process in the airways of horses with this disease.     

In vitro responses of bronchial spirals and parenchymal strips from healthy and heartworm-infected dogs

Bronchial spirals and pulmonary parenchymal strips from control (noninfected) and Dirofilaria immitis-infected dogs were tested for isometric contractile response to histamine and carbamylcholine. Responses of bronchial spirals from control dogs and from D immitis-positive groups did not differ in responses to histamine or carbamylcholine. Parenchymal strips from D immitis-positive dogs showed greater response to histamine than did those from controls; responses to carbamylcholine did not differ.     

Immunohistochemical determination of the expression of endothelin receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To immunohistochemically determine the expression of endothelin (ET) receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease (SPAOPD). SAMPLE POPULATION: Tissue specimens obtained from 8 healthy and 8 SPAOPD-affected horses. PROCEDURE: Horses were examined and assigned to healthy and SPAOPD groups. Horses were then euthanatized, and tissue specimens containing bronchi of approximately 4 to 8 mm in diameter were immediately collected from all lung lobes, fixed in zinc-formalin solution for 12 hours, and embedded in paraffin. Polyclonal primary antibodies against ET-A or ET-B receptors at a dilution of 1:200 and biotinylated IgG secondary antibodies were applied to tissue sections, followed by the addition of an avidin-biotin immunoperoxidase complex. Photographs of the stained slides were digitally recorded and analyzed by use of image analysis software to determine the intensity of staining. Two-way ANOVA was used for statistical analysis. RESULTS: The left diaphragmatic lung lobe of SPAOPD-affected horses had a significantly greater area of bronchial smooth muscle that immunostained for ET-A, compared with that for healthy horses. All lung lobes of SPAOPD-affected horses, except for the right diaphragmatic lobe, had significantly greater staining for ET-B receptors in bronchial smooth muscle, compared with results for healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed overexpression of ET-A and, in particular, ETB receptors in the bronchial smooth muscle of SPAOPD-affected horses, which suggested upregulation of these receptors. These findings improve our understanding of the role of ET-1 in the pathogenesis of SPAOPD.     

In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses

OBJECTIVE: To determine effects of cisapride and 5-hydroxytryptamine (5-HT) on the jejunum of horses. SAMPLE POPULATION: Jejunal muscle strips from 8 horses. PROCEDURE: Muscle strips were suspended in isolated muscle baths. Isometric stress responses to 5-HT and cisapride, with and without specific antagonists, were determined. RESULTS: Muscle strips incubated with atropine and tetrodotoxin responded to 5-HT and cisapride with an increase in contractile force. The 5-HT caused a concentration-dependent increase in contractile amplitude, with a maximum response (Emax) of 1,151+/-214 g/cm2 and a molar concentration that induces contractile force equal to 50% of maximum response (EC50) of 0.028+/-0.002 microM. Prior incubation with the 5-HT2 antagonist ketanserin decreased the Emax (626 +/-147 g/cm2) and potency (EC50, 0.307+/-0.105 microM) of 5-HT Prior incubation with the 5-HT3 antagonist tropisetron decreased the efficacy (Emax, 894+/-184 g/cm2) to 5-HT Cisapride also caused a concentration-dependent increase in contractile amplitude, with an Emax of 331+/-82 g/cm2 and an EC50 of 0.302+/-0.122 microM. Prior incubation with ketanserin decreased the Emax (55+/-17 g/cm2) and potency (EC50, 0.520+/-0.274 microM) of cisapride. CONCLUSION AND CLINICAL RELEVANCE: Stimulatory effects of 5-HT and cisapride on circular smooth muscle of equine jejunum are mediated primarily through a noncholinergic effect. The effects of 5-HT are mediated, at least partially, by 5-HT2 and 5-HT3 receptors, whereas the effects of cisapride are mediated primarily by 5-HT2 receptors. This may impact treatment of horses with postoperative ileus.     

Role of Endothelium and Nitric Oxide in the Response of Equine Colonic Arterial Rings to Vasoconstrictor Agents

Objective- To determine the in vitro contractile responses of equine colonic arteries to angiotensin II, histamine, serotonin, norepinephrine, prostaglandin F_2α, vasopressin, and a thromboxane-B₂-analogue. Study Design- The tension generated in colonic arterial rings placed in organ baths with oxygenated Tyrode's solution at 37°C after exposure to the previously mentioned chemical agents was measured using force-transducers interfaced with a polygraph. Sample Population- Large colon arterial rings collected from eight horses. Methods- The rings were allowed to equilibrate for 45 minutes after applying 2 g tension. Bath solution was replaced and tension reapplied at 15-minute intervals. Cumulative-concentration-responses were determined for concentrations ranging from 10^-8M to 10^-4M on three vessel groups namely endothelium intact, endothelium denuded, and L-NAME treated. The maximal response for each vessel was considered as 100%; responses to lower concentrations were calculated as a percentage of the maximum. The EC₅₀ value was determined for each concentration-response relationship of each agent. Results- Vessels with denuded endothelium or those incubated with L-NAME had greater contractile responses. Angiotensin, histamine, serotonin, and norepinephrine produced greater maximal responses than the other agents. Endothelium denuded rings had lower EC₅₀ values. Responses to norepinephrine and serotonin were affected less by denudation. Conclusion- Endothelium plays an important role in modulating responses of colonic arterial rings to contractile agents. Endothelium-derived vasodilators, other than nitric oxide, may modulate contractile responses of equine colonic arteries. Clinical Relevance- Endothelial damage associated with colonic vovulus may be a major factor for sustained reduced perfusion after surgical correction.     

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.     

Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature

REASONS FOR PERFORMING STUDY: Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. HYPOTHESIS: That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. OBJECTIVE: To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). METHODS: Horses were administered black walnut heartwood extract (BWHE) or water (control horses) via nasogastric intubation. After euthanasia, laminar vessels (100-800 microm internal diameter) were isolated and mounted on small vessel myographs to assess contractile function. RESULTS: Contractile responses to PE or 5-HT were identical in laminar arteries isolated from either control horses or those administered BWHE. In contrast, responses to PE or 5-HT were significantly reduced in laminar veins isolated from BWHE-administered horses when compared with laminar veins isolated from control horses. CONCLUSIONS AND POTENTIAL RELEVANCE: These results are consistent with the prodromal stages of laminitis being associated with selective dysfunction of laminar veins. Further studies are required to discern the precise nature of this dysfunction and its potential relevance to the pathogenesis of acute laminitis in the horse and possible therapeutic targets for treatment.     

Effect of histamine on lung contractile elements in growing cattle

OBJECTIVE: To determine the effect of histamine on the contractile elements of the respiratory tract in neonatal calves and young adult cattle. SAMPLE POPULATION: Samples of trachealis muscle, bronchi, and intrapulmonary arteries and veins dissected from the respiratory tracts of healthy bovids (2 to 8 days and 16 to 20 months old). PROCEDURE: Histamine cumulative concentration-effect curves (10(-6) to 10(-3) M) were constructed in duplicate smooth muscle samples mounted in organ baths. Contractile responses to histamine were compared with reference contractions elicited by methacholine (10(-5) M) for airways or KCl (127 mM) for vessels. RESULTS: In young adult cattle, trachealis muscle had a substantial contractile response to histamine (84% of methacholine-induced contraction), whereas bronchi reacted slightly (15 and 20% for large and small bronchi, respectively). Although contractile responses to KCl were comparable in arteries and veins, histamine-induced contractions were greater for intrapulmonary veins than for arteries (202 vs 48% of KCl-induced contraction). In neonatal calves, histamine-induced contraction of veins also exceeded that of arteries (230 vs 54% of KCl-induced contraction); however, unlike in young adult cattle, histamine produced notable contraction of large and small bronchi (48 and 60% of methacholine-induced contraction, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Compared with intrapulmonary arteries, intrapulmonary veins have greater contractile responses to histamine in neonatal and young adult cattle. Data suggest loss of histamine responsiveness in bronchial smooth muscle as neonatal calves grow to young adults. Venodilation may be useful in treatment of lung edema in cattle.     

Inflammatory mediators induce endothelium-dependent adherence of equine eosinophils to cultured endothelial cells

Accumulation of equine eosinophils at sites of parasite infestation or allergic inflammation depends upon their adherence to vascular endothelial cells and subsequent migration through the endothelium and extracellular matrix. This study has examined whether cytokines, which cause endothelial cell-dependent eosinophil adherence in other species, and histamine and substance P, which increase adherence of equine eosinophils to protein coated plastic, induce equine eosinophil adherence to cultured equine digital vein endothelial cell (EDVEC) monolayers. The EDVEC monolayers were stimulated with recombinant human (rh) interleukin (IL)-1beta, rhTNFalpha, substance P or histamine for different times and with a range of concentrations of mediators and the adherence of blood eosinophils from normal horses examined. All four mediators caused time- and concentration-dependent increases in adherence. However, neither the response to substance P, nor that to histamine, reached a maximum at the highest concentration tested (10-3 M: 10.6 +/- 2.6% and 4.5 +/- 0.6% adherent cells vs. background adherence of 1.9 +/- 0.4% and 1.1 +/- 0.2%; values for substance P and histamine, respectively, expressed as a percentage of total cells added initially; n=4). These data suggest that, as in other species, cytokines induce endothelial cell-dependent eosinophil adherence and mediators released during allergic inflammation may play a role in eosinophil recruitment by this mechanism.     

In vitro reactivity of digital arteries and veins to vasoconstrictive mediators in healthy horses and in horses with early laminitis

The in vitro reactivity of vasoconstrictive mediators that are implicated in acute laminitis was determined in palmar and plantar digital arteries and veins obtained from healthy horses and in palmar digital vessels of horses with early laminitis (Obel grade I). To obtain baseline reactivity data, 3 experiments were conducted, using healthy horses: (1) the reactivity of palmar and plantar digital arteries and veins to angiotensin II, norepinephrine, and 5-hydroxytryptamine (serotonin) were compared; (2) the direct effects of bacterial endotoxin on vascular reactivity were assessed; and (3) the reactivity of palmar digital arteries and veins to angiotensin II, norepinephrine, prostaglandin F2 alpha (PGF2 alpha), serotonin, and a thromboxane-endoperoxide analog (U46619) were determined. The vascular reactivity of these same 5 vasoconstrictors then was determined in horses with early laminitis and was compared with data from healthy (control) horses. Obel grade-I laminitis was experimentally induced in horses, using carbohydrate overload. Dose responses were conducted for each agent at concentrations between 10(-8)M and 10(-4)M. The potency of a drug was defined as the mean effective concentration necessary to induce 50% of maximal contraction (EC50). There were no differences in EC50 concentrations and in maximal contractions between forelimb and hind limb arteries and veins for angiotensin II, norepinephrine, and serotonin. Incubation with endotoxin had no effect on the reactivity of arteries and veins to angiotensin II, norepinephrine, and serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of activation of protein kinase C during agonist-induced constriction of veins isolated from the laminar dermis of horses

OBJECTIVE: To determine the effects of the protein kinase C (PKC) inhibitor, Ro-31-8220, on agonist-induced constriction of laminar arteries and veins obtained from horses. SAMPLE POPULATION: Laminar arteries and veins obtained from 8 adult mixed-breed horses. PROCEDURES: Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were then obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine, prostaglandin F(2), and endothelin-1. All responses were measured with or without the addition of Ro-31-8220 (3 microM). RESULTS: Laminar veins were more sensitive to vasoconstrictor agonists than laminar arteries, and incubation of laminar veins with Ro-31-8220 resulted in significantly smaller agonist-induced contractile responses for all agonists tested. In contrast, Ro-31-8220 had no effect on agonist-induced contractile responses of laminar arteries. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the study were consistent with activation of PKC being confined to agonist-induced contraction of laminar veins isolated from the laminar dermis of horses. Consequently, the possible involvement of PKC in the venoconstriction observed during the development of laminitis is worthy of further investigation.     

Response of equine airway smooth muscle to acetylcholine and electrical stimulation in vitro

Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contractile response to electrical stimulation was obtained at 18 V, 25 Hz, and 0.5 ms. Atropine (10(-6)M) or tetrodotoxin (3 x 10(-6)M) blocked the contraction, indicating that the contractile response was attributable to the release of neurotransmitter from cholinergic nerves. Cumulative concentration-response curves to acetylcholine (10(-9)M through 10(-4)M) were determined. Isometric contractile force increased as acetylcholine concentration increased. There was a significant (P less than 0.05) difference in the 50% effective dose for acetylcholine in tracheal smooth muscle and bronchial smooth muscle. The mean (+/- SD) contractile response to maximal electrical stimulus was 89% (+/- 7.4%) of that in response to 10(-4)M acetylcholine in tracheal smooth muscle and was 68% (+/- 10.4%) of the response to 10(-4)M acetylcholine in bronchial smooth muscle.     

Effects of xylazine on canine coronary artery vascular rings

OBJECTIVE: To determine the effects of xylazine on canine coronary artery smooth muscle tone. SAMPLE POPULATION: Hearts of 26 healthy dogs. PROCEDURE: Dogs were anesthetized with pentobarbital, and vascular rings of various diameters were prepared from the epicardial coronary arteries. Vascular rings were placed in tissue baths to which xylazine was added (cumulative concentrations ranging from 10(-10) to 10(-4) M), and changes in vascular ring tension were continuously recorded. Effects of the nitric oxide inhibitor NG-nitro-L-arginine methyl ester (L NAME; 5 mM), the alpha1-adrenoceptor antagonist prazosin (10 mM), and the alpha2-adrenoceptor antagonist atipamezole (10 mM) on xylazine-induced changes in vascular ring tension were determined. Results were expressed as percentage of maximal contraction for each vascular ring preparation. RESULTS: Xylazine induced vasoconstriction of small (< 500-microm-diameter) and medium (500- to 1,000-microm-diameter) vascular rings but not of large (> 1,000-microm-diameter) rings. For large vascular rings, L-NAME, atipamezole, and prazosin did not significantly affect the contractile response to xylazine. For small vascular rings, the contractile response following addition of xylazine to rings treated with L-NAME was not significantly different from the contractile response following addition of xylazine to control rings, except at a xylazine concentration of 10(-6) M. Xylazine-induced vasoconstriction of small vascular rings was blocked by atipamezole, but the addition of prazosin had no effect on xylazine-induced vasoconstriction. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that xylazine increases smooth muscle tone of small canine coronary arteriesand that this effect is predominantly mediated by stimulation of alpha2adrenoceptors.     

Correlation of clinical score, intrapleural pressure, cytologic findings of bronchoalveolar fluid, and histopathologic lesions of pulmonary tissue in horses with summer pasture-associated obstructive pulmonary disease

OBJECTIVE: To correlate clinical score, intrapleural pressure, cytologic findings of bronchoalveolar lavage fluid (BALF), and histologic lesions of pulmonary tissue in horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 8 adult horses affected with SPAOPD and 6 adult horses without evidence of respiratory tract disease. PROCEDURE: Clinical score, change in intrapleural pressure (deltaPpl) during tidal breathing, results of cytologic examination and bacteriologic culture of BALF, and results of histologic examination of pulmonary parenchyma were evaluated. RESULTS: Clinical scores for SPAOPD-affected horses (median, 5.75; range, 4.0 to 7.5) were significantly greater, compared with clinically normal horses (median, 2.0; range, 2.0 to 3.0). Cytologic examination of BALF from SPAOPD-affected horses revealed predominantly nondegenerate neutrophils. Histologic lesions were identified throughout pulmonary tissue and included severe accumulation of mucus and neutrophils within the small airways, metaplasia of bronchiolar goblet cells, and mild peribronchial infiltrate. Histologic examination of specimens collected via percutaneous biopsy was predictive of disease and corresponded to findings at postmortem examination. Clinical score and deltaPpl were highly correlated with mucus accumulation in the airways of affected horses. Peribronchial inflammatory infiltrate correlated with percentage of neutrophils in BALF of affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical scoring and deltaPpl provided valid estimates of disease severity. Findings from cytologic examination of BALF of SPAOPD-affected horses varied, although, in most instances, it was diagnostically useful. Severe mucus accumulation in the airways was the most remarkable histopathologic finding in SPAOPD-affected horses. Examination of biopsy specimens collected from pulmonary parenchyma was consistently useful in diagnosing SPAOPD.     

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.     

A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity

Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the reaction to Culicoides allergens that are different or absent in allergic horses.     

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.