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  • Author or Editor: Changaram S. Venugopal x
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Abstract

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 concentrationdependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentrationdependent 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. (Am J Vet Res 2001;62:154–159)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate 3 neurokinin-2 (NK2) receptor antagonists on the basis of their ability to block neurokinin A (NKA)-induced contractile responses in various regions of the guinea pig respiratory tract.

Animals—48 clinically normal guinea pigs.

Procedure—After euthanasia, the trachea and lungs were removed en bloc. The spirally cut trachea was divided into lower, middle, and upper portions. The main bronchus was spirally cut. A lung strip was cut from the edge of the lung. Tissue strips were mounted in organ baths containing Tyrode solution at 37°C and attached to force transducers interfaced with a polygraph. Lung strips were set at a tension of 1 g; other tissue strips were set at 2 g. After 45 minutes of equilibration, cumulative concentration-response (CR) relationships to graded concentrations of NKA were determined. In the treatment groups, tissues were incubated (30 minutes) with antagonists (MEN 10376, SR 48968, and SR 144190) at 3 concentrations (10–9, 10–7, and 10–5M) before CR relationships were determined. Effectiveness of SR 48968 against NKA was also tested in vivo.

Results—Lung strips failed to contract, but all others responded in a concentration-dependent manner. Bronchial spirals were most sensitive. SR 48968 had the highest pA2 value and effectively blocked NKA.

Conclusions and Clinical Relevance—The bronchial region where airflow resistance is high was the most sensitive to NKA, suggesting the importance of NKA in bronchoconstriction. Nonpeptide antagonists (SR 48968 and SR 144190) were more potent than the peptide antagonist (MEN 10376), indicating their greater therapeutic potential as antiasthmatic agents. ( Am J Vet Res 2004;65:984–991)

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in American Journal of Veterinary Research

Abstract

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.

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in American Journal of Veterinary Research

Abstract

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 concentrationresponse relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10–9 to 10–6M) 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 10–5M. 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 10–5M. 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. (Am J Vet Res 2005;66:1202–1208)

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in American Journal of Veterinary Research

Abstract

Objective—To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists.

Sample Population—Uteruses from 10 nongravid mares in anestrus.

Procedure—Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10–9, 10–7, and 10–5M) of BQ-123, IRL- 1038, or BQ-123 and IRL-1038 before concentrationresponse curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10–9 to 10–6M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose.

Results—ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue.

Conclusions and Clinical Relevance—ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility. (Am J Vet Res 2005;66:1094–1100)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature.

Sample Population—Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease.

Procedures—Endothelium-intact and -denuded arterial and venous rings were precontracted with 10–7 and 1.8 × 10–8 M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10–4 M Nω-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine triphosphate (10–8 to 10–3 M) was added in a noncumulative manner, and relaxation percentage versus time curves were generated. Areas under the curves (ie, percentage of relaxation time) were calculated.

Results—Relaxation response of arterial and venous rings to ATP was dose-dependent. Percentage of relaxation time in response to 10–4 and 10–3 M ATP was significantly greater, compared with that for rings not treated with ATP. Removal of endothelium attenuated but did not eliminate the relaxation response. Addition of L-NAME did not attenuate the relaxation response in arteries. At higher concentrations, the vascular response to ATP was biphasic.

Conclusions and Clinical Relevance—ATP applied to equine colonic arterial and venous rings with and without intact endothelium induced a biphasic response characterized by transient contraction followed by slow, substantial, and sustained relaxation. This ATP-induced response is possibly mediated by a mechanism other than NO. Adenosine triphosphate may be a useful treatment to modulate colonic vasomotor tone in horses with strangulating volvulus of the ascending colon. (Am J Vet Res 2001;62:1928–1933)

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in American Journal of Veterinary Research

Abstract

Objective—To compare responses of bronchial rings obtained from healthy horses and horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) to selected mediators of airway hyperreactivity in vitro.

Sample Population—Bronchial rings from 6 healthy horses and 6 horses affected with SPAOPD.

Procedure—Bronchial rings obtained from each group of horses were mounted in organ baths and attached to force transducers interfaced with a polygraph. After applying 2g of tension, each ring was allowed to equilibrate for 45 minutes in Tyrode's solution at 37 C. Cumulative concentration-response relationships to graded concentrations of selected mediators (10–8 to 10–4 M ) were determined and analyzed for significance at each concentration.

Results—Acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 induced concentrationdependent contractile responses in bronchial rings. Prostaglandin F induced weak and inconsistent contractile responses. The other 2 agents, norepinephrine and substance P, did not induce concentrationdependent responses. Considering the overall groupdrug effect, acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 were effective in inducing consistent concentration-dependent contractile responses in both groups. Only 5-hydroxytryptamine and histamine induced significant responses in contractility between groups. The response of bronchial rings from horses with SPAOPD to 5-hydroxytryptamine was significantly greater than those from control horses, whereas the response to histamine was significantly lower. Significant responses were evident at concentrations ranging from 10–6 to 10–4 M for both drugs.

Conclusions and Clinical Relevance—Because the airways of horses with SPAOPD had increased responsiveness to 5-hydroxytryptamine in vitro, treatment modalities using 5-hydroxytryptamine antagonists should be investigated to address this phenomenon. (Am J Vet Res 2001;62:259–263).

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in American Journal of Veterinary Research

Abstract

Objective—To identify differentially expressed genes in pulmonary tissues of horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD), which is a form of recurrent airway obstruction (RAO), compared with those of unaffected horses.

Animals—6 horses with SPAOPD-RAO and 6 unaffected (healthy) horses.

Procedures—Horses were assigned to 2 groups on the basis of medical history, clinical score, and transpleural pressure. Total RNA from each of the 5 lung lobes of each of the 6 SPAOPD-RAO–affected horses was extracted and pooled. Similarly, total RNA from unaffected horses was pooled. Differential display (DD) PCR assay was performed, and differentially expressed bands were purified and cloned into a plasmid vector. Plasmids were extracted from recombinant colonies, and purified DNA was sequenced. Genes of interest for RAO pathogenesis were identified. Real-time PCR assay was performed to confirm findings for the DD PCR assay.

Results—18 differentially expressed genes (17 upregulated and 1 downregulated) were identified. Three genes of particular interest were found to be altered (2 upregulated and 1 downregulated) in horses with SPAOPD-RAO by use of real-time PCR assay, and these findings matched the differential expression found by use of the DD PCR assay.

Conclusions and Clinical Relevance—SPAOPD-RAO in horses is a multifactorial, complex disease involving several genes. Upregulated genes, particularly β2-microglobulin, and the downregulated secretoglobin gene can serve as marker genes that may help to identify SPAOPD-RAO at an early age.

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in American Journal of Veterinary Research

Abstract

Objective—To compare plasma endothelin (ET)- like immunoreactivity between healthy horses and those with naturally acquired gastrointestinal tract disorders.

Animals—29 healthy horses and 142 horses with gastrointestinal tract disorders.

Procedure—Blood samples were collected from healthy horses and from horses with gastrointestinal tract disorders prior to treatment. Magnitude and duration of abnormal clinical signs were recorded, and clinical variables were assessed via thorough physical examinations. Plasma concentrations of ET-like immunoreactivity were measured by use of a radioimmunoassay for human endothelin-1, and CBC and plasma biochemical analyses were performed.

Results—Plasma ET-like immunoreactivity concentration was significantly increased in horses with gastrointestinal tract disorders, compared with healthy horses. Median plasma concentration of ET-like immunoreactivity was 1.80 pg/ml (range, 1.09 to 3.2 pg/ml) in healthy horses. Plasma ET-like immunoreactivity was greatest in horses with strangulating largeintestinal obstruction (median, 10.02 pg/ml; range, 3.8 to 22.62 pg/ml), peritonitis (9.19 pg/ml; 7.89 to 25.83 pg/ml), and enterocolitis (8.89 pg/ml; 6.30 to 18.36 pg/ml). Concentration of ET-like immunoreactivity was significantly associated with survival, PCV, and duration of signs of pain. However, correlations for associations with PCV and duration of pain were low.

Conclusions and Clinical Relevance—Horses with gastrointestinal tract disorders have increased plasma concentrations of ET-like immunoreactivity, compared with healthy horses. The greatest values were detected in horses with large-intestinal strangulating obstructions, peritonitis, and enterocolitis. This suggests a potential involvement of ET in the pathogenesis of certain gastrointestinal tract disorders in horses. (Am J Vet Res 2002;63:454–458)

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in American Journal of Veterinary Research