Progress 01/01/03 to 12/31/03
Outputs
This project is now under a "no cost extension" for an additional year. This study has 3 parts; pharmacological, immunochemistry and molecular. The Pharmacological study and its analysis have been completed. The imminohistochemistry study is completed but the analysis is in progress. In the molecular study, mRNA determination is completed, western blot is being done and analysis of results will be done within a couple of months. The Pharmacological studies indicated that both endothelin (ET) A and B receptors mediate contraction in equine airways. ETA receptors are more involved in physiological tone of airways and ETB receptors are up-regulated in hyperactive airway diseases. Significant increase in ETB up-regulation was noted in horses with summer pasture-associated obstructive pulmonary disease.
Impacts
The study indicated that ETB receptors are primarily involved in the hyperresponsiveness of airways in horses with hyperactive airway disease. Use of selective antagonists of ETB receptors may be a therepauetic approach to control the severity of the disease. We further intend to elucidate the signal transduction of ETB up-regulation, which will enhance the chances to control and prevent hyperactive airway disease.
Publications
- Polikepahad S, Moore RM, Vanden Heuvel J, Venugopal CS. Pharmcological characterization of endothelin receptors of bronchi in clinically healthy horses and those affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). FASEB J., 17(5): A1047 (653.7), 2003.
- Venugopal CS, Polikepahad S, Holmes EP, Leas TL, Vanden Heuvel, J & Moore, RM. 2004. Pharmacological and molecular evidence for endothelin-B receptor involvement in horses affected with summer pasture-associated obstructive pulmonary disease.(The final version of the manuscript is being circulated among the authors prior to submission to Am. J. Vet. Res).
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Progress 09/15/01 to 09/30/03
Outputs
The intent of the study was to characterize the endothelin (ET) receptors and to determine the role of those receptors (ETA and ETB) in the pathogenesis of equine summer pasture-associated obstructive pulmonary disease (SPAOPD). The concept of this study originated from the observation that response of bronchial rings collected from SPAOPD-affected horses to graded concentration of ET was greater than those of the healthy horses. The study began by classifying the horses into two groups (clincally healthy and SPAOPD) by their history, pulmonary function testing, clinical scoring, cytological examination of blood and broncho-alveolar lavage (BAL) fluid. The investigation consisted of three methological approaches namlely pharmacological studies, immunohistochemical staining and molecular studies. The pharmacological studies demonstrated that ET is a potent bronchoconstrictor agent in horses and caused greater contraction in bronchial rings from SPAOPD horses. The pA2
value determination studies showed that ETA receptors did not get altered significantly in SPAOPD, whereas ETB receptors showed a significant increase pA2 values suggestive of its alteration in affinity to ET. Immunohistochemical studies showed that ETA and ETB receptors are present in bronchial epithelium as well as in bronchial smooth muscles. However, bronchial smooth muscles showed a significant overexpression of ETB receptors in SPAOPD-affected horses. ETA receptors, on the other hand, showed only a trend in SPAOPD animals compared to healthy ones. The molecular studies showed that ETB receptor mRNA expression was singnificantly increased in SPAOPD animals. Westernblot analysis showed that ETB receptors protein was increased in SPAOPD animals. In summary the study demonstrated that ETB receptors are upregulated in SPAOPD-affected animals. It suggests that ETB receptor antagonists could have therapeutic potential for better management of SPAOPD affected horses.
Impacts
The study indicated that ETB receptors are primarily involved in the hyperresponsiveness of airways in horses with hyperactive airway disease. Use of selective antagonists of ETB receptors may be a therepauetic approach to control the severity of the disease. We further intend to elucidate the signal transduction of ETB up-regulation, which will enhance the chances to control and prevent hyperactive airway disease.
Publications
- Polikepahad, S., Moore, R.M., Krishnakumar, S., Van Den Heuvel, J. and Venugopal, C.S. Pharmacological characterization of endothelin receptor responses of bronchi in clinically healthy horses and those affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). FASEB J., 17(5): A1047 (653.7), 2003.
- Venugopal, C.S, Polikepahad, S., Holmes, E.P., Vanden Heuvel, J., Leas, T., Moore, R.M. Evidence for involvement of endothelin-B receptors in equine summer pasture-associated obstructive pulmonary disease (SPAOPD). FASEB J., 18(4): A 234 (172.3), 2004.
- Polikepahad, S., Moore, R.M., Koch, C.E., Holmes, E.P., Venugopal, C.S. Effects of epithelial denudation on endothelin-1 induced bronchoconstriction in clinically healthy and summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses. FASEB J. 18 (4): A 234 (172.1), 2004.
- Polikepahad, S., Paulsen, D., Moore, R.M., Costa, L., Venugopal, C.S. Immunohistochemical studies on expression of endothelin A and B receptors in the lungs of clinically healthy and SPAOPD-affected horses. FASEB J., 18(4): A 234 (172.2), 2004.
- Venugopal CS, Polikepahad S, Holmes EP, Vanden Heuvel J, Leas TL, Moore RM. Evidence for endothelin receptor involvement in the airway hyperreactivity in equine obstructive pulmonary disease. (Submitted to the Canadian Journal of Veterinary Research, 2005).
- Polikepahad, S., Moore, R.M., Venugopal, C.S. Endothelins and Airways. (Submitted to the Res.Commun.Molec.Pathol.Pharmacol., 2005).
- Polikepahad, S., Paulsen, D.B., Moore, R.M., Costa, L.R.R., Venugopal, C.S. Expression of endothelin receptors in the bronchi of clinically healthy and summer pasture-associated obstructive pulmonary disease (SPAOPD)-affected horses. (Submitted to AJVR, 2005).
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Progress 01/01/02 to 12/31/02
Outputs
Airway hyperresponsiveness is the most important characteristic in the pathogenesis of summer pasture associated obstructive pulmonary disease (SPAOPD, a form of COPD) in horses. Endothelin-1 (ET-1), an endothelium derived mediator, is a potent constrictor of human and animal airways. ET-1 causes the constriction of airways by acting through ETA and ETB receptors. In this study, we grouped the horses into clinically healthy and SPAOPD affected based on the pulmonary function testing and clinical scores. Bronchial rings were prepared from diaphragmatic lobe of lung collected from horses after euthanasia for in vitro studies. We used eight bronchial rings from each horse for pA2 value studies. Of the eight rings, two were used as controls. Three of the remaining six rings were incubated for 45 minutes with three different concentrations (0.001,0.1,10 uM) of ETA antagonist (BQ 123) and the other three with the same concentrations of ETB antagonist(IRL 1038). After
equilibration cumulative concentration-response relationship of all rings were determined to graded concentrations of ET-1 (0.01 to 1 uM). The results showed that ET-1 caused concentration dependent contractile responses of all rings. Comparison of control responses of the groups showed that the rings of SPAOPD affected horses contracted significantly greater than those of the healthy horses. We further examined the cause for the change in responsiveness. Since the concentrations used were the same in both groups, we hypothesized that there could be an alteration of ET receptors. Using the responses after the specific antagonists of the receptors, we determined the pA2 values of the antagonists by Schild plot. The pA2 value is a statistical constant, which indicates receptor alterations, particularly the changes in pharmacological affinity of receptors. Comparison of these pA2 values of the two groups indicated that the ETB receptors, which play no role in bronchoconstriction in
normal horses, become up-regulated contributing to the enhanced bronchoconstriction in SPAOPD horses. The affinity of ETA receptors, on the other hand, apparently remained unchanged. These findings open a new avenue for searching selective ETB receptor antagonists for the therapeutic approach of SPAOPD in horses. The progress we made is on schedule. We already completed 17 horses. We presented our findings as a poster presentation at Phi Zeta reserch emphesis day of the university. Another abstract submitted to FASEB is accepted for presentation in the Experimental Biology conference in April 2003 in San Diego. The results of the molecular studies of the samples of lung tissues we sent to Dr. Vanden Heuvel, Penn State university, will be available soon. The immunochemistry studies of the samples colleceted are targeted to be completed by the end of January, 2003. All the needed materials for immunostudies like paraffin blocks of tissues, polyclonal antibodies against ET receptors and
other reagents are ready. We also collected plasma from each animal for detection of ET-1 levels to determine the changes due to disease. The plasma studies will be completed in February, 2003.
Impacts
Endothelin (ET-1), an inflammatory mediator of airways, causes constriction of airways in humans and horses. ET-1 produces this effect by acting through its receptors. Two distinct receptors, ETA and ETB receptors, have been identified. In SPAOPD affected horses (a type of COPD), the contractile responsiveness of airways to ET-1 is significantly increased when compared to clinically healthy horses. When we investigated the reason for this increased responsiveness in SPAOPD horses, we found that ETB receptors are responsible for the hyperresponsiveness. In clinically healthy horses, the contraction is only through ETA receptors and not through ETB receptors. In SPAOPD horses the responsiveness of ETA receptors remaind the same whereas, ETB were significantly enhanced. This means that ETB receptors which were "silent receptors" in clincally healthy horses, are up-regulated in airways of SPAOPD affected horses. These findings indicate that blocking of both ETA and ETB
receptors should be considered as the target therapeutic approach in the treatment of this disease.
Publications
- Polikepahad S, Moore RM, Krishnakumar S, Heuvel JV and Venugopal C. Pharmacological characterization of enodothelin receptor responses of bronchi in clinically healthy horses and those affected with summer pasture associated obstructive pulmonary disease. FASEB J., 17: 2003.
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Progress 01/01/01 to 12/31/01
Outputs
Airway hyperresponsiveness and bronchoepithelial cell mucus hypersecretion are the most important characteristics of SPAOPD, a devastating respiratory disease of horses in the subtropical regions of the United States. It is seen during the late summer and early fall and is particularly associated with grazing in lush green grass. Similar to COPD it also causes edema and increased airway resistance to airflow in and out of lungs. Currently, endothelin (ET-1)is considered as an important chemical mediator involved in SPAOPD. Endothelin, a 21 amino acid peptide, is derived from endothelium which causes potent and profound bronchoconstriciton, pulmonary inflammation and increased mucus secretion. In humans, increased concentration of ET-1 is seen in bronchioalveolar lavage fluid. However, a higher level of circulating ET-1 is not always observed in hyperreactive airway diseases. This finding pauses a question that ET-1 levels need not be increased to produce a hyperactive
airway and it could be due to an alteration in the make up of the receptors. There are two types of receptors such as ET-A and ET-B receptors for ET-1. We were able to test the contracting ability of ET-1 on bronchial rings. ET-1 produced a dose-dependent contraction. The responsiveness of the rings from SPAOPD affected horses were greater than those of the clinically healthy horses suggesting an alteration of receptors occurs in disease. Using ETA and ETB receptor antagonists we observed that both ETA and ETB receptor stimulation caused contraction in equine bronchial rings.
Impacts
The study when completed will indicate an alteration of receptor is an important aspect of SPAOPD. It will also indicate either ETA or ETB receptor will have greter alteration to cause the symptoms. If that is proven, appropriate receptor antagonist will have a potential therapeutic use to control the intensity of the disease.
Publications
- No publications reported this period
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