Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jul;72(4):340-9.

Characterization of endothelin receptors in the peripheral lung tissues of horses unaffected and affected with recurrent airway obstruction

Sumanth Polikepahad  1 , Masudul Haque, Joseph Francis, Rustin M Moore, Changaram S Venugopal
Affiliations

Characterization of endothelin receptors in the peripheral lung tissues of horses unaffected and affected with recurrent airway obstruction

Sumanth Polikepahad et al. Can J Vet Res. 2008 Jul.

Abstract
in English, French

The purpose of the study was to determine and compare the expression of endothelin (ET) receptors in the peripheral lungs of healthy horses and those affected with recurrent airway obstruction (RAO) using reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, Western blot analysis, and immunohistochemical techniques. Two groups of horses (7 healthy and 7 RAO-affected) were selected from a pool of horses destined for euthanasia. The grouping of horses was based on the history, clinical scoring, and pulmonary function testing. After euthanasia, gross postmortem evaluation of the lungs was conducted, and lung samples were collected and either stored at -80 degrees C or fixed in zinc-formalin for 12 h. The RT-PCR was performed by using specific primers for ETA and ETB receptors, and beta-actin. To determine the relative gene expression real-time PCR was performed. To detect ET receptor protein expression, Western blotting and immunohistochemical studies were performed using polyclonal antibodies against ETA and ETB receptors and beta-actin. The ET receptor expression was determined by performing either densitometric analyses or scoring of immunostaining. Statistical analyses were performed to detect differences in receptor expression within and between the 2 groups. The results indicated that ET receptor expression, particularly ETB receptors, was significantly greater in the peripheral lungs of RAO-affected horses than in those of healthy horses. Clinical trials using ET receptor antagonists, particularly ETB antagonists might help in developing a therapeutic strategy to treat this career-ending disease.

L’objectif de la présente étude était de déterminer et comparer l’expression des récepteurs de l’endothéline (ET) dans les poumons périphériques de chevaux en santé et de chevaux souffrant d’obstruction récurrente des voies respiratoires (RAO) par réaction d’amplification en chaîne par la polymérase à l’aide de la transcriptase réverse (RT-PCR), par PCR en temps réel, par réaction d’immunobuvardage et par techniques immunohistochimiques. Deux groupes de chevaux (7 en santé et 7 atteints de RAO) ont été sélectionnés à partir d’un regroupement de chevaux destinés à l’euthanasie. Le groupement des chevaux s’est fait sur la base de l’anamnèse, le pointage clinique, et un test de fonction respiratoire. Après l’euthanasie, une évaluation des lésions macroscopiques des poumons a été faite, et des échantillons de poumons prélevés et soit entreposés à −80 °C ou fixé pendant 12 h dans une solution de zinc-formaline. L’épreuve de RT-PCR a été effectuée en utilisant des amorces spécifiques pour les récepteurs ETA et ETB, et β-actine. Afin de déterminer l’expression génique relative une épreuve de PCR en temps réel a été effectuée. Afin de déterminer l’expression des récepteurs ET, les épreuves d’immunobuvardage et immunohistochimiques ont été effectuées en utilisant des anticorps polyclonaux contre les récepteurs d’ETA et d’ETB ainsi que ceux pour β-actine. L’expression de récepteur ET a été déterminée en réalisant des analyses densitométriques ou en attribuant un pointage à l’immunocoloration. Les analyses statistiques ont été faites afin de détecter des différences dans l’expression des récepteurs à l’intérieur et entre les 2 groupes. Les résultats indiquent que l’expression des récepteurs ET, en particulier les récepteurs ETB, était significativement plus grande dans les poumons périphériques des chevaux atteints de RAO que ceux des chevaux en santé. Des essais cliniques utilisant des antagonistes des récepteurs ET, plus particulièrement les antagonistes ETB, pourraient aider au développement d’une stratégie pour traiter cette maladie qui met fin à une carrière.

(Traduit par Docteur Serge Messier)

PubMed Disclaimer

Figures

Figure 1
Figure 1
A — RT-PCR bands of ETA and ETB receptors and β-actin in healthy (n = 7) and RAO-affected horses (n = 7). The mean sizes of the RT-PCR bands for β-actin, and ETA and ETB receptors were 160, 190, and 220 base pairs, respectively. B — Mean ± standard error (Sx̄) for the mean intensity of the ETA and ETB receptor bands per mean intensity of β-actin bands in healthy and RAO-affected horses. * — statistically significant difference (P ≤ 0.05) in the mean intensities of ETB receptors between healthy and RAO-affected horses. † — statistically significant difference (P ≤ 0.05) between the mean intensities of ETA and ETB receptors in RAO-affected horses.
Figure 2
Figure 2
Real-time PCR showing the mean (±Sx̄) of relative ETA and ETB receptor gene expressions of the lungs of healthy and RAO-affected horses. * — statistically significant difference (P ≤ 0.05) and relative gene expression of ETB receptors between healthy and RAO-affected horses. † — a difference in the relative gene expression of ETA receptors between healthy and RAO-affected horses.
Figure 3
Figure 3
A — Western Blot bands of ETA and ETB receptors and β-actin in healthy and RAO-affected horses. The mean sizes of western blot bands for β-actin and ET-receptors were 40 and 60 kDa, respectively. B — Mean ± for the intensity of the ETA or ETB receptor bands per mean Sx̄ intensity of β-actin bands in healthy and RAO-affected horses. * — a statistically significant difference (P ≤ 0.05) in the intensities of ETB receptors between healthy and RAO-affected horses. † — a difference between ETA and ETB receptors in RAO-affected horses. ‡ — a difference in intensities of ETA receptors between healthy and RAO-affected horses.
Figure 4
Figure 4
Immunostaining of equine lung tissue (Bar = 500 μm). A — Immunostaining of equine lung tissue from an RAO-affected horse where the primary antibody was replaced with sheep gamma globulin as a negative control. B — Immunostaining of equine lung tissue from the same tissue sample as used for the negative control, but in which anti-ETB receptor antibody was applied. C — Immunostaining of ETB receptors in the luminal side of bronchial epithelium of the lung from an RAO-affected horse.
Figure 4D
Figure 4D
Mean (± Sx̄) of ETA and ETB receptor immunostaining in the lungs of healthy and RAO-affected horses. * — statistically significant differences (P ≤ 0.05) in the intensities of ETB receptors between healthy and RAO-affected horses. † — difference between the intensities of ETA and ETB receptors in RAO-affected horses. ‡ — difference in the intensities of ETA receptors between healthy and RAO-affected horses.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Robinson NE, Derksen FJ, Olszewski MA, Buechner-Maxwell VA. The pathogenesis of chronic obstructive pulmonary disease of horses. Br Vet J. 1996;152:283–306. - PubMed
    1. Seahorn TL, Beadle RE. Summer pasture-associated obstructive pulmonary disease. Equine pract. 1994;16:39–41. - PubMed
    1. Rubanyi GM, Polokoff MA. Endothelins: Molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev. 1994;46:325–415. - PubMed
    1. de Nucci G, Thomas R, D’Orleans-Juste P, et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci U S A. 1988;85:9797–9800. - PMC - PubMed
    1. Tsukahara H, Ende H, Magazine HI, Bahou WF, Goligorsky MS. Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase. J Biol Chem. 1994;269:21778–21785. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources