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R. R. Krishnamoorthy, V. Rao, R. Dauphin, G. Prasanna, C. Johnson, T. Yorio; Endothelin-1 Upregulates Endothelin B (ETB) Receptors to Promote Apoptosis of Rat Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4175.
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Endothelin-1 (ET-1) administration has been shown to produce optic nerve axonal loss and apoptosis of retinal ganglion cells, similar to that seen in glaucoma. However, the receptors through which ET-1 mediates these effects are not clearly known. The purpose of this study was to determine if ETB receptor activation contributes to cell death of retinal ganglion cells both in culture and in vivo in rats.
Wild-type and ETB-deficient rats were intravitreally injected with 2 nmole ET-1, sacrificed 48 hr post-injection and retina sections analyzed for apoptotic changes by TUNEL. In a separate set of experiments, ETB expression was analyzed by immunohistochemistry, in retinas of wild-type rats injected with ET-1. To study some of the signaling mechanisms contributing to ET-1 mediated cell death, virally transformed rat retinal ganglion cells (RGC-5 cells) were treated with 100 nM ET-1 for 24 hr and analyzed for phosphorylation of c-Jun N-terminal kinase (p-JNK). Additional tests to assess apoptotic changes were carried out by analyzing cytochrome c release into cytosol in RGC-5 cells treated with ET-1 for 24 hr. Apoptotic changes were also assessed by flow cytometry in RGC-5 cells treated with ET-1 in the presence or absence of endothelin receptor antagonists.
Intravitreal ET-1 treatment produced an appreciable increase in apoptotic cell death of retinal ganglion cells in wild-type rats. The ET-1 mediated increase in retinal ganglion cell death was attenuated in ETB-deficient rats. ET-1 mediated retinal ganglion cell death in wild-type rats was accompanied by increased expression of ETB receptors, particularly in the retinal ganglion cells. Measurements by flow cytometry showed that ET-1 mediated apoptosis of RGC-5 cells in culture, which was also blocked by pretreatment with the ETB receptor antagonist, BQ788. ET-1 (100 nM) treatment of RGC-5 cells for 24 hr produced an increase in phosphorylation of c-JNK and also promoted cytochrome c release into the cytosol, indicative of apoptotic changes mediated possibly through mitochondrial pathways.
Elevations in ocular endothelin concentrations (as seen in primary open angle glaucoma) produce increased ETB receptor expression, and contribute to apoptosis of retinal ganglion cells. These studies suggest the possibility of developing endothelin receptor antagonists as potential neuroprotective agents in attenuating retinal ganglion cell death seen in glaucoma.
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