Abstract
Purpose: :
Endothelin–1 (ET–1) administration has been shown to produce optic nerve axonal loss and apoptosis of retina ganglion cells, similar to that seen in glaucoma. However, the receptors mediating these deleterious effects and their signaling pathways are not identified. 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.
Methods: :
Brown Norway rats, intravitreally injected with 2 nmole ET–1, were sacrificed 48 h post–injection and retinas were isolated and processed for TUNEL assay to detect apoptosis. 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. 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. ETB receptor expression was studied in RGC–5 cells treated with different doses of ET–1 for 24 hr, by immunoblotting.
Results: :
Intravitreal ET–1 treatment produced an appreciable increase in apoptotic cell death of retinal ganglion cells, compared to vehicle–injected control eyes . The ET–1 mediated increase in retinal ganglion cell death was attenuated in ETB–deficient rats. ET–1 (100 nM) treatment 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. ET–1 treatment for 24 hr produced a dose–dependent increase in ETB receptor expression in RGC–5 cells
Conclusions: :
Elevations in ocular endothelin concentrations (as seen in primary open angle glaucoma) acting through ETB receptors, could 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.
Keywords: ganglion cells • apoptosis/cell death • retina