ETA receptor upregulation may be associated with ERK signaling in a rat model of glaucoma.

Purpose: The endothelin system has been shown to play a causative role in the neurodegenerative effects seen in animal models of glaucoma. However, the mechanisms leading to neurodegeneration need to be examined further. The goal of this study was to investigate the endothelin signaling pathway to determine the contribution of extracellular signal-regulated kinases 1 and 2 (ERK1/2) to endothelin-mediated cell death. Methods: Male retired breeder Brown Norway rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 and 4 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ETA receptor expression. In a separate set of experiments, western blots were performed on transformed 661W cells transiently transfected with either the ETA receptor or ETB receptor cDNA expression vector. Another set of experiments was performed with stable clones overexpressing the ETA receptor. The cells were grown on 100 mm dishes and treated for 24 hr with 100nM endothelin-1 (ET-1) or endothelin-3 (ET-3). Immunoblot analysis of levels of endothelin receptor and ERK1/2 phosphorylation was carried out. Results: An increase in immunostaining for ETA receptors was observed mainly in the inner plexiform layer and a modest increase was also observed in the RGC layer which was significant at 4 weeks of IOP elevation. Cell culture experiments showed an appreciable upregulation of ETB receptors following overexpression of ETA receptors and a reciprocal upregulation of ETA receptors following overexpression of ETB receptors. A 1.8-fold increase in ERK1/2 phosphorylation was observed in stable clones overexpressing ETA receptors, which was further elevated 2-3 fold after treating cells with either endothelin-1 or endothelin-3, compared to empty vector transfected cells. Conclusions: While the two endothelin receptors may have distinct functions, there is a significant overlap of the ETA and ETB receptor mediated signal transduction pathways and there appears to be some level of cross-talk between the two receptors. While there is a substantial body of evidence for the pro-survival role of ERKs, prolonged activation of ERK1/2 has been shown to be associated with cell death. While the mechanisms are not completely clear, the current study points to an association of ERK1/2 with cell death following overexpression of ETA and ETB receptors.