Endothelin receptors are targets for neuroprotection 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 underlying mechanisms are not completely understood. The goal of this study was to investigate the interaction between the ETA and ETB receptors and the involvement of the MAP kinase pathways in endothelin mediated cell death and determine if the dual ETA/ETB receptor antagonist, macitentan could attenuate neurodegenerative changes following IOP elevation in Brown Norway rats. Methods: Cultured transformed 661W cells were transiently transfected with a plasmid DNA encoding the ETA receptor and treated with ET-1 or ET-3 for 24 h. Phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was determined by immunoblotting. Wild type and ETB-deficient Wistar-Kyoto rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ERK1/2 and JNK and their phosphorylation. Brown Norway rats subjected to IOP elevation in one eye were either untreated or treated with macitentan (10 mg/kg body wt) for 1 month. Retinal flatmounts obtained from these rats were used to determine RGC counts following staining with the RBPMS antibody. Results: Cell culture experiments showed an appreciable upregulation of pERK1/2, while pJNK levels were not appreciably altered, following overexpression of the ETA receptor in 661W cells. ETB-deficient rats showed increased immunostaining for pERK1/2 in the nerve fiber layer (NFL), ganglion cell layer (GCL) and inner plexiform layer (IPL), compared to wild type rats. Following IOP elevation, ERK1/2 phosphorylation was greatly reduced in wild type rats, while ETB-deficient rats showed better preservation of pERK1/2 levels. Conversely, immunostaining for pJNK in wild type rats was increased in the NFL following IOP elevation, but was attenuated in ETB-deficient rats. Rats fed with macitentan displayed increased RGC survival by 25 to 42% following IOP elevation, compared to untreated rats. Conclusions: There is a substantial body of evidence for the pro-survival role of ERKs, and the pro-death role of JNKs. The current study points to an involvement of ERK1/2 and JNK signaling with endothelin receptor expression following IOP elevation. Blocking both ETA and ETB receptors has neuroprotective effects on RGCs during ocular hypertension.