Endothelin-1 (ET-1) Causes Death of Retinal Neurons Through Activation of Nitric Oxide Synthase (NOS) and Production of Superoxide Anion

Abstract

Purpose:: To determine whether ET-1 causes death of retinal neurons consisting mainly of amacrie cells, and its interaction with nitric oxide synthase (NOS) and superoxide production.

Methods:: Cultured retinal neurons from fetal rats were exposed to various doses of ET-1 (0.1, 1.0, 10 and 100 nM). Neuronal toxicity of ET-1 was assessed by trypan blue exclusion, Hoechst 33258 staining and TUNEL assay at different times after the exposure. Intracellular levels of nitric oxide (NO), superoxide and peroxynitrite were semiquantitatively determined by confocal microscope using specific fluorogenic compounds of DAF2-DA, hydroethidine and dihydrorhodamine-123, respectively. Effects of adding SOD (100U/ml) and L-NAME on these changes were evaluated. In addition, the receptor mechanism was elucidated using BQ-123 and BQ-788 for ET_A and ET_B receptor antagonists respectively.

Results:: ET-1 caused death of retinal neurons in a dose and time dependent way. Cell death caused by ET-1 (100 nM) was significantly suppressed by SOD and L-NAME. Fluorometric analyses revealed ET-1 caused intracellular NO formation in a time and dose dependent manner. ET-1 also enhanced intracellular superoxide and peroxynitrite formation 24 hours after the ET-1 (100 nM) exposure, which was suppressed by SOD and L-NAME. These ET-1-induced alterations were significantly suppressed when both BQ123 and BQ-788 were simultaneously added.

Conclusions:: These results indicate that neuronal death caused by ET-1 is possibly mediated by activation of NOS in association with superoxide and peroxynitrite formation.

Keywords: nitric oxide • retinal culture • retina: proximal (bipolar, amacrine, and ganglion cells)