Endothelin-1 (ET-1) causes death of retinal neurons through activation of nitric oxide synthase (NOS) and production of superoxide anion

Endothelin-1 (ET-1) is the most potent and long-acting vasoconstricting peptide presently known. In addition to its vascular effects, endothelin signaling pathway exists in the central nervous system (CNS), which is deeply related to neuronal degeneration. In the present study, we evaluated the effect of ET-1 on death of retinal neurons consisting mainly of amacrine cells, and its interaction with nitric oxide synthase (NOS) and superoxide production. 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 33,258 staining and TUNEL assay at different times. Intracellular levels of nitric oxide (NO), superoxide and peroxynitrite were determined semiquantitatively by DAF2-DA, hydroethidine and dihydrorhodamine-123, respectively. The effects of adding SOD (100 U/ml) and L-NAME with ET-1 on these changes were evaluated. In addition, the receptor mechanisms involved in these reactions were determined by BQ-123 and BQ-788, receptor antagonists for ETA and ETB receptors, respectively. Exposure of cultured retinal neurons to ET-1 reduced the percentage of living cells in a dose- and time-dependent way, and the percentage of living cells was significantly increased by addition of SOD and L-NAME. Fluorometric analyses revealed that ET-1 increased the intracellular NO level in a dose- and time-dependent manner. The intracellular superoxide and peroxynitrite levels were also significantly increased 24 h after incubation with 100 nM of ET-1, and this elevation was suppressed by SOD and L-NAME. These ET-1-induced alterations were significantly suppressed when both BQ-123 and BQ-788 were added simultaneously with ET-1 to the medium. These results indicate that the neuronal death caused by ET-1 is most likely mediated by the activation of NOS in association with the formation of superoxides and peroxynitrite.