Induction of Cellular Toxicity in Cultured Porcine Corneal Keratocytes by Endothelin-1

Abstract

The effect of endothelin-1 (ET-1) on corneal cells is not well understood. We investigated the biochemical changes of cultured porcine corneal keratocytes under exposure to ET-1. The results indicate that ET-1 has remarkable effects to inhibit corneal keratocytes on ³H-thymidine, ³H-leucine, ³H-uridine uptakes and cellular migration. It is in a dose-dependent manner at concentrations ranging from 10^-7 M to 10^-9 M. The 50% inhibitory dose (ID₅₀) for ET-1, as measured by ³H-thymidine uptake, ³H-uridine uptake and ³H-leucine uptake, were 10^-7 M, 10^-0.52 M and 10^-11.8 M, respectively. The dead and living cells were estimated with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of ET-1, the cellular MTT values were also decreased. The ID₅₀ for ET-1 with cell migration assay and MTT assay were measured at 10^-7.86 M and 10^-5.1 M. Endothelin-1 (10^-6 M) promptly changed cellular morphology and attenuated adhesion observed with laser scanning cytometer. Endothelin-1-induced characteristic apoptosis cells were observed using a TUNEL assay that detected fragmented DNA of apoptosis. Western blot assay revealed that endothelin-1 induced proteolysis and decreased in fibronectin protein. These findings indicate that endothelin-1 may lead keratocytes to death resulting from induction of apoptosis and functional loss.