Induction of cellular toxicity in cultured porcine corneal keratocytes by endothelin-1.
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.
Wu
K Y
KY
Department of Ophthalmology, Kaohsiung Medical University, Taiwan, Republic of China. hong6657@ms5.hinet.net
Hong
S J
SJ
Wang
H Z
HZ
Lin
C P
CP
Lai
Y H
YH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects*; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects; D005353:Fibronectins / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D013552:Swine
D019332:Endothelin-1; D005353:Fibronectins; D004247:DNA