Purchase this article with an account.
Shay-Whey M. Koh; Ciliary Neurotrophic Factor Released by Corneal Endothelium Surviving Oxidative Stress Ex Vivo. Invest. Ophthalmol. Vis. Sci. 2002;43(9):2887-2896.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
purpose. To demonstrate that corneal endothelial (CE) cells that survive oxidative stress in corneas release ciliary neurotrophic factor (CNTF), which does not possess the secretion signal sequence.
methods. CNTF and CNTF receptor α subunit (CNTFRα) in CE cells and cell-conditioned medium (H2O2 and PBS placed in bovine corneal cups for 30 minutes at 37°C) in explant cultures were demonstrated by Western blot (WB) and immunoprecipitation (IP). The number of dead CE cells was determined microscopically with a viability kit, by an observer uninformed of the explants’ identities. CNTF and CNTFRα synthesis and release by CE cells in 35S-methionine–labeled (0.1 mCi/mL for 8 hours at 37°C) corneal cups were shown by autoradiography and WB.
results. CE cells in fresh bovine eyes expressed a 25-kDa CNTF that was recognized by three different antibodies. CE cells expressed a 61-kDa CNTF-immunoreactive molecule (IM), which disappeared from the CE cells in H2O2-conditioned corneal cups, concomitant with the appearance of the 25-kDa CNTF in the conditioned medium. Corneal cups containing 0, 0.006, 0.012, 0.023, 0.045, 0.09, 0.18, and 0.35 mM H2O2 demonstrated relative levels of CE cell 61-kDa CNTF-IM of 100%, 84%, 77%, 61%, 52%, 39%, 35%, and 35%, respectively, whereas levels of 25-kDa CNTF in the conditioned medium were 23%, 32%, 39%, 63%, 80%, 90%, 100%, and 63%, respectively. CE cells expressed a 53-kDa CNTFRα that, along with trace amounts of a 61-kDa CNTFRα-IM, appeared concomitantly with the 25-kDa CNTF in the conditioned medium. H2O2 (0–0.56 mM) did not affect the viability of CE cells (15 dead cells per 600 cells). CE cells in 35S-methionine–labeled corneal cups synthesized and released a 35S 61-kDa molecule that was both CNTF- and CNTFRα-immunoreactive in an H2O2-dependent manner, whereas 25-kDa CNTF was detected in the 35S-methionine labeling medium.
conclusions. CE cells release autocrine CNTF under sublethal oxidative stress by a mechanism that involves CNTFRα and the formation of a 61-kDa CNTF/CNTFRα-IM.
This PDF is available to Subscribers Only