The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5-1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50-80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1alpha. CoCl2 (100-200 microM), a chemical inducer of HIF-1alpha, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1alpha mediated expression and secretion of protective factors is involved.

Previous studies indicated that adenosine can increase [cAMP] i and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP] Western blot (WB) confirmed the presence of A 2b (∼50 kDa) and A 1 (∼40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP] i by 2·7-fold over control and this was inhibited 66% by 10 μM alloxazine, a specific A 2b blocker. A 1 activation with 1 μM N 6 -CPA (a specific A 1 agonist) or 100 nM adenosine decreased [cAMP] i by 23 and 6%, respectively. Adenosine had no effect on [Ca 2+ ] i mobilization. Indirect immunofluorescence localized A 2b receptors to the lateral membrane and A 1 to the apical surface in cultured BCE. Adenosine significantly increased apical Cl − permeability by 2·2 times and this effect was nearly abolished by DMPX (10 μM), a general A 2 blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A 1 and A 2b adenosine receptors. A 1 , preferentially activated at <1 μM adenosine, acts to decrease [cAMP] i and A 2b , activated at >1 μM adenosine, increase [cAMP] i .