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S. Li, K. Tan, J. A. Bonanno; Soluble Adenylyl Cyclase-Mediated Protection of Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2825.
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Cyclic AMP produced by transmembrane adenylyl cyclase (tmAC) plays a role in cell survival in many cell types. An additional increase of [cAMP] by soluble adenylyl cyclase (sAC) has been observed in primary cultured bovine corneal endothelial (BCE) cells. sAC is a cytosolic enzyme that can be activated by HCO3-. We hypothesize that elevated [cAMP] by sAC in response to increased [HCO3-] may contribute to protecting endothelial cells from apoptotic stressors. This study was designed to test whether the protective effect of bicarbonate on corneal endothelial cells is mediated by sAC.
Corneal endothelial cells isolated from fresh bovine eyes were primarily cultured and extended to passage 3. In treatment experiments, the cells were grown in either bicarbonate free (BF) medium and maintained in 0% CO2, or bicarbonate rich (BR) medium that contains 40 mM HCO3- and maintained in 5% CO2. Both the media are serum free. The cells were treated with either 0.1 µM staurosporine (SP), an apoptosis inducer, 5 µM 2-hydroxyestradiol (2HE), a sAC inhibitor, and 10 µM H89, a PKA inhibitor, or a combination of these reagents. A siRNA sequence specific to sAC was also transfected into the cells to knock down this enzyme. Induced cell apoptosis was assessed by flow cytometry with annexinV-FITC staining. The sAC gene knock down was examined by western blotting. Two intrinsic apoptotic signals, Cytochrome C release and Caspase3 activation, were also examined by western blotting and colorimetric assay respectively.
A small, but statistically insignificant increase in apoptosis was observed in BCE cells grown in BF medium comparing to BR. However, when treated with SP, apoptosis was 2-fold higher in BF. This finding suggests that HCO3- exerts a protective function when corneal endothelial cells undergo stress. Cellular [cAMP] was decreased ~40% by 2-HE. Apoptosis was increased 2-, 2.5-, and 3-fold by 2HE, H89, and sAC siRNA treatment alone. In SP treated cells, 2-HE or H89 abrogated all protection by BR. The results from examinations of Cytochrome C and Caspase3 provided further confirmation of sAC/HCO3--dependent protection. These results are consistent with the notion that apoptotic rates are inversely related to cytosolic [cAMP].
Our findings indicate that soluble adenylyl cyclase activity is HCO3- dependent, and bicarbonate has a modest anti-apoptotic effect on corneal endothelial cells. However when stress is applied to cells, bicarbonate-dependent protection via sAC is significant. Increasing cellular [cAMP] by sAC in response to high [HCO3-] provides an important protective mechanism in corneal endothelial cells.
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