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K. Tan-Allen, J. A. Bonanno; Protection of Corneal Endothelium by HCO3- Is Through cAMP Signaling Generated by Soluble Adenylate Cyclase (sAC). Invest. Ophthalmol. Vis. Sci. 2008;49(13):3957. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous in vitro studies have shown that HCO3- increases [cAMP]I in corneal endothelial (CE) cells and that HCO3- protects the endothelium from staurosporine toxicity. Here we show that cAMP is protective and that sAC activity is the source of the protection in HCO3- media.
Cultured bovine CE were incubated in either BR (40 mM HCO3-, pH 7.4, 25 mM HEPES), 5% CO2, 37°C or BF (0 mM HCO3-, pH 7.4, 25 mM HEPES), 37°C air equilibrated DMEM for 48 hours, then stressed with 50 nM staurosporine for 17 hrs. Cells were either lysed for Western blot assays or trypsinized, and labeled with Annexin V-FITC / PI and analyzed by flow cytometry. Flow cytometry of BF cells co-incubated with staurosporine (ST) and 50 µM rolipram, a phosphodiesterase 4 (PDE4) inhibitor which caused [cAMP]I elevation, was also included as a positive test for cAMP mediated protection. cAMP ELISA and flow cytometry of cells with sAC siRNA knockdown were also done to further evaluate the roles of cAMP and sAC in protection. Protein expression of pBcl2 was also performed.
Background Annexin-V positive (An+) labeling was 3.5% for cells incubated in BF and 3.0% in BR. Following ST stress An+ labeling was 9.9% in BF and 6.6% in BR (n=8, p<0.005). Following 48 hrs incubation in a scrambled sequence siRNA (siCon) there were 10% An+ cells in BF and 6% in BR indicating some toxicity. ST treatment of SiCon cells increased An+ labeling to 17.25% in BF and 10.25% in BR. sAC siRNA transfection alone produced 16.5% An+ cells in BF and 8% in BR, while ST induced 26.5% in BF and 26.3% in BR (n=5). BR medium raised [cAMP]I from 20 to 35 pmoles cAMP/mg protein in untreated cells. sAC siRNA transfection decreased BR [cAMP]I from 16.1+/-3.3 (n=2) in siControl treated cells to 8.3+/-2.12 (n=2) pmoles cAMP/mg protein in sAC siRNA treated cells. In BF, [cAMP]I dropped from 6.7+/-1.9 (n=2) in siControl treated cells to 5.85 +/-0.25 (n=2) pmoles cAMP/mg protein in sAC siRNA treated cells. In BF, rolipram raised [cAMP]I to 49.5 pmoles/mg protein and significantly reduced staurosporine-induced An+ cells in BF from 9.9% to 7.0% (n=5, p=0.018). Normalized phospho-Bcl2 level was 1.9 fold higher in BR than in BF (n=2).
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