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K. Tan-Allen, J. A. Bonanno; Soluble Adenylate Cyclase (sAC) Protects Corneal Endothelium from Staurosporine-Induced Apoptosis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):548.
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Recent studies have shown that adenosine protects corneal endothelium (CE) from apoptotic stressors through a cAMP mediated mechanism. Here we examined whether cAMP produced by soluble adenylate cyclase (sAC), a HCO3- dependent AC expressed in CE, could also contribute to CE stress protection.
Cellular [cAMP] was varied by incubating cultured bovine CE in either 40 mM HCO3- DMEM (HCO3-rich, BR, pH 7.4, 25 mM HEPES), 5%CO2, 37°C incubation or 0 mM HCO3- DMEM (HCO3- free, BF, pH 7.4, 25 mM HEPES), 37°C air equilibrated incubation. The BR medium raised [cAMP] to 35 from 20 pmoles cAMP/mg protein in BF medium. CE was stressed with 50 µM staurosporine for 17 hrs and nuclei evaluated for condensation by DAPI staining. Levels of pCREB and transcription of cAMP sensitive pro-survival genes BCL2 and BCL-xL were examined by Western Blot and semi-quantitative RT-PCR. Specific inhibition of sAC activity was accomplished by 48 hour incubation in BF, application of 2-hydroxyestradiol (2-HE, a commercial sAC inhibitor) or KH7 (a newly developed specific sAC antagonist).
DAPI staining showed that 50 µM staurosporine induced fewer condensed and fragmented nuclei typical of apoptotic cells in BR incubated cultures compared to BF. Quantitative counting of apoptotic DAPI-stained nuclei showed that the BR sample had 21% fewer apoptotic cells than BF. Increased [cAMP]I in BR medium also resulted in a higher level of phospho-CREB and BCL2 mRNA transcription compared to BF. No significant difference was detected in BCL-xL semi-quantitative RT-PCR. Preliminary quantitative DAPI analysis showed that co-incubation of BR cells with 2-HE (25 uM) and 50 nM staurosporine produced 45% more apoptotic cells than staurosporine treatment alone. Cell treatment with 25 uM KH7 and staurosporine caused a 2.6 fold increase in apoptotic population over staurosporine sample. There was no significant toxicity associated with either 2-HE or KH7 incubation relative to control vehicle (DMSO) treatment.
The results suggest that cAMP produced by increased sAC activity, induced by HCO3-, provides protection to CE from staurosporine stress. This is consistent with increased p-CREB and BCL2 mRNA transcription from HCO3-activated sAC.
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