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K. Tan–Allen, J.A. Bonanno; Protection of Cultured Bovine Corneal Endothelium (CBCE) From Staurosporine Induced Cell Death by Adenosine Receptor Activation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1613.
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© ARVO (1962-2015); The Authors (2016-present)
Cell stress can induce adenosine production, which is associated with cytoprotection. We examined whether A1 and A2b adenosine receptor activation in CBCE could be protective.
CBCE were pulsed for 30 minutes with either an A1 agonist (N6–CPA, 1 µM) or 10 µM of the general adenosine receptor (AR) activators, adenosine or NECA (for A1 and A2b activation) followed by 1 hour wash. This was repeated before application of the cell death inducer staurosporine (100 nM, 17 hours). To test for endogenous adenosine protection, cells were pre–incubated in AMPCP (0.3 mM, 30 minutes), an antagonist of CD73 (an ecto–nucleotidase), before co–incubation with staurosporine and AMPCP (17 hours). Apoptotic cells were then quantitated using flow cytometry (Annexin V staining). Nuclear condensation was detected by DAPI staining. Statistical results are expressed as mean ± S.D.
(a) AR agonists and AMPCP had no effect on background levels of apoptosis. (b) Staurosporine alone produced 4.1%±1.3 Annexin V positive cells. (c) AMPCP + staurosporine produced 39.0%±4 Annexin V positive cells, a near 10 fold increase over non–AMPCP treated cells. (d) N6–CPA reduced staurosporine mediated Annexin V staining to 3.5% ±0.5. (e) 10 µM NECA reduced staurosporine mediated Annexin V staining to 2.8±0.7. (f) 10 µM NECA and N6–CPA preconditioned samples had fewer apoptotic features (condensed nuclei, nuclear fragmentation) and cell loss relative to control. NECA PC showed 4.2%±0.9 apoptotic cells versus 28.6 %±2.4 in control; N6–CPA PC exhibited 8.8%±2.8 apoptotic cells; AMPCP–staurosporine treatment yielded 19.2%±4.1 apoptotic population over 12.8%±2.9 in control.
Endogenous adenosine is protective. Transient exogenous A1 and/or A2b activation prior to a staurosporine stress load is also protective.
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