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R.H. W. Funk, C. Roehlecke, E.–M. Kniep; A Blocker of Carbonic Anhydrase Protects Retinal Neural Cells From Apoptotic Damages . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2058.
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© ARVO (1962-2015); The Authors (2016-present)
Apoptotic degeneration of retinal neurons is often triggered by advanced glycation end products (AGEs) and reactive oxygen species (ROS). To alleviate the toxic effects of these agents appears a promising step in neuroprotection
Cells of the retinal neural cell line E1A–NR3 were treated with the AGE intermediates glyoxal and methylglyoxal, or with ROS inducing hydrogen peroxide to induce apoptosis. Different manifestations of apoptotic changes were investigated.
All three substances led to exposure of phosphatidylserine on plasma membranes (measured by binding of annexinV), acidification of cytosol (measured with the pH indicator dye BCECF AM), breakdown of mitochondrial membrane potential (determined with the potential sensitive dye JC–1), augmentation of ROS (monitored by oxidation of CM–H2DCF–DA) and DNA fragmentation (determined by measuring the amount of subdiploid DNA). In addition, we tested the cells for CML–modified proteins and active caspase–3 using appropriate antibodies. These latter manifestations of apoptosis were solely detected after incubating the cells with glyoxal.
Since decreased cytosolic pH is a phenomenon observed frequently concomitant with apoptosis we speculated whether cytosolic acidification might be the prerequisite, which triggers the events of the apoptosis cascade. Carbonic anhydrase generates protons by hydration of newly formed CO2, we therefore investigated if blockers of this enzyme stabilize the cytosolic pH after induction of cell stress. We applied the carbonic anhydrase blocker dorzolamide together with the stress agents glyoxal, methylglyoxal, or hydrogen peroxide, respectively. The result was a distinct inhibition of intracellular acidification. Beyond this, all apoptotic changes mentioned above were reduced when dorzolamide was present in the incubation mixtures together with the stress inducers. Consequently, dorzolamide acts as a neuroprotectant in our cell model.
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