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A. Dibas, M. Yang, J. Bobich, T. Yorio; Neuronal Aquaporin in Retinal Ganglion Cell–Line and Human Optic Nerve: Changes Under Hypoxia and Apoptosis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3751.
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© ARVO (1962-2015); The Authors (2016-present)
It has been proposed that changes in cell volume precede cell death. It is unclear if water channels, aquaporins, play a role in this process. The following study tests the presence of aquaporin in retinal ganglion cell–line RGC–5 and human optic nerve tissue and study changes in expression under hypoxic insults.
Changes in cell volume was assessed following excitation of fura–2–labeled cells at 360 nm. Western blotting using anti–aquaporin–9 antibodies was used to test the presence of neuronal AQP9 in RGC–5 cells and human optic nerve. Real–time PCR was used for measuring changes in AQP9 and beta–actin under hypoxia. Hypoxia was initiated by flushing a mixture of 95% N2 and 5% CO2 for 15 minutes in a controlled atmosphere culture chamber. Apoptosis assay was performed by measuring the levels of released mitochondrial cytochrome c in the cytosol.
RGC–5 cells subjected to a hypotonic solution showed a decrease in fluorescence and swelling as a result of water influx, a response that was blocked by known inhibitors of AQP such as phloretin or cyano–hydroxycinnamic (40 µM) acid pretreatment. Western blotting detected the presence of a doublet AQP9 protein bands (33 and 50 KDa) in the plasma membrane fractions of both RGC–5 cells and human optic nerve. Interestingly, under hypoxia, there was an initial decrease in AQP9 (1hr) then up–regulation at 3–6 hr post hypoxia as judged by Western blotting and RT–PCR. Up–regulation of AQP9 was accompanied by the release of mitochondrial cytochrome c in the cytosol as judged by Western blotting.
This is the first report showing that retinal ganglion cells have neuronal aquaporin–9. Changes in AQP9 expression correlated with apoptosis of retinal ganglion cells suggesting a novel role in retinal ganglion cellular death.
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