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Jonathan M. Beckel, Arthur J. Argall, Jason C. Lim, Mohammad Shahidullah, Edward J. Macarak, Alan M. Laties, Nicholas A. Delamere, Claire H. Mitchell; Optic Nerve Astrocytes Release ATP upon Mechanical Strain. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1819.
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Astrocytes in the optic nerve have been identified as key mediators of mechanosensitive change in glaucoma. We have previously shown that increased intraocular pressure triggers a release of ATP from the retina, and in other neuronal systems, astrocytes display mechanosensitive ATP release. This study thus asked if optic nerve astrocytes released ATP in response to mechanical strain and began to probe for components of purinergic signaling pathways in these cells.
Astrocytes obtained from the neonatal rat optic nerve were cultured. For stretch experiments, astrocytes were seeded on a silicon substrate and subjected to a 5% equilateral strain at 0.3 Hz for 2 min using a specially designed pneumatic piston. Samples of the extracellular media were taken before and after stretch and analyzed for ATP concentration using the luciferase assay. For swelling experiments, astrocytes grown in 96-well plates were exposed to hypotonic solution and ATP determined on line. RT-PCR of astrocytes was performed using standard procedures.
While stretching astrocytes in control solution did not produce a detectable release of ATP, stretching in the presence of ecto-ATPase inhibitors ARL67156 and β,γ-mATP increased extracellular levels of ATP 5 fold. Swelling was partially inhibited by 10 µM of the pannexin blocker carbenoxolone. Osmotic swelling increased ATP release 3.5 fold, with an analogous block observed with carbenoxolone. RT-PCR indicated that astrocytes express mRNA for the pannexin-1 hemichannel, the ecto-ATPase NTPDase1 and the ionotropic ATP receptor P2X7.
Our results demonstrated that both mechanical stretch and osmotic swelling of rat optic nerve astrocytes resulted in ATP release, most likely through pannexin hemichannels. This suggests ATP release is a generalized response to mechanical strain by optic nerve astrocytes. The high ecto-ATPase activity of these astrocytes, coupled with their expression of NTPDase1 and P2X7, suggest that purines may play an important role in the pressure-induced response of optic nerve astrocytes in glaucoma.
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