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Jonathan M. Beckel, Jingsheng Xia, Jason C. Lim, Edward J. Macarak, Claire H. Mitchell; Reduced GFAP Expression in Optic Nerve Head Astrocytes Linked To Pannexin-mediated Release Of ATP. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2011.
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Astrocytes in the optic nerve head have been identified as key mediators of mechano-sensitive change in glaucoma, and levels of activation marker GFAP can rise in glaucoma. We have previously shown that astrocytes display mechano-sensitive ATP release through a non-vesicular mechanism. This study further examined the mechanisms of mechano-sensitive ATP release from rat optic nerve head astrocytes (RONHA) and investigated the role purinergic signaling plays in astrocyte activation.
RONHA from neonatal rats (PD 3-7) were cultured on 96 well plates, with ATP concentrations determined using a luciferin-luciferase assay and a plate reader. Stretched RONHA were seeded on a silicon substrate and subjected to a 5% equilateral strain at 0.3 Hz for 4 hours using a specially designed pneumatic piston. RNA was extracted 20 hours later and qPCR was performed using standard procedures. Regulatory volume decrease (RVD) measurements were determined from cells filled with calcein-AM and imaged on a fluorescent microscope.
The application of P2 receptor analog ATPγS (100 µM) reduced the expression of GFAP mRNA in RONHA when levels were measured 16 hrs after stimulation. Application of mechanical strain to astrocytes by either stretching or swelling the cells, led to a rapid release of ATP. The pannexin inhibitors probenecid (1mM) and carbenoxolone (10µM) reduced this mechano-sensitive release of ATP, suggesting release involved pannexin hemichannels. In contrast, neither 18α-glycyrrhetinic acid (50µM), GdCl3 (50µM) nor the TRPV4 antagonist HC067047 (1-10µM), inhibited the swelling-induced release of ATP. While cells normally restored their volume soon after swelling, this regulatory volume decrease was prevented by probenecid (1mM) and the ATPase apyrase (1U/ml), implying that auto-stimulation of P2 receptors by released ATP is involved in maintaining homeostasis after mechanical strain.
Mechanical strain triggers ATP released from RONHA through pannexin hemichannels. Released ATP may act in an autocrine/paracrine manner on astrocyte purinergic receptors to maintain homeostasis and lower GFAP levels.
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