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Mohammad Shahidullah, Amritlal Mandal, Nicholas A. Delamere; Hyposmotic Stress Causes ATP Release and Stimulates Na,K-ATPase Activity in Porcine Lens. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3411.
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Reports of purinergic receptors in the lens epithelium suggest lens function can be altered by chemical signals coming either from aqueous humor or from the lens itself. Here, we tested whether hyposmotic shock triggers ATP release from the intact lens. Na,K-ATPase activity was examined because purinergic agonists previously were found to stimulate Na,K-ATPase activity in the lens epithelium.
Intact porcine lenses were exposed to hyposmotic solution (200 mOsm) for 5 - 15 min and ATP release in the bathing medium was measured by luciferin-luciferase bioluminescence assay. To determine Na,K-ATPase activity, the epithelium was removed immediately after 10 min exposure to hypotonic solution and the amount of ATP hydrolysis in the epithelial homogenate was quantified by a colorimetric assay.
Exposure of the intact lens to hyposmotic solution released ATP into the bathing medium and increased Na,K-ATPase activity by ~ 2 fold. Hypotonicity-induced release of ATP and the increase in Na,K-ATPase activity both were partially suppressed by a connexin hemichannel inhibitor, 18α glycyrrhetinic acid (AGA, 100µM) but abolished by AGA + probenecid (1 mM), a pannexin hemichannel blocker. The hypotonicity-induced stimulation of Na,K-ATPase activity was abolished by the P2Y purinergic receptor antagonist, Reactive Blue-2 (30 µM). The increase in Na,K-ATPase activity also was prevented by apyrase (20 U/ml), an enzyme that catalyses ATP breakdown.
The findings suggest hyposmotic shock triggers ATP release from the intact porcine lens in a response that appears to involve connexin and pannexin hemichannels. ATP released from the intact lens in this fashion was sufficient to stimulate Na,K-ATPase activity. The response was consistent with a previous report that purinergic agonists stimulate Na,K-ATPase activity by a Src kinase-dependent mechanism. The hyposmotic responses might signify an autoregulatory loop in which a change of osmolarity, or the mechanical stress of osmotic swelling, triggers ATP release followed by purinergic receptor-mediated change of lens epithelium function.
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