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D. Reigada, C.H. Mitchell; ATP Release Across the Apical Membrane of Fresh Bovine RPE . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2242.
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Purpose: Stimulation of ATP receptors on bovine RPE cells has been linked to increased transport of Cl- and fluid towards the choroid. ATP is released from cultured human cells, but it is not known whether release can occur from fresh RPE cells, and the polarity of any such release is unclear. This study was undertaken to determine whether fresh bovine RPE cells can release ATP across their apical membrane in response to stimuli. Methods:: ATP release was quantified by measuring light released using the luciferin/luciferase assay. Release was determined in two ways. Sheets of RPE/choroid clamped in an Ussing-type chamber and mounted with the apical bath facing a photomultiplier tube were perfused with test solutions containing luciferin/luciferase. The second protocol involved removing the retina from the RPE eyecup, bathing the apical membrane of the RPE with the indicated solution, removing an aliquot of extracellular solution at a given time and assaying ATP content off-line. Results: Extracellular ATP levels were elevated in both the Ussing-type chamber and the RPE eyecup systems by solutions made 50% hypotonic by the addition of water. Extracellular ATP concentrations were also elevated by 1 mM UTP and by 1 mM H2O2 . The ATP release triggered by hypotonicity was transient and began to decrease within 15 min of stimuli addition. The elevation by UTP and H2O2 was sustained and levels continued to rise > 20 min after agonist addition. Conclusions: Fresh bovine RPE cells release ATP across their apical membrane in response to a variety of stimuli. This is consistent with release into sub-retinal space in vivo and suggests a mechanism by which changes in ionic strength or oxidation state could alter fluid movement across the RPE.
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