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Feng HE, Melina A. Agosto, Theodore G. Wensel; Characterization Of Phosphoinositides In The Light- And Dark-adapted Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1599.
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The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but the roles of phosphoinositides in vertebrate phototransduction remain unclear. In our previous studies, reconstitution of phosphodiesterase 6 (PDE6) and activated transducin on the surface of large unilamellar vesicles containing PI(4,5)P2 resulted in PDE activity nearly 4-fold above the level observed with vesicles containing no phosphoinositides. In this study, the effect of light adaptation on phosphoinositide levels in mouse rod outer segments (ROS) was investigated.
Wild-type C57BL/6 mice were adapted to dark or room light conditions for ≥12 hours, after which ROS were purified by sucrose gradient centrifugation. Phosphoinositides were extracted from the purified ROS and quantified using a modified ELISA assay. PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 were detected by binding of purified recombinant 1D4-tagged pleckstrin homology (PH) domains from hepatocyte growth factor-regulated tyrosine kinase substrate, PI(4)P adaptor protein-1, tandem PH domain-containing protein 1, phospholipase C delta, and general receptor for phosphoinositides 1, respectively, followed by incubation with anti-1D4 monoclonal antibody, horseradish peroxidase-conjugated secondary antibody, and a fluorescent substrate.
We developed a sensitive method for detection of phosphoinositides in mouse ROS. The detection limit was approximately 0.05-0.1 pmol of phosphoinositide. Specificity of the purified recombinant PH domains for their target phosphoinositides was verified using a panel of synthetic phosphoinositides. ROS from light-adapted mice were found to contain much higher levels of PI(4,5)P2 and PI(3)P than ROS from dark-adapted mice, whereas PI(4)P was present at similar levels in both. PI(3,4)P2 and PI(3,4,5)P3 were not detectable in ROS from either light- or dark-adapted mice.
Levels of PI(3)P and PI(4,5)P2 are significantly different in light- and dark adapted mice, suggesting activation of PI-4 kinases or PI-5 kinases, inhibition of degradatory enzymes, or both. The elevation of PI(3)P while PI(3,4,5)P3 remains undetectable implies that the previously-reported light activation of Type I PI-3 kinase is not involved in PI(3)P increases.
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