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Ekaterina Bikovtseva, Rajan Deep Adhikari, M Carter Cornwall, Jeannie Chen, Sowmya Bekshe Lokappa, Rikard Frederiksen; Requirement of Lactate for Rhodopsin Dephosphorylation and Dark Adaptation in Mouse Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4507.
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Bright light exposure activates rhodopsin (R*) and leads to rhodopsin phosphorylation and arrestin binding. Recovery of dark sensitivity requires that these latter steps be reversed. Our previous studies showed an impaired rate of rhodopsin dephosphorylation in isolated mouse retinas kept under conditions commonly used in electrophysiological experiments. We hypothesize that these in vitro conditions lack certain metabolites which promote normal rhodopsin dephosphorylation. Here, we report that the addition of millimolar concentrations of lactate to the medium bathing these preparations is sufficient to restore dephosphorylation activity.
After overnight dark adaptation, retinas from wild type mice (c57bl6) were isolated, exposed to 70% bleach and incubated for up to 3 hours in darkness in Ames’ media containing 11-cis retinal and various concentrations of Na L-lactate. These samples were then prepared for isoelectric focusing (IEF) to separate the different phosphorylated species, followed by western blot to visualize rhodopsin. Band intensities were quantified using NIH ImageJ. Unbleached dark-adapted retina samples were used as controls.
Isolated retinas that were not supplemented with lactate showed little rhodopsin dephosphorylation during the incubation course of 3 hours. Addition of 4 mM Na L-lactate to Ames’ solution containing 11-cis retinal rescues and facilitates the dephosphorylation of the six Ser/Thr residues of rhodopsin C-terminus. This dephosphorylation was nearly complete in 3 hours. In experiments where we used different concentrations of lactate in the incubation media we found that 4 mM promotes dephosphorylation to a larger degree compared with samples containing either higher or lower concentration.
Rhodopsin dephosphorylation depends on the presence of lactate normally present in the extracellular matrix. At a concentration of 4 mM, the rate of rhodopsin dephosphorylation in isolated retinas is comparable to that observed in vivo experiments.
This is a 2020 ARVO Annual Meeting abstract.
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