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Miduturu Srinivas, Junyuan Gao, Richard Mathias, NEFELI SLAVI; Imaging the glutathione redox potential in the intact lens. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3069.
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© ARVO (1962-2015); The Authors (2016-present)
The maintenance of a reducing environment in the lens core is critical for its transparency. The concentrations of glutathione, the major anti-oxidant in the lens show a regional variation with high levels in surface cells to lower levels in the lens center. However, little is known about the depth-dependent variation of the glutathione redox potential (EGSH) in the intact lens. The recent development of redox-sensitive fluorescent probe (Grx1-roGFP2) by Gutscher et al (Nat. Methods, 5 (2008)) allows real-time imaging of EGSH. Here, we used Grx1-roGFP2 to measure the radial profile of EGSH in the intact mouse lens.
Purified recombinant Grx1-roGFP2 was injected into individual lens fiber cells at different depths. The membrane potential was monitored after each injection to ensure that there was no damage to fiber cells. Fluorescence emission ratios at 520nm were acquired with 405 and 488nm excitation wavelengths and were used to calculate EGSH. The 405/488 ratios and EGSH in the lens were measured as a function of the normalized distance from the lens center (r/a), where a is the radius. The effect of L-buthionine (S,R)-sulfoximine (BSO; 1 mM), a highly specific inhibitor of GSH biosynthesis on EGSH was also determined.
The 405/488 fluorescence ratios were low at the lens surface (r=a), but began to increase at r/a values between 0.8 and 0.6 and reached a maximum value at the lens center. Similarly, EGSH values increased from -315 mV at the lens surface to -280 mV at r/a values < 0.7. Incubation of the lens with BSO caused a significant increase in the 405/488 ratio in all regions of the lens indicating an oxidative shift in EGSH.
These measurements indicate a highly reducing environment in the surface cells, and a more oxidized environment in central cells, consistent with the regional variation in levels of GSH. Because gap junctions formed by Cx46 are important for the delivery of glutathione from the outer cortex to inner fiber cells, it is of interest to examine how EGSH is modulated by changes in Cx46 coupling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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