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Wenlin Zhang, Joseph A Bonanno; Ammonia toxicity attenuates ATP production and activates autophagy in human corneal endothelium.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1449.
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© ARVO (1962-2015); The Authors (2016-present)
We recently described increased ATP production secondary to glutamine metabolism in corneal endothelium. The NH3:2H+ co-transporter SLC4A11 is essential in maintaining the homeostasis of this glutaminolysis. In Slc4a11-/- mouse corneal endothelium, we observed ammonia-related toxicity as well as elevated autophagy. Thus, here we examined the acute effect of ammonia toxicity on human corneal endothelial cells, looking for signs of energy metabolism disruption and autophagy activation.
Immortalized human corneal endothelium (HCEC) were treated for 12 hrs and 24 hrs with various concentrations (0, 1, 5, 10, 20, 50 mM) of NH4Cl in culture. Cellular ATP content was measured by luciferin-luciferase assay (Invitrogen). Cell morphology images were acquired with an Infinity I camera (Lumenera Corp.) attached to an inverted phase contrast microscope. Western blot and immunostaining of LC3 was performed with anti-LC3 antibody (PM036, MBL International Cooperation). Fluorescence images were acquired with an AxioImager M1 fluorescence microscope (Zeiss) and the AxioCam MRm camera (Zeiss). Data are presented as mean ± SEM.
Ammonia (NH4+/NH3) dose dependently (p < 0.0001) and time dependently (p = 0.0001) decreased the ATP content in HCEC. ATP level (nmol/106 cells) is highest (4.040 ± 0.209) without NH4Cl treatment and lowest in 24hrs with 50 mM NH4Cl treatment condition (1.581 ± 0.112). NH4Cl significantly activated autophagy in a dose dependent manner: there were increased numbers of vacuoles and increased LC3 fluorescence staining along vacuole walls as NH4Cl treatment concentration went up. The dose dependent manner of autophagy activation by NH4Cl was verified via Western Blot analysis of LC3 I/II ratio.
Ammonia toxicity directly attenuates ATP production in human corneal endothelium, and autophagy activation may serve as a compensatory mechanism to sustain the energy level.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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