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Diego G Ogando, Wenlin Zhang, Shimin Li, Joseph A Bonanno; Ammonia permeability is decreased and autophagy activated in corneal endothelium of Slc4a11 KO mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1196.
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© ARVO (1962-2015); The Authors (2016-present)
SLC4A11 is a basolateral membrane transporter with permeability to H+ and NH3. Mutations in SLC4A11 cause corneal endothelial dystrophies. Slc4a11 KO mice show corneal edema at birth and a decrease in the corneal endothelial (CE) cell density as they age. Lactate accumulates in the stroma of Slc4a11 KO mice indicating a failure of the corneal endothelial pump in this model (ARVO 2014#2029). We tested the following hypothesis: 1) NH3 permeability and pHi are altered in KO CE. 2) As a result of NH3i increase, autophagy is induced in KO CE.
<br /> CE pHi was measured with BCECF in ex vivo mouse corneas mounted in a perfusion chamber (12 weeks of age, n=10). Autophagy was quantified by LC3B immunofluorescence (12 and 40 weeks of age, n=6). Values are expressed as mean ± s.e.m. Independent t-test was used for comparisons.
The rate and extent of NH4Cl induced alkalinization was higher in WT compared to KO CE (Rate: WT: 0.362±0.035, KO: 0.204±0.035 pH/min, p=0.005; Extent: WT: 0.154±0.011, KO: 0.092±0.010 pH units, p=0.001), indicating Slc4a11 NH3 permeability. The acidification during NH4Cl perfusion was faster in WT than KO (WT: 0.023±0.001, KO: 0.018±0.001 pH/min, p=0.043). Moreover, the acidification following NH4Cl removal was faster in WT with respect to KO (WT: 0.219±0.018 vs KO: 0.157±0.011 pH units, p=0.015), consistent with Slc4a11 having H+ permeability and the steady-state pHi in WT cells was lower than in KO in the absence of bicarbonate (WT: 6.980±0.014 vs KO: 7.058±0.014, p=0.001), consistent with net NH3 efflux. Lastly, LC3B immunofluorescence was significantly increased in KO respect to WT at 12 weeks of age (WT: 7,090±1,059, KO: 71,224±7,686 integrated intensity/cell, p=7.2 x 10-7) and at 40 weeks of age (WT: 9,326±1,213, KO: 64,248±13,188, p=0.003) , indicating increased autophagy in KO CE.
(1) Slc4a11 KO corneal endothelial cells have reduced NH3 permeability and increased pHi. (2) Autophagy is induced in KO CE cells that may be a consequence of NH3 accumulation.
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