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T. T. Nguyen, C. Liu, J. Bonanno; The Role of Bicarbonate and Carbonic Anhydrase in Lactic Acid Transport Across Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):697.
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The cornea produces a significant amount of lactate, a potent osmolyte, that needs to be efficiently removed in order to avoid corneal edema. We asked whether bicarbonate and carbonic anhydrase (CA) activity facilitate the transport of lactic acid in corneal endothelium (CE).
Physiological experiments were performed in vitro using fresh rabbit CE mounted in a double-sided perfusion chamber. CE cells were loaded with the intracellular pH sensitive fluorescent dye BCECF-am. Change in the rate of lactate-induced acidification (LIA) was measured in the presence of HCO3- free (BF) and HCO3- rich (BR) medium and in the presence and absence of 100uM acetazolamide, a carbonic anhydrase inhibitor. Endogenous lactate efflux was also determined in BF and BR ringer using bovine CE cells grown to confluence on tissue culture inserts. To examine physiological function in vivo, 11 white New Zealand rabbits were injected intracamerally in one eye with shRNA directed against NBCe1 cotransporter and the other with scrambled shRNA delivered by lentiviral vector. Corneal thickness (CT) was measured before and after challenge with the topical CAI Azopt. Two weeks post-injection, corneas were harvested and processed for western blotting or RT-PCR and lactate analysis. Seven separate rabbits were treated topically with Azopt in one eye and PBS in the other. CT measurements were taken every hr for 4-5 hrs. Subsequently, the rabbits were euthanized and corneas were processed for lactate analysis. Lastly, 4 rabbits were injected intracamerally with 25µl 1mM ouabain in one eye. Similarly, CT measurements were taken and corneas were subjected to lactate analysis.
pHi decreased (~.05) following exposure to lactate on the basal and apical surface in tissues perfused in BF ringer. In BR ringer, there was a smaller decrease (~.02) in pHi (BL surface) & no decrease apically. In the presence of HCO3-, acetazolamide increased the rate of LIA on the BL surface (from .035 pH units/min to .101 pH units/min). Endogenous lactate efflux was 30% greater in the presence of HCO3-. Six rabbits had moderate to significant (50-98%) NBCe1 knockdown (KD). Lactate concentration in these 6 rabbits were 38+58% greater in the KD eye, although not statistically significant. Topical application of Azopt induced a 10+ 2% increase in CT. Corneal lactate concentration was 23+19 % greater in the eyes treated with Azopt (n=7, p=.015). Ouabain induced an average of 28+ 6% increase in CT and 43+ 40% in lactate concentration (n=4, p=0.15).
Transport of lactate from the corneal stroma to the anterior chamber is facilitated by HCO3- /CA and active transport activity of the CE.
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