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T. T. Nguyen, M. Cui, J. Bonanno; Lactate Transport in Cultured Bovine Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3936.
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Previous studies done in various tissues have shown that bicarbonate (HCO3-) along with carbonic anhydrase (CA) facilitates lactate flux. Because of the presence of HCO3- transporters, lactate:H+ cotransport, and several CAs, a similar mechanism could be present in the corneal endothelium (CE). We will test the hypothesis that HCO3- together with CA activity facilitates lactate:H+ flux from the basolateral to apical surface of CE.
Bovine CE cells were grown to confluence on tissue culture inserts and placed in a 24 well plate. 500 µl of 5mM (L)-lactate in either bicarbonate free (BF) or bicarbonate rich (BR) ringer solution was added to the basolateral compartment and 350 µl of lactate-free BF or BR ringer to the apical compartment. Samples were taken from the apical compartment at various time points (0, 1, 3, 5, 10, 20 minutes) after addition of lactate. Lactate concentration was analyzed using the BioVision Lactate Assay Kit. Endogenous lactate was collected from the basolateral and apical compartment using lactate free BR (5% CO2, pH7.5) and BF (Air, pH7.5) ringer’s on both sides at 37°C. Lactate-dependent H+ flux was determined in CE cultured on coverslips perfused with BF or BR ±100uM acetazolamide, a carbonic anhydrase inhibitor by measuring the the rate of change in intracellular pH (pHi) upon addition of 20mM (L)-lactate. Relative changes in cell buffering capacity for each condition was determined by measuring the pHi change to a 5 mM NH4Cl pulse.
In BR media, basolateral to apical lactate flux was 60% (n=2) faster compared to BF media. In the endogenous lactate test, BR induced a 30% (n=2) greater lactate accumulation in the apical compartment compared to BF. Cell buffering capacity was unaffected by acetazolamide in BF, but reduced 35% in BR. Whereas lactate-dependent pHi changes were faster in BF, lactate-dependent proton flux (j=β*dpHi/dt) was 3 times faster in BR media compared to BF. Proton flux was unaffected by acetazolamide in BF. However, in BR media, proton flux decreased by 55% in the presence of acetazolamide.
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