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Tracy T. Nguyen, Joseph A. Bonanno; Bicarbonate, NBCe1, NHE, and Carbonic Anhydrase Activity Enhance Lactate-H+ Transport in Bovine Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2011;52(11):8086-8093. doi: 10.1167/iovs.11-8086.
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To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO3 −, sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux.
MCT1–4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pHi were measured in BCEC in HCO3 −-free or HCO3 −-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na+/H+ exchange blocker), disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells.
MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pHi measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpHi/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO3 − but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO3 −, was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA.
MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H+ transport. Lactate dependent proton flux can activate Na+/H+ exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO3 −, HCO3 − transport, NHE and CA activity.
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