title: Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.
abstract: OBJECTIVE
  To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO(3)(-), sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux.
  
  
  METHODS
  MCT1-4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pH(i) were measured in BCEC in HCO(3)(-)-free or HCO(3)(-)-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.
  
  
  RESULTS
  MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pH(i) 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 (dpH(i)/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO(3)(-) but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO(3)(-), 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.
  
  
  CONCLUSIONS
  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 HCO(3)(-), HCO(3)(-) transport, NHE and CA activity.
authors: School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
authors: Tracy T
authors: TT
authors: Nguyen
authors: Joseph A
authors: JA
authors: Bonanno
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D015153:Blotting, Western; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000378:metabolism*; D027501:Monocarboxylic Acid Transporters / Q000378:metabolism*; D009544:Niflumic Acid / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D027501:Monocarboxylic Acid Transporters; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D019344:Lactic Acid; D009544:Niflumic Acid; D000584:Amiloride; D002256:Carbonic Anhydrases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride