Investigative ophthalmology & visual science · 2011
Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.
СтатьяПоддержка NIH: внешняя программаПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Nguyen Tracy T, Bonanno Joseph A
Аннотация
PURPOSE: 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.
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.
Предметные рубрики MeSH
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic AcidAmilorideAnimalsBicarbonatesBlotting, WesternCarbonic Anhydrase InhibitorsCarbonic AnhydrasesCattleCells, CulturedEndothelium, CornealFluorescent Antibody Technique, IndirectHydrogen-Ion ConcentrationLactic AcidMonocarboxylic Acid TransportersNiflumic AcidReal-Time Polymerase Chain ReactionSodium-Bicarbonate SymportersSodium-Hydrogen Exchangers