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Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.
- Source :
-
Investigative ophthalmology & visual science [Invest Ophthalmol Vis Sci] 2011 Oct 17; Vol. 52 (11), pp. 8086-93. Date of Electronic Publication: 2011 Oct 17. - Publication Year :
- 2011
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Abstract
- 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.<br />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.<br />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.<br />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.
- Subjects :
- 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid pharmacology
Amiloride analogs & derivatives
Amiloride pharmacology
Animals
Blotting, Western
Carbonic Anhydrase Inhibitors pharmacology
Cattle
Cells, Cultured
Endothelium, Corneal drug effects
Fluorescent Antibody Technique, Indirect
Hydrogen-Ion Concentration
Niflumic Acid pharmacology
Real-Time Polymerase Chain Reaction
Bicarbonates metabolism
Carbonic Anhydrases metabolism
Endothelium, Corneal metabolism
Lactic Acid metabolism
Monocarboxylic Acid Transporters metabolism
Sodium-Bicarbonate Symporters metabolism
Sodium-Hydrogen Exchangers metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1552-5783
- Volume :
- 52
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Investigative ophthalmology & visual science
- Publication Type :
- Academic Journal
- Accession number :
- 21896839
- Full Text :
- https://doi.org/10.1167/iovs.11-8086