Lactate-H<sup>+</sup>Transport Is a Significant Component of the In Vivo Corneal Endothelial Pump

Nguyen, Tracy; Bonanno, Joseph A.

doi:10.1167/iovs.12-9475

Cited by 29 publications

(43 citation statements)

References 43 publications

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“…Given the pH i regulatory activity demonstrated here, SLC4A11 could add significant cellular buffering capacity that would facilitate the removal of lactic acid from the highly glycolytic cornea. Loss of SLC4A11 function in the corneal endothelium could thereby slow the removal of lactate from the cornea, which will produce corneal edema (18).…”

Section: Discussionmentioning

confidence: 99%

SLC4A11 is an EIPA-sensitive Na⁺ permeable pH_i regulator

Ogando

Jalimarada

Zhang

et al. 2013

American Journal of Physiology-Cell Physiology

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Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na+-B(OH)4− (borate) cotransporter and a Na+-2OH− cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na+-HCO3− cotransporter, 2) Na+-OH−(H+) transporter, and/or 3) Na+-B(OH)4− cotransporter. CO2/HCO3− perfusion yielded no significant differences in rate or extent of pHi changes or Na+ flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3−, acidification on removal of Na+ and alkalinization on Na+ add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na+-HCO3− cotransport activity. In the absence of CO2/HCO3−, SLC4A11-transfected cells showed higher resting intracelllular Na+ concentration ([Na+]i; 25 vs. 17 mM), increased NH4+-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na+ efflux and influx were faster (80%) following Na+ removal and add back, respectively, indicative of Na+-OH−(H+) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na+-OH−(H+) transporter and not an activator of NHEs. SLC4A11-mediated H+ efflux is inhibited by 5-( N-ethyl- N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/d t or ΔpH during a Na+-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na+-OH−(H+) and NH4+ permeability.

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Section: Discussionmentioning

confidence: 99%

SLC4A11 is an EIPA-sensitive Na⁺ permeable pH_i regulator

Ogando

Jalimarada

Zhang

et al. 2013

American Journal of Physiology-Cell Physiology

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“…20,21 Primary antibodies to CD147 and MCT1, -2, and -4 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-b-actin, anti-mouse IgG, and anti-rabbit IgG were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).…”

Section: Western Blottingmentioning

confidence: 99%

“…As described in previous publications, 20,21 freshly peeled corneal endothelium was placed with apical surface (anterior chamber facing) up and basolateral side (stromal facing) down on a glass slide, and fixed with 2% paraformaldehyde solution containing 75 mM lysine, 10 mM sodium periodate, and 45 mM sodium phosphate, pH 7.4. Endothelial cells were permeabilized using 0.01% saponin-0.1% Triton X-100 for 10 minutes.…”

Section: Immunofluorescencementioning

confidence: 99%

“…7,8 Lactate along with a H þ is cotransported through the plasma membrane of many cell types via stereospecific, pH-dependent monocarboxylate transporters (MCT1-4). [9][10][11][12][13][14][15][16][17][18] Recently, we have identified MCT1, -2, and -4 in human, 19 bovine, 20 and rabbit 21 corneal endothelium. Interference with primary or secondary active transporters that regulate intracellular pH (pH i ), reduced buffering by removal of HCO 3 À , or inhibition of carbonic anhydrases slowed lactate fluxes, led to corneal lactate accumulation, and increased corneal thickness, indicating that lactate efflux is a significant component of the endothelial pump.…”

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confidence: 99%

“…Interference with primary or secondary active transporters that regulate intracellular pH (pH i ), reduced buffering by removal of HCO 3 À , or inhibition of carbonic anhydrases slowed lactate fluxes, led to corneal lactate accumulation, and increased corneal thickness, indicating that lactate efflux is a significant component of the endothelial pump. 20,21 CD147, an evolutionary conserved member of the immunoglobulin superfamily, has gained considerable attention for its multiple functions involved in reproduction, neural function, inflammation, and tumor invasion. It is also described in the literature as EMMPRIN, basigin (Bsg), TCSF, M6, OK, 5A11, Gp42, CE9, and neurothelin.…”

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CD147 Required for Corneal Endothelial Lactate Transport

Li¹,

Nguyen²,

Bonanno³

2014

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. CD147/basigin is a chaperone for lactate:H þ cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema.METHODS. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity.RESULTS. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 6 0.81 to 1.5 6 0.37 nmol/min, increased corneal [lactate] from 19.28 6 7.15 to 56.73 6 8.97 nmol/mg, acidified endothelial cells (pH i ¼ 6.83 6 0.07 vs. 7.19 6 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 6 0.0005 to 0.0012 6 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 6 0.9 lm at 28 days; Azopt increased CCT to 24.4 6 3.12 vs. 12.0 6 0.48 lm in control, and corneal [lactate] was 47.63 6 6.29 nmol/mg in shCD147 corneas and 17.82 6 4.93 nmol/mg in paired controls.CONCLUSIONS. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump.Keywords: CD147, lactate transport, MCT1, MCT4, corneal endothelial pump T he cornea's energy needs are supplied predominantly from glucose.1 Glucose enters the cornea from the aqueous humor across the corneal endothelium and diffuses through the stroma to the corneal epithelium. Corneal epithelial cells are very glycolytic, converting 85% of glucose to lactate. 2-4Therefore, for every 100 glucose molecules entering the cornea, 170 lactate molecules are produced. The accumulation of lactate within the cornea can create an osmotic load that will increase stromal hydration and corneal thickness. 5 To maintain corneal hydration and transparency, the large quantity of lactate must be simultaneously exported out. Since the surface corneal epithelial cells are impermeable to lactate, lactate efflux must take place across the endothelium. Efflux is driven by the 2-fold [lactate] gradient from cornea to anterior chamber. 5,6 Facilitated transport of lactate has been extensively studied in rabbit corneal endothelium.7,8 Lactate along with a H þ is cotransported through the plasma membrane of many cell types via stereospecific, pH-dependent monocarboxylate transporters (MCT1-4).9-18 Recently, we have identified MCT1, -2,...

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