May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Is the Human Retinal Pigment Epithelium (RPE) Basolateral Membrane a Barrier to CO2?
Author Affiliations & Notes
  • J. Adijanto
    SERPD, NEI/NIH, Bethesda, Maryland
    Dep of Chemical and Biomolecular Engineering, UMD, College park, Maryland
  • S. Jalickee
    SERPD, NEI/NIH, Bethesda, Maryland
  • T. Banzon
    SERPD, NEI/NIH, Bethesda, Maryland
  • S. S. Miller
    SERPD, NEI/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  J. Adijanto, None; S. Jalickee, None; T. Banzon, None; S.S. Miller, None.
  • Footnotes
    Support  NEI intramural
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5549. doi:
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      J. Adijanto, S. Jalickee, T. Banzon, S. S. Miller; Is the Human Retinal Pigment Epithelium (RPE) Basolateral Membrane a Barrier to CO2?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5549.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The photoreceptors produce significant amounts of CO2 in the light and dark (Wangsa-Wirawan and Linsenmeier, Arch Ophthalmol, 2003). A significant part of this metabolic CO2 diffuses across the RPE and enters the choroidal blood supply. In this study, we examined CO2-induced changes in RPE physiology.

Methods: : All experiments were performed with confluent monolayers of cultured human fetal RPE (hfRPE) grown on transwells (Maminishkis et al, IOVS, 2006). We used pH-sensitive fluorescence dye (BCECF) to monitor intracellular pH (pHi) while simultaneously recording transepithelial potential (TEP) and total epithelial resistance (RT). We also used the capacitance-probe technique to measure fluid-transport (JV) across the epithelia. To change CO2 level, we equilibrated normal Ringer (5% CO2) with either 13% or 1% CO2 gas and perfused these solutions into the compartments bathing the hfRPE apical or basolateral membranes.

Results: : Compared with the apical membrane, replacement of basal bath Ringer with either 1% CO2 or 13% CO2 equilibrated Ringer caused an approximately 10-fold smaller ΔpHi. In addition, CO2/HCO3 free Ringer alkalinized the cell when perfused onto the apical membrane but acidified the cell when perfused onto the basal membrane. These experiments suggest that CO2 permeates the apical membrane of the RPE more freely than the basolateral membrane. An earlier study concluded that aquaporin 1 (AQP1) facilitates CO2 transport across cell membranes (Endeward et al, FASEB, 2006). However, our experiments show that pCMBS, an AQP1 inhibitor, did not reduce the 1% or 13% CO2 induced pH-changes, arguing against that conclusion. Evidence is presented to indicate that HCO3 is mainly transported across the hfRPE basolateral membrane by a Cl/HCO3 exchanger with a possible contribution from a Na/HCO3 co-transporter and that Cl is recycled at the basolateral membrane via Cl channels (CFTR). We show that the 13% CO2-load at the apical membrane caused intracellular acidification that strongly inhibited the Cl/HCO3 exchanger. Fluid-transport experiments show that 13% CO2 increased, and 1% CO2 decreased ion-linked fluid-absorption across the hfRPE.

Conclusions: : 13% CO2-induced inhibition of the exchanger increases net Cl absorption by increasing the amount of Cl recycled through CFTR. The relatively small 13 % CO2-induced ΔpHi at the basolateral membrane suggests the presence of a CO2-barrier. Cytosolic carbonic anhydrase would convert this CO2 into HCO3, increase the driving force of the Na/HCO3 co-transporter, and increase solute linked (NaCl + NaHCO3) JV.

Keywords: retinal pigment epithelium • pH regulation/protons • carbon dioxide 
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