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CO2-induced ion and fluid transport in human retinal pigment epithelium.

Adijanto J, Banzon T, Jalickee S, Wang NS, Miller SS - J. Gen. Physiol. (2009)

Bottom Line: Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area.The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide.This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.

ABSTRACT
In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 +/- 0.05 to 7.14 +/- 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase-mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 +/- 3.3 to 24.0 +/- 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 +/- 1.6 to 6.7 +/- 2.3 microl x cm(-2) x hr(-1) (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H(2)O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

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pH sensitivity of basolateral membrane Cl/HCO3 exchanger. Low (1 mM) Cl Ringer was perfused into the apical bath to record the initial control response. This maneuver was then repeated in (A) 13% or (B) 1% apical bath CO2. After returning to control Ringer, low basal bath [Cl]-induced control response was obtained. Solid bars above the graphs represent solution changes from control Ringer as described in the legend to Fig. 2.
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fig7: pH sensitivity of basolateral membrane Cl/HCO3 exchanger. Low (1 mM) Cl Ringer was perfused into the apical bath to record the initial control response. This maneuver was then repeated in (A) 13% or (B) 1% apical bath CO2. After returning to control Ringer, low basal bath [Cl]-induced control response was obtained. Solid bars above the graphs represent solution changes from control Ringer as described in the legend to Fig. 2.

Mentions: To assess basolateral membrane Cl/HCO3 exchanger activity, basal bath [Cl] was reduced from 126 to 1 mM, which alkalinized the cell by ≈0.22 (Fig. 6). In three experiments, this alkalinization (ΔpHi = 0.18 ± 0.05) was abolished by 0.5 mM of basal DIDS (ΔpHi = 0.02 ± 0.01; n = 3; P < 0.05), but this effect was not reversible. Next, we tested the pHi dependence of the Cl/HCO3 exchanger by comparing the basal bath Δ[Cl]-induced pHi response in 5 versus 13% apical bath CO2 (Fig. 7 A). The steady-state pHi in 5 and 13% apical bath CO2 differed significantly, which required us to use the total buffering capacity of the hfRPE to calculate equivalent H+ fluxes. In the presence of 13% CO2-equilibrated Ringer in the apical bath, the basal bath Δ[Cl]-induced change in H+ flux was 2.3 ± 1.0 mM × min−1, approximately fourfold smaller than the H+ flux in 5% CO2 (9.0 ± 4.5 mM × min−1; n = 7; P < 0.01); this effect was fully reversible. Fig. 7 B summarizes a parallel experiment in which basal bath [Cl] was reduced in the presence of 1% CO2-equilibrated Ringer in the apical bath. In this case, the basal bath Δ[Cl]-induced proton flux was 27.4 ± 10.8 mM × min−1, or approximately fivefold larger than the flux in 5% CO2 (5.9 ± 6.5 mM × min−1; n = 5; P = 0.01). These experiments indicate that the DIDS-sensitive basolateral membrane Cl/HCO3 exchanger in hfRPE is pHi dependent.


CO2-induced ion and fluid transport in human retinal pigment epithelium.

Adijanto J, Banzon T, Jalickee S, Wang NS, Miller SS - J. Gen. Physiol. (2009)

pH sensitivity of basolateral membrane Cl/HCO3 exchanger. Low (1 mM) Cl Ringer was perfused into the apical bath to record the initial control response. This maneuver was then repeated in (A) 13% or (B) 1% apical bath CO2. After returning to control Ringer, low basal bath [Cl]-induced control response was obtained. Solid bars above the graphs represent solution changes from control Ringer as described in the legend to Fig. 2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2713148&req=5

fig7: pH sensitivity of basolateral membrane Cl/HCO3 exchanger. Low (1 mM) Cl Ringer was perfused into the apical bath to record the initial control response. This maneuver was then repeated in (A) 13% or (B) 1% apical bath CO2. After returning to control Ringer, low basal bath [Cl]-induced control response was obtained. Solid bars above the graphs represent solution changes from control Ringer as described in the legend to Fig. 2.
Mentions: To assess basolateral membrane Cl/HCO3 exchanger activity, basal bath [Cl] was reduced from 126 to 1 mM, which alkalinized the cell by ≈0.22 (Fig. 6). In three experiments, this alkalinization (ΔpHi = 0.18 ± 0.05) was abolished by 0.5 mM of basal DIDS (ΔpHi = 0.02 ± 0.01; n = 3; P < 0.05), but this effect was not reversible. Next, we tested the pHi dependence of the Cl/HCO3 exchanger by comparing the basal bath Δ[Cl]-induced pHi response in 5 versus 13% apical bath CO2 (Fig. 7 A). The steady-state pHi in 5 and 13% apical bath CO2 differed significantly, which required us to use the total buffering capacity of the hfRPE to calculate equivalent H+ fluxes. In the presence of 13% CO2-equilibrated Ringer in the apical bath, the basal bath Δ[Cl]-induced change in H+ flux was 2.3 ± 1.0 mM × min−1, approximately fourfold smaller than the H+ flux in 5% CO2 (9.0 ± 4.5 mM × min−1; n = 7; P < 0.01); this effect was fully reversible. Fig. 7 B summarizes a parallel experiment in which basal bath [Cl] was reduced in the presence of 1% CO2-equilibrated Ringer in the apical bath. In this case, the basal bath Δ[Cl]-induced proton flux was 27.4 ± 10.8 mM × min−1, or approximately fivefold larger than the flux in 5% CO2 (5.9 ± 6.5 mM × min−1; n = 5; P = 0.01). These experiments indicate that the DIDS-sensitive basolateral membrane Cl/HCO3 exchanger in hfRPE is pHi dependent.

Bottom Line: Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area.The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide.This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.

ABSTRACT
In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 +/- 0.05 to 7.14 +/- 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase-mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 +/- 3.3 to 24.0 +/- 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 +/- 1.6 to 6.7 +/- 2.3 microl x cm(-2) x hr(-1) (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H(2)O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

Show MeSH
Related in: MedlinePlus