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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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CA II dependence of apical membrane Na/2HCO3 cotransporter. Low HCO3 (2.62 mM) Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 250 µM of apical DZA. After DZA washout, low apical bath [HCO3]-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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fig5: CA II dependence of apical membrane Na/2HCO3 cotransporter. Low HCO3 (2.62 mM) Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 250 µM of apical DZA. After DZA washout, low apical bath [HCO3]-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: CA II catalyzes the interconversion of CO2 and HCO3 in the cytosol, and CA inhibition by DZA may affect apical Na/2HCO3 cotransport activity. We tested this notion by decreasing apical bath [HCO3] (10-fold) and compared the resultant pHi and TEP responses in the presence of 250 µM of apical DZA to that in control (Fig. 5). In five experiments, DZA decreased apical bath Δ[HCO3]-induced TEP response by 60% (from 2.25 ± 0.81 to 0.89 ± 0.29 mV; P < 0.01) and increased the pHi response from 0.11 ± 0.01 to 0.19 ± 0.01 (P < 0.01). The effect of DZA on the pHi and TEP responses was partially reversible after a 5-min washout in control Ringer (ΔTEP = 1.27 ± 0.46 mV; ΔpHi = 0.17 ± 0.02). The reduced apical bath Δ[HCO3]-induced TEP response in the presence of DZA indicates inhibition of apical Na/2HCO3 cotransport activity. On the other hand, the apical bath Δ[HCO3]-induced acidification was larger in the presence of DZA because CA II inhibition reduces intracellular CO2/HCO3 buffering capacity, which compromises the ability of the RPE to buffer the acidification caused by HCO3 efflux from the apical membrane.


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)

CA II dependence of apical membrane Na/2HCO3 cotransporter. Low HCO3 (2.62 mM) Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 250 µM of apical DZA. After DZA washout, low apical bath [HCO3]-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

fig5: CA II dependence of apical membrane Na/2HCO3 cotransporter. Low HCO3 (2.62 mM) Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 250 µM of apical DZA. After DZA washout, low apical bath [HCO3]-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: CA II catalyzes the interconversion of CO2 and HCO3 in the cytosol, and CA inhibition by DZA may affect apical Na/2HCO3 cotransport activity. We tested this notion by decreasing apical bath [HCO3] (10-fold) and compared the resultant pHi and TEP responses in the presence of 250 µM of apical DZA to that in control (Fig. 5). In five experiments, DZA decreased apical bath Δ[HCO3]-induced TEP response by 60% (from 2.25 ± 0.81 to 0.89 ± 0.29 mV; P < 0.01) and increased the pHi response from 0.11 ± 0.01 to 0.19 ± 0.01 (P < 0.01). The effect of DZA on the pHi and TEP responses was partially reversible after a 5-min washout in control Ringer (ΔTEP = 1.27 ± 0.46 mV; ΔpHi = 0.17 ± 0.02). The reduced apical bath Δ[HCO3]-induced TEP response in the presence of DZA indicates inhibition of apical Na/2HCO3 cotransport activity. On the other hand, the apical bath Δ[HCO3]-induced acidification was larger in the presence of DZA because CA II inhibition reduces intracellular CO2/HCO3 buffering capacity, which compromises the ability of the RPE to buffer the acidification caused by HCO3 efflux from the apical membrane.

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