Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of apical bath CO2 on apical membrane Na/2HCO3 cotransporter. (A) 0.5 mM DIDS was added to the apical bath to obtain initial control response. The DIDS-induced response was then obtained in the presence of 13% apical bath CO2. After washout with control Ringer, DIDS was added to the apical bath to obtain the final control response. (B) 13% CO2-equilibrated Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 0.5 mM of apical DIDS. After DIDS washout, 13% apical CO2-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
getmorefigures.php?uid=PMC2713148&req=5

fig4: Effect of apical bath CO2 on apical membrane Na/2HCO3 cotransporter. (A) 0.5 mM DIDS was added to the apical bath to obtain initial control response. The DIDS-induced response was then obtained in the presence of 13% apical bath CO2. After washout with control Ringer, DIDS was added to the apical bath to obtain the final control response. (B) 13% CO2-equilibrated Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 0.5 mM of apical DIDS. After DIDS washout, 13% apical CO2-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: Apical bath CO2 may be converted into HCO3 by transmembrane CAs on the apical membrane surface, thus stimulating apical Na/2HCO3 cotransport activity. Therefore, we tested the effect of altering apical bath CO2 on apical Na/2HCO3 cotransport activity by comparing apical DIDS (0.5 mM) -induced pHi and TEP responses in control Ringer (5% CO2) to that in 1 or 13% CO2-equilibrated Ringer (Fig. 4 A). In four experiments, apical DIDS-induced pHi and TEP responses in control Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.52 ± 0.33 mV) were the same as that in 13% CO2-equilibrated Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.57 ± 0.67 mV; P > 0.05). Similarly, the apical DIDS-induced pHi and TEP responses in control Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.66 ± 0.59 mV) were the same as that in 1% CO2-equilibrated Ringer (ΔpHi = 0.06 ± 0.02; ΔTEP = 1.31 ± 0.78 mV; n = 5; P > 0.05). To further test the pHi sensitivity of the apical membrane Na/2HCO3 cotransporter, we perfused 13% CO2-equilibrated Ringer into the apical bath in the presence or absence of 0.5 mM of apical DIDS (Fig. 4 B). If increasing apical bath CO2 increases apical Na/2HCO3 cotransport activity, 13% apical CO2 should cause a larger acidification in the presence of apical DIDS compared with control. However, in the presence of apical DIDS, the 13% CO2-induced acidification (ΔpHi = 0.22 ± 0.03) was the same as control (ΔpHi = 0.22 ± 0.02; n = 4; P > 0.05). Collectively, these results lead to the conclusion that apical Na/2HCO3 cotransport activity is not affected by apical CO2-induced alterations in pHi.


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)

Effect of apical bath CO2 on apical membrane Na/2HCO3 cotransporter. (A) 0.5 mM DIDS was added to the apical bath to obtain initial control response. The DIDS-induced response was then obtained in the presence of 13% apical bath CO2. After washout with control Ringer, DIDS was added to the apical bath to obtain the final control response. (B) 13% CO2-equilibrated Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 0.5 mM of apical DIDS. After DIDS washout, 13% apical CO2-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

fig4: Effect of apical bath CO2 on apical membrane Na/2HCO3 cotransporter. (A) 0.5 mM DIDS was added to the apical bath to obtain initial control response. The DIDS-induced response was then obtained in the presence of 13% apical bath CO2. After washout with control Ringer, DIDS was added to the apical bath to obtain the final control response. (B) 13% CO2-equilibrated Ringer was perfused into the apical bath to record the initial control response. This maneuver was repeated in the presence of 0.5 mM of apical DIDS. After DIDS washout, 13% apical CO2-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: Apical bath CO2 may be converted into HCO3 by transmembrane CAs on the apical membrane surface, thus stimulating apical Na/2HCO3 cotransport activity. Therefore, we tested the effect of altering apical bath CO2 on apical Na/2HCO3 cotransport activity by comparing apical DIDS (0.5 mM) -induced pHi and TEP responses in control Ringer (5% CO2) to that in 1 or 13% CO2-equilibrated Ringer (Fig. 4 A). In four experiments, apical DIDS-induced pHi and TEP responses in control Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.52 ± 0.33 mV) were the same as that in 13% CO2-equilibrated Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.57 ± 0.67 mV; P > 0.05). Similarly, the apical DIDS-induced pHi and TEP responses in control Ringer (ΔpHi = 0.05 ± 0.02; ΔTEP = 1.66 ± 0.59 mV) were the same as that in 1% CO2-equilibrated Ringer (ΔpHi = 0.06 ± 0.02; ΔTEP = 1.31 ± 0.78 mV; n = 5; P > 0.05). To further test the pHi sensitivity of the apical membrane Na/2HCO3 cotransporter, we perfused 13% CO2-equilibrated Ringer into the apical bath in the presence or absence of 0.5 mM of apical DIDS (Fig. 4 B). If increasing apical bath CO2 increases apical Na/2HCO3 cotransport activity, 13% apical CO2 should cause a larger acidification in the presence of apical DIDS compared with control. However, in the presence of apical DIDS, the 13% CO2-induced acidification (ΔpHi = 0.22 ± 0.03) was the same as control (ΔpHi = 0.22 ± 0.02; n = 4; P > 0.05). Collectively, these results lead to the conclusion that apical Na/2HCO3 cotransport activity is not affected by apical CO2-induced alterations in pHi.

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