Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Abstract

Corneal transparency and hydration control are dependent on HCO(3)(-) transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na(+)-3HCO(3)(-) cotransporter (NBC1) in addition to a basolateral 1Na(+)-2HCO(3)(-) cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO(3)(-) permeability and whether the cotransporter participates in transendothelial net HCO(3)(-) flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5-15 nM siRNA decreased NBC1 expression by 80-95%, 4 days posttransfection. Apical and basolateral HCO(3)(-) permeabilities were determined by measuring the rate of pH(i) change when HCO(3)(-) was removed from the bath under constant pH or constant CO(2) conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO(3)(-) permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO(3)(-) permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO(3)(-) flux was 0.707 +/- 0.009 mM.min(-1).cm(2) in the basolateral-to-apical direction and increased to 1.74 +/- 0.15 when cells were stimulated with 2 muM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO(3)(-) flux to 0.236 +/- 0.002. NBC1 siRNA treatment or 100 muM ouabain also eliminated steady-state HCO(3)(-) flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO(3)(-) permeability, basolateral-to-apical fluxes, and net HCO(3)(-) flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO(3)(-) flux and is functional only at the basolateral membrane.

Fig. 1

Effect of $1{\mathrm{Na}}^{+}-3{\mathrm{HCO}}_3^{-}$ cotransporter (NBC1) small interfering RNA (siRNA) on NBC1 expression. A: Western blot analysis of NBC1 in bovine corneal endothelial cells (BCEC). Total cellular protein was extracted from BCEC treated or untreated with different concentrations of NBC1 siRNA and examined at different time points. Protein (30 μg) was subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes overnight and stained with enhanced chemiluminescence (ECL) using rabbit polyclonal anti-NBC1. The major band position of NBC1 is at ~130 kDa. B: densitometric analyses of protein expression from experiments as shown in A. Data were quantified relative to control band intensity, respectively (n = 3). C: decrease in NBC1 protein 4 days after 15 nM siRNA treatment examined using immunofluorescence. Green, NBC1; red, ZO-1; blue, nuclei.

Fig. 2

Western blot analysis of NBC1 and Na⁺-K⁺-ATPase. Total cellular proteins were extracted from BCEC treated or untreated (control, Con) with different concentrations of NBC1 siRNA or siCONTROL siRNA (si-Con), and Western blot analysis was performed using NBC1 antibody, α-subunit of Na⁺-K⁺-ATPase antibody, or β-actin antibody as detailed in MATERIALS AND METHODS.

Fig. 3

Effect of NBC1 siRNA treatment on basolateral and apical ${\mathrm{HCO}}_3^{-}$ fluxes. A: cells untreated or treated with 5 nM NBC siRNA were perfused in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$-rich Ringer on both sides. Basolateral ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ was totally removed under the constant-pH protocol (1: BF, bicarbonate free), and then, after reequilibration, basolateral ${\mathrm{HCO}}_3^{-}$ concentration was reduced from 28.5 to 2.85 mM under the constant-CO₂ protocol (2: LB, low bicarbonate). This sequence was then repeated on the apical side using the same protocols (3 and 4). 1′–4′ indicate the same procedures using siRNA-treated cells. B: maximum change in intracellular pH (pHi) over the initial 20 s (−dpHi/dt). Values are means ± SE (n = 6). **P ≤ 0.001 vs. control.

Fig. 4

Effect of siCONTROL siRNA on basolateral and apical ${\mathrm{HCO}}_3^{-}$ fluxes. A: cells untreated or treated with 5 nM siCONTROL siRNA were perfused in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$-rich Ringer on both sides. The basolateral and then apical ${\mathrm{HCO}}_3^{-}$ concentration was reduced from 28.5 to 2.85 mM under the constant-CO₂ protocol. B: maximum change in pH_i over the initial 20 s. Values are means ± SE.

Fig. 5

Effect of NBC1 siRNA on non-steady-state transendothelial net ${\mathrm{HCO}}_3^{-}$ flux. A: cells treated or untreated with 5 nM NBC1 siRNA were initially perfused in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$-rich Ringer on the basolateral and apical sides. LB solution without HEPES containing 1 μM 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) free acid was then quickly exchanged on the apical side followed by clamping the exit tube on that side. After the initial rise in pH was recorded, the apical solution was replaced with ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$-rich ringer. The chamber was then flipped over, and the same experiment was done for the basolateral side. For forskolin or ouabain experiments, LB solution containing 2 μM forskolin or 100 μM ouabain was perfused across the cells for 2 min before clamping on apical or basolateral side as described above. B to A, basolateral to apical flux; A to B, apical to basolateral. B: maximum slope (ΔpH/s) over the first 20 s. Values are means ± SE (n = 6). *P ≤ 0.005 vs. control. **P ≤ 0.001 vs. control. C: transendothelial net ${\mathrm{HCO}}_3^{-}$ flux. Values are means ± SE (n = 6). *P ≤ 0.005 vs. control. **P ≤ 0.001 vs. control.

Fig. 6

Effect of NBC1 siRNA on steady-state net ${\mathrm{HCO}}_3^{-}$ flux. Cells cultured on Anopore membrane tissue culture inserts were treated or untreated with 15 nM siRNA. Four days later when cells were confluent, the inserts were washed and 200 μl of DMEM containing 1 μM BCECF free acid were placed in the apical compartment, 300 μl were placed on the basolateral side, and cells were incubated for 6 h. Similarly, non-siRNA-treated inserts were prepared in the same manner but were incubated with 100 μM ouabain for 24 h. Samples from apical and basolateral compartments were collected and pH was measured as described in MATERIALS AND METHODS. ΔpH = apical pH − basolateral pH. Values are means ± SE (n = 6). **P ≤ 0.001 vs. control.