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

Abstract

Ϫ transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na ϩ -3HCO 3 Ϫ cotransporter (NBC1) in addition to a basolateral 1Na ϩ -2HCO 3 Ϫ cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO 3 Ϫ permeability and whether the cotransporter participates in transendothelial net HCO 3 Ϫ 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 Ϫ permeabilities were determined by measuring the rate of pHi change when HCO 3 Ϫ was removed from the bath under constant pH or constant CO2 conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO 3 Ϫ permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO 3 Ϫ permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO 3 Ϫ flux was 0.707 Ϯ 0.009 mM ⅐ min Ϫ1 ⅐ cm 2 in the basolateral-to-apical direction and increased to 1.74 Ϯ 0.15 when cells were stimulated with 2 M 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 Ϫ flux to 0.236 Ϯ 0.002. NBC1 siRNA treatment or 100 M ouabain also eliminated steady-state HCO 3 Ϫ flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO 3 Ϫ permeability, basolateral-to-apical fluxes, and net HCO 3 Ϫ flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO 3 Ϫ flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport THE ION AND FLUID TRANSPORT PROPERTIES of the corneal endothelium are responsible for maintaining the hydration and transparency of the cornea. Corneal endothelial fluid transport requires the presence of HCO 3 Ϫ (15, 18, 29) and Cl Ϫ (44), is sensitive to carbonic anhydrase inhibitors (19,22,29), and is completely eliminated by ouabain, a Na ϩ -K ϩ -ATPase inhibitor. Net stroma to anterior chamber HCO 3 Ϫ flux is responsible for the measured short-circuit current and the small, negative transendothelial potential (19), suggesting that HCO 3 Ϫ is the primary secreted anion. Although significant progress has been made in identifying and locating plasma membrane transporters in the corneal endothelium, the contribution of the transporters to net HCO 3 Ϫ transport is largely unknown. In this study we have examined the role of the sodium bicarbonate cotransporter (NBC1) in transendothelial HCO 3 Ϫ transport. Previous studies have shown that the uptake of HCO 3 Ϫ across the basolateral membrane of corneal endothelial cells occurs by a potent Na ϩ -dependent, Cl Ϫ -independent, DIDSsensitive, and electrogenic Na ϩ -2HCO 3 Ϫ cotransporter (5, 8, 21...

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

“…Recent studies (Diecke et al, 2004;Li et al, 2005) have confirmed that is secreted from the basolateral to apical side. This secretion is currently hypothesized to be the source of the ion-coupled osmotic fluid pumping.…”

“…This secretion is currently hypothesized to be the source of the ion-coupled osmotic fluid pumping. Uptake of at the basolateral membrane is by co-transport via NBC1b (Li et al, 2005;Sun and Bonanno, 2003;Sun et al, 2000). Apical permeability however, is 3-4 times smaller than basolateral (Bonanno et al, 1999).…”

“…We have previously shown that expression of the basolateral cotransporter (NBC1b) in BCEC could be significantly reduced by this approach (Li et al, 2005). Three small interference RNAs were synthesized according to the sequence 583-603 (RNAi-583), 989-1009 (RNAi-989), 1498-1518 (RNAi-1498) of bCLCA1 ORF.…”

Dependence of cAMP meditated increases in Cl− and HCO3− permeability on CFTR in bovine corneal endothelial cells

“…A recent study showed that siRNA knockdown of NBC significantly reduced basolateral HCO 3 -permeability and eliminated net basolateral to apical fluxes (Li et al 2005). Interestingly, studies using cultured cells indicate that basolateral HCO 3 -permeability is 3-4 times greater than apical permeability (Bonanno et al 1999).…”

Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders

“…This strategy has been used to explore the function of microsomal glutathione S-transferase 1 in protection of RPE cells from oxidative stress, 29 the effect of loss of mitochondrial DNA sequences on RPE cells, 30 the role of NeuroD in photoreceptor formation, 31 the role of stem cell factor/c-Kit in tumorigenesis of uveal melanocytes, 32 the role of aldehyde dehydrogenase isozymes or methionine sulfoxide reductase A in oxidative defense of lens cells, 33,34 the role of eukaryotic translation initiation factor 5A on tumor necrosis factora-induced apoptosis of lamina cribosa cells, 35 survivalpromoting effects of hepatocyte growth factor on corneal epithelial cells, 36 the role of 1Na + -3HCO 3 À cotransporter in HCO 3 À flux in corneal endothelial cells, 37 TGFb2-induced production of extracellular matrix by astrocytes derived from optic nerve, 38 and regulation of eye development by transcriptional control of CCCTC binding factor. 39 …”