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Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

ABSTRACT: Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.

SUBMITTER: Sun XC

PROVIDER: S-EPMC4106483 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

Sun Xing Cai XC Bonanno Joseph A JA

Experimental eye research 20030901 3

Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on ...[more]

PMID: 12907161

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Project description:Key pointsAccording to the HCO3- metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1-A speeds transport by regenerating/consuming HCO3- . The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed in Xenopus oocytes. We confirm the essential elements of the previous experimental observations, taken as support for the HCO3- metabolon hypothesis. However, using our own experimental protocols or those of others, we find that NBCe1-A function is unaffected by cytosolic CAs. Previous conclusions that cytosolic CAs do stimulate NBCe1-A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na-H exchanger. Theoretical analyses show that, although CAs could stimulate non- HCO3- transporters (e.g. Na-H exchangers) by accelerating CO2 / HCO3- -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO3- , CO3= , or NaCO3- ion pairs.AbstractThe HCO3- metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes HCO3- replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol with Xenopus oocytes expressing eGFP-tagged NBCe1-A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1-A carboxy terminus affects background-subtracted NBCe1 slope conductance (GNBC ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (Vh ) far from NBCe1-A's reversal potential (Erev ) for prolonged periods - found that bCA increases total membrane current (ΔIm ), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1-A, injected with bCA and clamped to -40 mV, CO2 / HCO3- exposures markedly decrease Erev , producing large transient outward currents persisting for >10 min and rapid increases in [Na+ ]i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔIm and d[Na+ ]i /dt, it does not reduce GNBC . In oocytes not expressing NBCe1-A, CO2 / HCO3- triggers rapid increases in [Na+ ]i that both hCA II and bCA enhance in concentration-dependent manners. These d[Na+ ]i /dt increases are inhibited by EZA and blocked by EIPA, a Na-H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1-A and injected with bCA, EIPA abolishes the EZA-dependent decreases in ΔIm and d[Na+ ]i /dt. Thus, CAs/EZA produce their ΔIm and d[Na+ ]i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of HCO3- transport, supporting the conclusion that an NBCe1-A- HCO3- metabolon does not exist in oocytes.

Dataset Information

Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

Publications

Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

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