Abstract
Purpose: :
Mutation of the gene that encodes the Human analog of the borate transporter, NaBC1 has been reported to cause various corneal hereditary endothelial dystrophies (CHED) and reduced expression is associated with Fuchs Dystrophy. The function of NaBC1 in corneal endothelium has not been investigated. In this study we determined the expression of NaBC1 in BCEC with an aim to characterize its functions.
Methods: :
BCEC were cultured in DMEM with 10% serum. mRNA and protein expression was determined by PCR and SDS PAGE followed by western blotting, respectively. Cellular localization was determined by immuno-fluorescence (IF) microscopy. Effects of different concentrations (0.1, 1.0, 10 mM) of borate on the viability of BCEC cells after incubation for 18h were determined by MTT assay. Transport function of NaBC1 was tested by measuring changes in intracellular pH by incubating cells grown on coverslips with BCECF-AM (2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester), a pH sensitive fluorescent dye and recording fluorescence changes in response to 2 and 10 mM borate in Ringer solution (pH7.5).
Results: :
Using NaBC1 specific primers, PCR yielded a single band of the predicted size. NaBC1 in BCEC was found to be a ~90kDa protein and was comparable to that in a Human CE cell line (positive control). IF suggests that NaBC1 is a basolateral membrane protein. Up to 10mM, borate did not induce significant toxicity (<5% death) compared to control (n=6). Physiological studies showed that exposure to 2 mM borate to the apical cell surface produced an initial cytosolic acidification (0.01 units) followed by an alkalinization (0.025 units). The extent of acidification and alkalinization was greater with 10mM borate, 0.015 & 0.04 pH units, respectively.
Conclusions: :
NaBC1 is expressed in BCEC cells. BCEC forms a suitable in-vitro model to investigate the functional importance of NaBC1 in corneal endothelium and corneal dystrophies. Short term exposure to borate does not induce toxicity. Physiological data are consistent with rapid entry of boric acid followed by Na-dependent borate uptake.
Keywords: ion transporters • cornea: endothelium • stress response