Differential expression of Na : K : 2Cl cotransporter, glucose transporter1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues

Little is known about whether culturing corneal limiting layers causes chan ges in the expression of their membrane transporter proteins from those pre sent in fresh tissues. Accordingly, we compared mRNA abundance of three wel l-described types of transporters: water channel aquaporin 1 (AQP1), glucos e transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC p rotein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, an d NKCC was quantified by a lysate nuclease protection assay. NKCC transcrip tion was further characterized by Northern blotting. All data were normaliz ed to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Ex pression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. Aft er the third passage, the endothelial cell mRNA abundance in each case decr eased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there w as a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA prob e hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins wit h apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, m embrane transporter function in culture may not be always indicative of the ir role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithellal and endothelial cells, the re is expression of two different proteins and mRNAs that possibly encode f or secretory (NKCC1) and absorptive (NKCC2) isoforms.