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N. Nieto Rementeria, N. Macias, A. Acera, T. M. Suarez-Cortes, E. Berra; Expression and Enzymatic Activity Regulation of Carbonic Anhydrases in Human Non-Pigmented Epithelium Cell Line, Dependence on Cell Density. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6103.
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© ARVO (1962-2015); The Authors (2016-present)
The Carbonic Anhydrases (CAs) form a family of enzymes that catalyze the reversible hydration of CO2 to HCO3-. They are compromised in aqueous humor production in Non-Pigmented Epithelium (NPE). CAII and CAIV have a main role in aqueous humor synthesis, and CAXII over-expression has been described in glaucoma patients. The main aim of this study is to characterize the expression of CAII, CAIV, CAIX and CAXII isoforms, evaluate their enzymatic activity in a human NPE established cell line, and to analyze the effect of cell confluence.
The NPE established human cell line, named HCE developed by Dr. M Coca-Prados (Carré DA. et al., 1997), was used as cellular model. For cell density-dependent CA regulation, cells were grown at low (60%) and high confluence (90%). Reverse transcription and polymerase chain reaction (RT-PCR) were performed to analyze mRNA expression of each CA isoform. Both, chemiluminiscent and immunofluorescent Western blots were performed to detect CA isoforms. CA enzymatic activity was indirectly determined based on the p-nitrophenyl acetate (pNPA) assay which determines the CA esterase activity in enzymatic extracts. Additionally, selective inhibitors of CAs were used to evaluate the specific enzymatic activity of different isoforms.
RT-PCR results indicated that CA IX and CAXII are higher expressed on high confluence cell cultures. In contrast, the expression of both CAII and CAIV is not cell-density dependent. Western blot analyses confirmed that CAIV protein expression is not dependent of cell density. The results of p-nitrophenyl acetate (pNPA) assays showed an increased esterase activity in high confluence cultures compared to low confluence cultures.
In conclusion, although enzymatic overall activity was increased in dense cultures, we showed different expression pattern among analyzed CAs, suggesting they could be regulated by different mechanisms and reflecting a different role. CAIX and CAXII induction was associated to dense NPE cultures, probably to counteract acidosis through the regulation of the intra- and extracellular pH, confirming previous observations in tumors. However, CAII and CAIV were not found to be cell-density dependent in NPE cells.
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