Sun Xing Cai, Li Jinhua, Cui Miao, Bonanno Joseph A
Indiana University, School of Optometry, Bloomington, Indiana 47405, USA.
Invest Ophthalmol Vis Sci. 2008 Mar;49(3):1048-55. doi: 10.1167/iovs.07-1188.
Carbonic anhydrase activity has a central role in corneal endothelial function. The authors examined the role of carbonic anhydrase IV (CAIV) in facilitating CO(2) flux, HCO(3)(-) permeability, and HCO(3)(-) flux across the apical membrane.
Primary cultures of bovine corneal endothelial cells were established on membrane-permeable filters. Apical CAIV was inhibited by benzolamide or siRNA knockdown of CAIV. Apical CO(2) fluxes and HCO(3)(-) permeability were determined by measuring pH(i) changes in response to altering the CO(2) or HCO(3)(-) gradient across the apical membrane. Basolateral to apical (B-to-A) HCO(3)(-) flux was determined by measuring the pH of a weakly buffered apical bath in the presence of basolateral bicarbonate-rich Ringer solution. In addition, the effects of benzolamide and CAIV knockdown on steady state DeltapH (apical-basolateral compartment pH) after 4-hour incubation in DMEM were measured.
CAIV expression was confirmed, and CAIV was localized exclusively to the apical membrane by confocal microscopy. Both 10 microM benzolamide and CAIV siRNA reduced apparent apical CO(2) flux by approximately 20%; however, they had no effect on HCO(3)(-) permeability or HCO(3)(-) flux. The steady state apical-basolateral pH gradient at 4 hours was reduced by 0.12 and 0.09 pH units in benzolamide- and siRNA-treated cells, respectively, inconsistent with a net cell-to-apical compartment CO(2) flux.
CAIV does not facilitate steady state cell-to-apical CO(2) flux, apical HCO(3)(-) permeability, or B-to-A HCO(3)(-) flux. Steady state pH changes, however, suggest that CAIV may have a role in buffering the apical surface.
碳酸酐酶活性在角膜内皮功能中起核心作用。作者研究了碳酸酐酶IV（CAIV）在促进二氧化碳通量、碳酸氢根（HCO₃⁻）通透性以及HCO₃⁻跨顶端膜通量方面的作用。
在可透膜滤器上建立牛角膜内皮细胞原代培养。通过苯并酰胺或CAIV的小干扰RNA（siRNA）敲低来抑制顶端CAIV。通过测量因顶端膜两侧二氧化碳或HCO₃⁻梯度改变而引起的细胞内pH（pH(i)）变化来测定顶端二氧化碳通量和HCO₃⁻通透性。通过在富含碳酸氢根的基底外侧林格溶液存在的情况下测量弱缓冲顶端浴液的pH来测定从基底外侧到顶端（B到A）的HCO₃⁻通量。此外，还测量了苯并酰胺和CAIV敲低对在DMEM中孵育4小时后的稳态ΔpH（顶端-基底外侧区室pH）的影响。
通过共聚焦显微镜证实了CAIV的表达，且CAIV仅定位于顶端膜。10微摩尔的苯并酰胺和CAIV siRNA均使表观顶端二氧化碳通量降低了约20%；然而，它们对HCO₃⁻通透性或HCO₃⁻通量没有影响。在苯并酰胺和siRNA处理的细胞中，4小时时的稳态顶端-基底外侧pH梯度分别降低了0.12和0.09个pH单位，这与细胞到顶端区室的净二氧化碳通量不一致。
CAIV不促进细胞到顶端的稳态二氧化碳通量、顶端HCO₃⁻通透性或B到A的HCO₃⁻通量。然而，稳态pH变化表明CAIV可能在缓冲顶端表面方面起作用。
