Bonanno J A, Srinivas S P, Brown M
Morton D. Sarver Center for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley 94720, USA.
Exp Eye Res. 1995 Apr;60(4):425-34. doi: 10.1016/s0014-4835(05)80099-5.
Carbonic anhydrase inhibitors are known to inhibit fluid transport by the corneal endothelium. This phenomenon could be due to a combination of effects involving disruption of intracellular pH regulation, reduced gradients for diffusion of CO2, substrate limitation to HCO3- transport systems or direct inhibition of membrane HCO3- transport. We examined the effects of the carbonic anhydrase inhibitor, Acetazolamide (ACTZ), on intracellular pH (pHi) and HCO3- transport in cultured bovine corneal endothelium. The pHi was measured utilizing the pH sensitive fluorescent dye, BCECF. Na+:HCO3- cotransport and Cl-/HCO3- exchange activities were studied by measuring the HCO3(-)-dependent flux of Na+ and Cl-, respectively. Na+ and Cl- fluxes were measured using the ion-sensitive dyes SBFI and SPQ, respectively. Application of 100 or 500 microM ACTZ to cells perfused under HCO3(-)-rich conditions, significantly reduced steady-state pHi by 0.06 +/- 0.01 (n = 14, P < 0.05). ACTZ also eliminated rapid pHi transients due to CO2 diffusion, significantly slowed the initial rate of pHi changes (50 +/- 10% of control, n = 7, P < 0.05) secondary to Na+:HCO3- cotransport or Cl-/HCO3- exchange (37 +/- 1% of control, P < 0.05, n = 7). However, the flux of the cotransported ions, Na+ and Cl-, and the steady-state levels of these ions were not affected by ACTZ. We conclude that the drop in steady-state pHi, the elimination of CO2 induced pHi transients and the slowed pHi changes secondary to HCO3- transport were due to inhibition of cytosolic carbonic anhydrase by ACTZ, i.e. slowing the equilibrium among CO2, HCO3- and H+ and not due to limitation of substrate availability or direct inhibition of the membrane transporters.
已知碳酸酐酶抑制剂可抑制角膜内皮细胞的液体转运。这种现象可能是多种效应共同作用的结果，包括细胞内pH调节紊乱、二氧化碳扩散梯度降低、碳酸氢根转运系统的底物限制或膜碳酸氢根转运的直接抑制。我们研究了碳酸酐酶抑制剂乙酰唑胺（ACTZ）对培养的牛角膜内皮细胞内pH（pHi）和碳酸氢根转运的影响。利用对pH敏感的荧光染料BCECF测量pHi。通过分别测量依赖于碳酸氢根的钠离子和氯离子通量，研究了钠离子：碳酸氢根共转运和氯离子/碳酸氢根交换活性。分别使用离子敏感染料SBFI和SPQ测量钠离子和氯离子通量。在富含碳酸氢根的条件下向灌注的细胞中施加100或500微摩尔的ACTZ，可使稳态pHi显著降低0.06±0.01（n = 14，P < 0.05）。ACTZ还消除了因二氧化碳扩散引起的快速pHi瞬变，显著减慢了继发于钠离子：碳酸氢根共转运或氯离子/碳酸氢根交换的pHi变化的初始速率（为对照的50±10%，n = 7，P < 0.05）（为对照的37±1%，P < 0.05，n = 7）。然而，共转运离子钠离子和氯离子的通量以及这些离子的稳态水平不受ACTZ影响。我们得出结论，稳态pHi的下降、二氧化碳诱导的pHi瞬变的消除以及继发于碳酸氢根转运的pHi变化减慢是由于ACTZ抑制了胞质碳酸酐酶，即减缓了二氧化碳、碳酸氢根和氢离子之间的平衡，而不是由于底物可用性的限制或膜转运体的直接抑制。
