Wu Q, Pierce W M, Delamere N A
Department of Pharmacology and Toxicology, University of Louisville, Kentucky 40292, USA.
J Membr Biol. 1998 Mar 1;162(1):31-8. doi: 10.1007/s002329900339.
Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pHi) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pHi was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pHi. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pHi 0.52 +/- 0.10 pH units. In the presence of DBI, the magnitude of pHi reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 +/- 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by approximately 40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pHi alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pHi changes that might alter the function of other ion transporters and channels in the NPE.
碳酸酐酶（CA）抑制剂可降低房水（AH）向眼内的分泌速率。不同的CA同工酶在这一反应中可能发挥不同作用。在此，我们使用一种与葡聚糖结合的CA抑制剂（DBI），在源自兔非色素上皮（NPE）的细胞系中选择性抑制膜相关CA，研究了碳酸酐酶抑制剂对细胞质pH（pHi）调节的影响。使用荧光染料BCECF测量pHi，并比较pHi对细胞可渗透的CA抑制剂乙酰唑胺（ACTZ）和DBI的反应。ACTZ显著抑制了由去除和重新添加碳酸氢盐/二氧化碳引起的快速pHi变化，但DBI在这方面无效，这与DBI无法进入细胞并抑制细胞质CA同工酶一致。单独添加时，ACTZ和DBI使基线pHi产生类似程度的降低（0.2个pH单位）。我们考虑CA-IV是否可能通过钠-氢交换促进氢离子排出。钠-氢交换抑制剂氨氯地平（1 mM）使pHi降低0.52±0.10个pH单位。在DBI存在的情况下，氨氯地平引起的pHi降低幅度显著（P<0.05）降至0.26±0.09个pH单位。ACTZ同样降低了pHi降低的幅度。DBI还使氯化铵（20 mM）预脉冲酸化的细胞中pHi恢复速率降低了约40%；ACTZ和氨氯地平也导致pHi恢复速率降低。DBI未能改变由提高外部钾浓度引起的pHi碱化反应，该反应对氨氯地平不敏感，但对ACTZ敏感。这些观察结果与在DBI或ACTZ存在下钠-氢交换活性降低一致。我们认为，CA-IV同工酶可能催化质膜外未搅动层中氢离子和碳酸氢根与二氧化碳的快速平衡，防止氢离子局部积累，否则氢离子会与钠离子竞争相同的外部钠-氢交换结合位点。抑制CA-IV可能产生pHi变化，进而可能改变NPE中其他离子转运体和通道的功能。
