Morgan Patricio E, Supuran Claudiu T, Casey Joseph R
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Mol Membr Biol. 2004 Nov-Dec;21(6):423-33. doi: 10.1080/09687860400014872.
Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + H+. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- exchange assays. Transport was measured by following changes of intracellular [Cl-] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2',7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM celecoxib inhibited AE1 by 62+/-4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.
碳酸酐酶（CA，EC 4.2.1.1.）催化二氧化碳可逆水合形成碳酸氢根离子（HCO3-）和氢离子（H+）。碳酸氢根转运蛋白催化不能透过细胞膜的碳酸氢根的跨膜转运，与碳酸酐酶协同发挥作用。由于碳酸酐酶和碳酸氢根转运体共用底物碳酸氢根，我们研究了新型碳酸酐酶竞争性抑制剂是否也对碳酸氢根转运体有直接抑制作用。我们将人红细胞膜氯/碳酸氢根交换体AE1在转染的HEK293细胞中表达，作为碳酸氢根转运体模型。在独立于碳酸酐酶活性的氯/硝酸根交换试验以及氯/碳酸氢根交换试验中评估AE1活性。分别使用细胞内荧光报告染料6-甲氧基-N-（3-磺丙基）喹啉鎓和2'，7'-双-（2-羧乙基）-5-（及-6）羧基荧光素，通过跟踪细胞内[Cl-]和pH的变化来测量转运。我们研究了16种不同的碳酸酐酶抑制剂对AE1转运活性的影响。其中12种是新报道的化合物；两种是临床使用的非甾体抗炎药（塞来昔布和伐地考昔），两种是抗惊厥药（托吡酯和唑尼沙胺）。塞来昔布和4种新型化合物显著抑制AE1氯/硝酸根交换活性，EC50值在0.22 - 2.8 microM范围内。显然，体积更大的化合物对AE1的抑制效力更强。使用40 microM的每种化合物进行的最大抑制仅为AE1转运活性的22 - 53%，可能是因为试验是在存在竞争性底物的情况下进行的。在依赖功能性碳酸酐酶产生转运底物的氯/碳酸氢根交换试验中，40 microM塞来昔布使AE1的活性抑制了62±4%。我们得出结论，一些碳酸酐酶抑制剂，包括临床使用的塞来昔布，在临床显著浓度下会抑制碳酸氢根转运。
