Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl⁻/HCO₃⁻ exchanger, AE1

Abstract

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 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM 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 μM 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.

• Cl−/HCO3− anion exchange
• carbonic anhydrase
• sulfonamide
• carbonic anhydrase inhibitor
• pH regulation