The role of carbonic anhydrase inhibitors on anion permeability into ox red blood cells.

Abstract

SUMMARY1. Organic anion permeability in ox red blood cell was measured by studying steady-state self-exchange of oxalate, chosen as a prototypical substrate of the organic anion transport system previously described; chloride self-exchange measured the inorganic anion permeability.2. Carbonic anhydrase inhibitors of the sulphonamide class inhibit both organic anion self-exchange (A-/A-) and chloride self-exchange (Cl-/Cl-) although carbonic anhydrase plays no role in these exchanges. These results confirm the conclusions already published that sulphonamides can act directly on the cellular membrane as specific inhibitors of anion transport.3. There is a correlation between the chemical structure of the sulphonamides and their capacity for inhibiting transmembrane anionic exchange. It is of significance that N-sulphamyl substitution, which abolishes the carbonic anhydrase inhibitory potency, does not destroy anionic inhibitory capacity and may even increase it.4. For each sulphonamide the capacities for inhibiting chloride transport and oxalate transport are strictly identical. Inhibition appears non-competitive.5. The temperature sensitivity of oxalate self-exchange is exactly the same as that of chloride self-exchange. From this, and from the nature of their inhibition by sulphonamides, it is proposed that chloride and organic anions share the same transport mechanism.6. In the light of the present results the chloruretic action of sulphonamides in various tissues, in particular the kidney, is discussed.

Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

“…6A). These results resemble those for AE1-mediated sulfate exchange in erythrocytes (47). Thus, acetazolamide could be used in Xenopus oocytes, as in 293 cells (20), to inhibit endogenous CA2 activity without direct inhibition of kAE1 function.…”

“…Although earlier pharmacological evidence was interpreted to suggest that oocytes lack carbonic anhydrase activity (52), acetazolamide partially inhibited AE1-mediated Cl Ϫ /HCO 3 Ϫ exchange, but not Cl Ϫ /Cl Ϫ exchange in oocytes (Fig. 6) or in red cells (47). This observation led to detection in oocytes of endogenous CA2 polypeptide (Fig.…”

A Transport Metabolon

“…This is because of the high activity of CAII relative to the transport activity of AE1 in red cells requiring doses of acetazolamide in the mM range. Acetazolamide does not directly inhibit AE transport activity because it cannot inhibit chloride/ bicarbonate (25) or chloride/chloride or oxalate self-exchange (48). Thus, the observed decrease in AE transport activity in the presence of acetazolamide was due to the inhibition of CA activity.…”

“…This observation may be due to the fact that endogenous carbonic anhydrase activity of AE1-transfected HEK293 cells is much higher than the total anion exchange activity. Thus, no reduction of anion exchange activity measured in our assay would be expected until CAII was nearly completely inhibited, as found in the red cell system (24,48).…”

“…Effect of CAII Inhibition on Cl Ϫ /HCO 3 Ϫ Exchange Activity-To determine the role of CAII in facilitating AE1 transport activity, we compared the Cl Ϫ /HCO 3 Ϫ exchange activity of cells transiently transfected with AE1 cDNA before and after incubation with acetazolamide, a sulfonamide that inhibits CA enzymatic activity without direct effect on anion exchange (25,48). To measure AE1 transport activity, HEK293 cells grown on coverslips were transiently transfected with AE1 cDNA, loaded with BCECF-AM, a pH-sensitive fluorescent dye, and mounted in a fluorescence cuvette.…”