Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined.
The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.
D000086:Acetazolamide / Q000494:pharmacology; D000818:Animals; D002256:Carbonic Anhydrases / Q000302:isolation & purification / Q000378:metabolism; D002956:Citrulline / Q000096:biosynthesis*; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005016:Ethoxzolamide / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D008970:Molecular Weight; D013449:Sulfonamides / Q000494:pharmacology*; D014508:Urea / Q000096:biosynthesis*
D007527:Isoenzymes; D013449:Sulfonamides; D002956:Citrulline; D014508:Urea; D002256:Carbonic Anhydrases; D000086:Acetazolamide; D005016:Ethoxzolamide