Rat renal proximal tubular gluconeogenesis: Possible involvement of nonmitochondrial carbonic anhydrase isozymes

Abstract

The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 m m pyruvate by tubules incubating in 25 m m HCO 3 − but not in 50 m m HCO 3 −: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO 3 − for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO 2 (CO 2 + HCO 3 −). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 m m pyruvate in 25 m m HCO 3 − buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 μ m AZ or with 450 μ m BZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 m m; including 600 μ m BZ or 188 μ m AZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CAV. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 m m malate the rate of glucose synthesis was unaffected by less than 100 μ m AZ or 400 μ m BZ and was decreased maximally by 40 and 20%, respectively, by 125 μ m AZ, 450 μ m BZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 m m increased the rate of glucose synthesis; 600 μ m BZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 m m but 188 μ m AZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incubated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 μ m BZ was included. It is concluded that only when pyruvate is carboxylated does BZ inhibition of CA V affect mitochondrial production of oxaloacetate. It is further concluded that the maximum decrease of glucose synthesis observed with BZ results from a block in gluconeogenesis after phospho enolpyruvate carboxykinase (PEPCK). Greater inhibition of the rate of glucose synthesis was always observed with 188 μ m AZ. This inhibition was independent of substrate concentration, indicating a posterior block; however, the rate of glucose synthesis varied with each substrate, indicating an additional block before PEPCK. It is concluded that AZ and BZ inhibit enzymes involved in gluconeogenesis other than CA V. Since 95% of the CA activity of rat kidney tubules is nonmitochondrial it is suggested that this inhibition may be a secondary effect of inhibition of other CA isozymes.