Inhibition of CA V decreases glucose synthesis from pyruvate

Abstract

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 °C in Krebs-Henseleit with 95% O 2/5% CO 2 at pH 7.1 with 5 m m pyruvate, 5 m m lactate, 3 m m ornithine, 10 m m NH 4Cl, 1 m m oleate; with these inclusions both urea and glucose synthesis start with HCO 3 −-requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated K i for each approximately 0.1 m m. In other experiments hepatocytes were incubated at 37 °C in Krebs-Henseleit with 95% O 2/5% CO 2 at pH 7.1 with 10 m m glutamine, 1 m m oleate; with these inclusions glucose synthesis no longer starts with a HCO 3 −-requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated K i of 0.1 m m, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 °C, with 7.5 m m pyruvate, 3 m m ATP, and 10 m m NaHCO 3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.