Mitochondrial carbonic anhydrase is involved in rat renal glucose synthesis

At 37 degrees C, pH 7.4, carbonic anhydrase activity (kenz) of disrupted rat renal proximal tubules and cortical mitochondria was 2.5 +/- 0.8 (n = 3) and 0.15 +/- 0.40 (n = 3) ml.mg-1.s-1, respectively. Turnover number for renal mitochondrial carbonic anhydrase (CA V) was 24,000 s-1. CA V activity of intact mitochondria was completely inhibited by 0.15 microM ethoxzolamide (EZ). Intact proximal tubules, prepared from 48-h starved male rats, were incubated at 37 degrees C in 10 mM pyruvate in Krebs-Henseleit bicarbonate saline buffer, 5% CO2-95% O2. The rate of glucose synthesis over 60 min was reduced 50% by including 0.6 microM EZ in the incubation solution. The concentration of NaHCO3 was doubled to 50 mM (with a corresponding decrease in NaCl) and the solution gassed with 10% CO2-90% O2; 2.4 microM EZ no longer decreased glucose synthesis. It was concluded that inhibition of glucose synthesis by EZ was directly a result of inhibiting the carbonic anhydrases. The rate of glucose production was subsequently determined with tubules incubating in a HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) buffer; this rate was decreased 50% by 0.6 microM EZ. These data support the hypotheses that CA V provides HCO3- for pyruvate carboxylase and that CO2 can be provided by tubular metabolism. Intact tubules were incubated in from 5 to 20 mM pyruvate in either 25 or 50 mM HCO3-; in either buffer, the rate of glucose synthesis was similar, increasing with increasing pyruvate concentration. At no pyruvate concentration was there a change in the rate of glucose production when tubules were incubated in 50 mM HCO3- buffer with 1.6 microM EZ. These data also support the hypothesis that CA V provides the HCO3- substrate for pyruvate carboxylation when there is a high rate of intracellular CO2 production and external CO2 is low. It is further concluded that the cytosolic carbonic anhydrase (CA II) and the membrane-bound carbonic anhydrase (CA IV) are not involved in glucose synthesis from pyruvate.