Inhibition of gluconeogenesis and urea synthesis in isolated rat hepatocytes by acetazolamide

Citrulline generation in isolated rat liver mitochondria is markedly inhibited by the carbonic anhydrase inhibitor acetazolamide (Dodgson et al., 1983). This finding suggests that hepatic mitochondrial-matrix carbonic anhydrase may exert an important controlling effect on ureogenesis by supplying bicarbonate for carbamoyl phosphate synthesis. Three different carbonic anhydrase isoenzymes have been identified in mammals and designated CAI, CAII and CAIII. In liver CAII is uniquely sensitive to inhibition by acetazolamide and is present in a 4-fold higher concentration in the hepatocyte cytosol of female rats than in males (Jeffrey et al., 1984). with higher cytosol concentration of CAIII being found in males (Shiels et al., 1984). Sex differences in mitochondrial carbonic anhydrase have not been reported. We have extended these findings by examining the effect of acetazolamide on rates or ureogenesis and gluconeogenesis in isolated rat hepatocytes from male and female rats. Isolated rat hepatocytes were prepared from 48 h-starved male and female SpragueDawley rats by collagenase perfusion and suspended in Krebs bicarbonate buffer (Krebs & Henseleit, 1932) gassed with O-,/CO, (19 : 1). Samples (2ml) of cell suspension (30-60mg wet wt.) were incubated with I .8mM-NH4CI. SmM-L-( +)-lactate and 0.2mM-ornithine in the presence or absence of different concentrations of acetazolamide. Incubations were terminated after either 10 or 20min by the additions of 4.5ml of 0.6M-HCI0,. Urea and glucose were determined in the deproteinized supernatant by standard spectrophotometric methods. Rates of ureogenesis and gluconeogenesis were determined between the 10 and 20min time points. Ureogenesis and gluconeogenesis in male rat hepatocytes were markedly inhibited by acetazolamide (Fig. 1 ) . Maximum inhibition of ureogenesis (7506) occurred at acetazolamide concentrations between 0.5 and I .OmM, with 25% inhibition evident at 0.005 mM. Gluconeogenesis was inhibited by a maximum of 400; at I.0rnM-acetazolamide. with l2:$ inhibition at 0.05mM. An identical degree of inhibition of ureogenesis was obtained with hepatocytes from female rats. These data suggest that inhibition of ciirbonic anhydrase by acetazolamide may decrease the supply of intramitochondrial bicarbonate for both the carbamoyl-phosphate synthetase and pyruvate carboxylase reactions. I t is thus possible that bicarbonate availability may be a limiting factor in both urea and glucose production. This conclusion is supported by the demonstrations of inhibition of ureogenesis by metabolic acidosis at a site proximal to citrulline synthesis (Monson r t ul.. 1984). The observed similarity between acetarolamide inhibition of urea production in hepatocytes from mule and female rats suggests the presence of similar mitochondria1 concentrations in males and females of one or more susceptible carbonic anhydrase isozymes. The possibility of mechanisms other than carbonic anhydrase inhibition being responsible for the effects of acetazolc 20