Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats.
The contents of adenine nucleotides as well as steady-state concentrations of a number of glycolytic, pentose phosphate-pathway and tricarboxylic acid-cycle intermediates were measured in extracts of livers from normal and phenobarbital-treated rats that were perfused with p-nitroanisole. Metabolites were measured in livers that were freeze-clamped during periods of maximal rates of drug metabolism. Treatment of rats with phenobarbital increased rates of p-nitroanisole O-demethylation approx. fivefold. The concentrations of lactate, xylulose 5-phosphate and ribulose 5-phosphate were increased by phenobarbital treatment, whereas that of fructose 1,6-bisphosphate declined. Perfusion of livers with p-nitroanisole produced significant increases in 6-phosphogluconate and ribulose 5-phosphate in livers from phenobarbital-treated rats, but not in livers from control rats. Treatment of rats with phenobarbital caused [NADP(+)]/[NADPH] to change in the direction of more oxidation, as calculated from measured concentrations of 6-phosphogluconate and ribulose 5-phosphate; however, the [NADP(+)]/[NADPH] ratio calculated from ;malic' enzyme was not changed. Additions of p-nitroanisole produced a reduction of NADP(+) as calculated from 6-phosphogluconate dehydrogenase activity, but did not alter the [NADP(+)]/[NADPH] ratio calculated from substrates assumed to be in equilibrium with ;malic' enzyme. Activities of both glucose 6-phosphate dehydrogenase and ;malic' enzyme were increased by phenobarbital treatment. NAD(+) became more reduced as a result of phenobarbital treatment; however, perfusion of livers with p-nitroanisole did not cause a change in the oxidation-reduction state of this nucleotide. Concentrations of adenine nucleotides in livers were not altered significantly by treatment of rats with phenobarbital; however, a significant decline in the [ATP]/[ADP] ratio occurred during mixed-function oxidation of p-nitroanisole in livers from phenobarbital-treated rats, but not in livers from normal rats. Perfusion of livers with two other substrates for mixed-function oxidation, hexobarbital and aminopyrine, produced an increase in the [NADP(+)]/[NADPH] ratio calculated from ;malic' enzyme. In contrast with livers perfused with p-nitroanisole, there was no significant change in adenine nucleotides in livers exposed to hexobarbital or aminopyrine. Addition of 2,4-dinitrophenol (25mum) to the perfusate containing aminopyrine decreased the [ATP]/[ADP] ratio and tended to prevent the oxidation of NADPH observed with aminopyrine alone. Thus in the presence of an uncoupler of oxidative phosphorylation, NADPH generation may exceed its utilization via mixed-function oxidation.
Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats. The contents of adenine nucleotides as well as steady-state concentrations of a number of glycolytic, pentose phosphate-pathway and tricarboxylic acid-cycle intermediates were measured in extracts of livers from normal and phenobarbital-treated rats that were perfused with p-nitroanisole. Metabolites were measured in livers that were freeze-clamped during periods of maximal rates of drug metabolism. Treatment of rats with phenobarbital increased rates of p-nitroanisole O-demethylation approx. fivefold. The concentrations of lactate, xylulose 5-phosphate and ribulose 5-phosphate were increased by phenobarbital treatment, whereas that of fructose 1,6-bisphosphate declined. Perfusion of livers with p-nitroanisole produced significant increases in 6-phosphogluconate and ribulose 5-phosphate in livers from phenobarbital-treated rats, but not in livers from control rats. Treatment of rats with phenobarbital caused [NADP(+)]/[NADPH] to change in the direction of more oxidation, as calculated from measured concentrations of 6-phosphogluconate and ribulose 5-phosphate; however, the [NADP(+)]/[NADPH] ratio calculated from ;malic' enzyme was not changed. Additions of p-nitroanisole produced a reduction of NADP(+) as calculated from 6-phosphogluconate dehydrogenase activity, but did not alter the [NADP(+)]/[NADPH] ratio calculated from substrates assumed to be in equilibrium with ;malic' enzyme. Activities of both glucose 6-phosphate dehydrogenase and ;malic' enzyme were increased by phenobarbital treatment. NAD(+) became more reduced as a result of phenobarbital treatment; however, perfusion of livers with p-nitroanisole did not cause a change in the oxidation-reduction state of this nucleotide. Concentrations of adenine nucleotides in livers were not altered significantly by treatment of rats with phenobarbital; however, a significant decline in the [ATP]/[ADP] ratio occurred during mixed-function oxidation of p-nitroanisole in livers from phenobarbital-treated rats, but not in livers from normal rats. Perfusion of livers with two other substrates for mixed-function oxidation, hexobarbital and aminopyrine, produced an increase in the [NADP(+)]/[NADPH] ratio calculated from ;malic' enzyme. In contrast with livers perfused with p-nitroanisole, there was no significant change in adenine nucleotides in livers exposed to hexobarbital or aminopyrine. Addition of 2,4-dinitrophenol (25mum) to the perfusate containing aminopyrine decreased the [ATP]/[ADP] ratio and tended to prevent the oxidation of NADPH observed with aminopyrine alone. Thus in the presence of an uncoupler of oxidative phosphorylation, NADPH generation may exceed its utilization via mixed-function oxidation.