Oxidative demethylation of p-nitroanisole, a cytochrome P450-linked mixed-function oxidation, was evaluated in isolated perfused rat and rabbit lungs. The product, p-nitrophenol, was monitored continuously in the lung effluent by spectrophotometric measurement. Pulmonary p-nitrophenol production in mumol/h per g dry wt was 6.2+/-0.4 by rabbits and 2.0+/-0.3 by rats (mean+/-SE). Maximal activity of the reaction required pulmonary perfusion rates in excess of 60-80 ml/min per g of dry lung. The half-maximal rate of p-nitrophenol production was observed with p-nitroanisole concentration of 13 micron. Pretreatment of rabbits with chlorpromazine increased p-nitroanisole O-demethylation activity by 63% but phenobarbital pretreatment had no effect. Ventilation with 75% carbon monoxide plus 20% O2 reversibly inhibited the reaction. Specific activity of p-nitroanisole demethylase in the microsomal fraction was 0.5 nmol/min per mg protein in rabbit lungs and 0.1 nmol/min per mg protein in rat lungs. Other rabbit lung subcellular fractions compared with microsomes had significantly lower specific activity. This study demonstrates that p-nitroanisole O-demethylation can be continuously monitored in the intact lung and describes conditions necessary for maximal activity of this pathway.
A B S T R A C T The relationship between alveolarPo2 and the rate of 0-demethylation of p-nitroanisole, a model substrate for cytochrome P-450-linked mixedfunction oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically. The Po2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical 02 analyzer. In control lungs ventilated with 5% CO2 in air, the rate ofp-nitrophenol production was -3.1+0.04 (mean+SE; n = 9) umol/h per g dry wt. p-Nitrophenol production was unaltered when 02 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar 02 was 0.1% or less and was abolished during ventilation with 0.005% 02. The rate of the reaction was inhibited by 50% when alveolar Po2 was 0.3 mm Hg representing an intracellular [021 of -0.4 uM. In the presence of metyrapone (0.1-1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07-0.19 ,umol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% 02 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/02 = 0.5). These results indicated that 0-demethylation ofp-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar Po2 is <1 mm Hg.
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