Molecular Pharmacology, Vol 13, 196-215, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Interaction of Epinephrine Metabolites with the Liver Microsomal Electron Transport System

¹ Institute of Pharmacology, University of Milan, and Consiglio Nazionale delle Ricerche Center of Cytopharmacology, Milan, Italy

3,4-Benzpyrene hydroxylase activity, NADPH-neotetrazolium reductase activity, and the NADPH-dependent lipid peroxidation reaction were inhibited by 100 µM epinephrine about 70%, 50%, and 70%, respectively, using either NADPH or an NADPH-generating system consisting of NADP and glucose 6-phosphate dehydrogenase. On the other hand, inhibition of benzpyrene hydroxylase and NADPH-neotetrazolium reductase activity by epinephrine was abolished using NADP and the soluble fraction as NADPH-generating system. Epinephrine itself did not have an inhibitory effect on these two enzyme activities; inhibition was due partly to electron transfer to epinephrine through superoxide-anion radical (O₂[unknown]) at the level of NADPH-cytochrome c reductase and partly to the adrenochrome produced by epinephrine oxidation. Moreover, it was proved that adrenochrome itself was metabolized by liver microsomes consuming NADPH and transformed into leucoadrenochrome, probably via adrenochrome semiquinone, which may also have an inhibitory effect on benzpyrene hydroxylase even though it was much less effective than adrenochrome. It was found that the soluble fraction contained a heat-stable protein factor that blocked adrenochrome formation, thus reducing the inhibition of benzpyrene hydroxylase and NADPH-neotetrazolium reductase activities by epinephrine. This heat-stable protein factor was purified by a fairly simple method and identified as superoxide dismutase (hepatocuprein) from its spectrophotometric and electron paramagnetic resonance spectra and its capacity to scavenge O₂[unknown]. The purified hepatocuprein alone, like the crude heated soluble fraction, had no inhibitory effect on the microsomal hydroxylation reaction and could not prevent the inhibitory action of adrenochrome. Like the heated soluble fraction, it only reduced the inhibitory action of epinephrine on microsomal benzpyrene hydroxylase activity. The addition of purified hepatocuprein alone significantly inhibited the microsomal NADPH-dependent lipid peroxidation reaction at high ionic strength. However, it could not block the inhibitory effect of epinephrine on the lipid peroxidation reaction as did the crude soluble fraction. Thus it is suggested that another mechanism is operative for the lipid peroxidation reaction, because inhibition was not affected by either the soluble fraction or the purified hepatocuprein. The inhibitory mechanism of epinephrine metabolites toward the microsomal mixed-function oxidase system and the mode of action of hepatocuprein are discussed.

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ACKNOWLEDGMENTS We are grateful to Dr. Fabio Campadelli at the Research Center Bollate, Montecatini Edison, S.p.A., Milan, for performing the EPR analysis, and to Dr. Sergio Saracchi of the Faculty of Agriculture, Milan University, for amino acid analyses. We would like to thank Mr. Mario Fittipaldi and Miss Paola Adamoli at the Provincial Laboratory of Public Health, Milan, for their kind help in the atomic absorption studies. We also thank Professor Toshio Yamano of the Department of Biochemistry, Osaka University Medical School, for many valuable suggestions. The expert assistance of Misses Alessandra Favero and Clara Varicchio is gratefully acknowledged.