Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: In relation to covalent binding of its reactive intermediate

Yasuhiro Masubuchi, Shigeki Igarashi, Tokuji Suzuki, Toshiharu Horie, Shizuo Narimatsu

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Abstract

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4- hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis- Menten kinetics, and the K(m) value (1.1 αM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti <Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2- hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti- CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2- hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.

Original languageEnglish
Pages (from-to)724-731
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume279
Issue number2
Publication statusPublished - Nov 1996
Externally publishedYes

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Imipramine
Liver Microsomes
Cytochrome P-450 Enzyme System
Hydroxylation
NADP
Enzymes
Microsomes
Proteins
Wistar Rats
N Demethylating Oxidoreductases
Cytochrome P-450 CYP2D6
Desipramine
Quinidine
Antibodies
Propranolol
Sodium Dodecyl Sulfate
Oxides
Polyacrylamide Gel Electrophoresis
Anti-Idiotypic Antibodies
Animal Models

ASJC Scopus subject areas

  • Pharmacology

Cite this

Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes : In relation to covalent binding of its reactive intermediate. / Masubuchi, Yasuhiro; Igarashi, Shigeki; Suzuki, Tokuji; Horie, Toshiharu; Narimatsu, Shizuo.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 279, No. 2, 11.1996, p. 724-731.

Research output: Contribution to journalArticle

Masubuchi, Yasuhiro ; Igarashi, Shigeki ; Suzuki, Tokuji ; Horie, Toshiharu ; Narimatsu, Shizuo. / Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes : In relation to covalent binding of its reactive intermediate. In: Journal of Pharmacology and Experimental Therapeutics. 1996 ; Vol. 279, No. 2. pp. 724-731.
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abstract = "Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4- hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis- Menten kinetics, and the K(m) value (1.1 αM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti <Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2- hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti- CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2- hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.",
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AU - Horie, Toshiharu

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