Tamihide Matsunaga

Distribution and characterization of anandamide amidohydrolase in mouse brain and liver

Anandamide (N-Arachidonoylethanolamine) amidohydrolase catalyzing hydrolysis of anandamide was characterized in mice. The enzymatic activity was highest in the liver, followed by the brain and testis. Negligible activity was found in heart, lung and spleen. The activity in brain and liver was mainly localized in the microsomal fractions. Kinetic experiments demonstrated that Km (microM) and Vmax (nmol/min/mg protein) for the brain microsomes were 9.3 and 2.58, respectively, while those for the hepatic microsomes were 180 and 18.9, respectively. The activity in the microsomes from the liver and brain was markedly inhibited by Cu2+, Hg2+, Se4+, phenylmethylsulfonylfluoride and sodium dodecylsulfate. Brain but not hepatic microsomal enzyme activity was inhibited by delta9-tetrahydrocannabinol, cannabidiol and cannabinol. Kinetic parameters demonstrated that the inhibition by the cannabinoids was competitive in nature. Relatively high distribution of the enzyme activity in brain suggests an importance of the enzyme in the central nervous system to regulate the neuromodulatory fatty-acid amides.

A cytochrome P450 isozyme having aldehyde oxygenase activity plays a major role in metabolizing cannabinoids by mouse hepatic microsomes

A cytochrome P450 (designated P450 MUT-2) which catalyses the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid has been purified from hepatic microsomes of untreated male mice. Analysis of NH2-terminal sequence suggests that the isozyme is a member of the P450 2C gene subfamily. P450 MUT-2 exhibited aldehyde oxygenase activity for 11-oxo-delta 8-TH, 11-oxo-delta 9-THC, 11-oxo-cannabinol (11-oxo-CBN) and 9-anthraldehyde together with high activity for the hydroxylation of cannabinoids at the 11-position. Antibody against P450 MUT-2 significantly inhibited the microsomal formation of delta 8-THC-11-oic acid from 11-oxo-delta 8-THC, but not that of 9-anthracene carboxylic acid from 9-anthraldehyde. Major metabolic reactions of delta 8-THC, delta 9-THC and CBN with mouse hepatic microsomes were the 11-hydroxylation (all cannabinoids), 7 alpha-(delta 8-THC) or 8 alpha-hydroxylation (delta 9-THC) and epoxide formation (delta 8- and delta 9-THC). All these reactions except for 7 alpha-hydroxylation of delta 8-THC and alpha-epoxide formation from delta 9-THC were also markedly inhibited by the antibody. These results indicate that P450 MUT-2 is a major enzyme for metabolizing cannabinoids by mouse hepatic microsomes.

cDNA cloning and sequence of CYP2C29 encoding P-450 MUT-2, a microsomal aldehyde oxygenase

A cDNA clone encoding a cytochrome P-450 (P-450) isozyme was isolated from a mouse liver cDNA library. This P-450, designated CYP2C29, the first member of the mouse CYP2C subfamily to be reported, contained the complete coding region of 490 amino acid residues. The deduced amino acid sequence of CYP2C29 exhibited 83% identity with that of the rat CYP2C7 and had an N-terminal sequence identical to that of P-450 MUT-2, a microsomal aldehyde oxygenase. Two peptides, derived from BrCN cleavage of P-450 MUT-2, were also identical to the cDNA deduced protein of CYP2C29. These results indicate that CYP2C29 cDNA encodes P-450 MUT-2.

Structure and Regulation of P450s in the Rat P450iia Gene Subfamily

The rat P450IIA subfamily was characterized at the protein, cDNA, and gene level. The purified IIA1 and IIA2 P-450s displayed distinct positional specificities toward hydroxylation of the prototype substrate testosterone. The IIA1 and IIA2 genes were also regulated differently during development; IIA1 was expressed within 1 week after birth in both males and females, and was specifically suppressed in males at puberty, while IIA2 was not expressed in females and was activated at puberty in males. The cDNA-deduced amino acid sequences of these enzymes showed 88% similarity with interspersed regions of high and low similarity indicative of former gene conversion events. Both the IIA1 and IIA2 genes contained nine exons.

Catalytic activity of cytochrome P450 isozymes purified from rat liver in converting 11-oxo-Δ8-tetrahydrocannabinol to Δ8-tetrahydrocannabinol-11-oic acid

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

Analysis and pharmacotoxicity of feruloyltyramine as a new constituent and p-coumaroyltyramine in Cannabis sativa L.

Feruloyltyramine (FT), a new amide compound, together with p-coumaroyltyramine (p-CT) was isolated and identified in ethanol extract of cannabis seeds. FT and p-CT were also detected in the roots, leaves and resin of Cannabis sativa L. The intracerebroventricular injection of these amides caused hypothermia and motor incoordination in mice, and the maximal effects were caused 160 to 240 min after the injection. Furthermore, p-CT also exhibited cataleptogenic effect in mice, although FT did not show any effect. These results suggest that these amide compounds may be responsible for some pharmacotoxicity of marihuana.

Mouse hepatic microsomal oxidation of aliphatic aldehydes (C8 to C011) to carboxylic acids

Addition of saturated and alpha, beta-unsaturated aliphatic aldehydes (C8 to C11) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from alpha, beta-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.

Fluorometric assay of microsomal aldehyde oxygenase activity: 9-anthraldehyde as a substrate

With regard to hepatic microsomal oxidation of 9-anthraldehyde (9-AA), a fluorometric method for determination of 9-anthracene carboxylic acid (9-ACA) is described. 9-AA was incubated with hepatic microsomes prepared from male ddN mice. 9-ACA formed was fluorometrically (excitation and emission wavelengths of 255 and 458 nm, respectively) quantitated after the separation from 9-AA by an alkali extraction and ethyl acetate reextraction. Hepatic microsomes less than 0.1 mg protein were enough to assay the microsomal aldehyde oxidation. The enzyme in the microsomes that catalyzes the oxidation of 9-AA to 9-ACA has been characterized by this method.

A Novel Metabolite, an Oxepin Formed from Cannabidiol with Guinea‐pig Hepatic Microsomes

The metabolic formation of an oxepin derivative, 3‐pentyl‐6, 7, 7a, 8, 9, 11a‐hexahydro‐1, 7‐dihydroxy‐7, 10‐dimethyldibenzo‐[b,d]‐oxepin, from cannabidiol was studied in‐vitro using guinea‐pig hepatic microsomes.

Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of Δ9-tetrahydrocannabinol by guinea pig liver microsomes

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.