| Co-Investigator(Kenkyū-buntansha) |
HIROI Toyoko Osaka City University, Medical School, Associate Researcher, 医学部, 助手 (30305643)
IMAOKA Susumu Osaka City University, Medical School, Associate Professor, 医学部, 助教授 (60145795)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
It was very difficult to purify each human and rat CYP2D isoform. Full length cDNAs of human and rat CYP2D isoforms (CYP2D1, 2D2, 2D3, 2D4 and CYP2D6) were isolated, and then these isoforms were expressed in yeast cells. It was investigated whether CYP2D isoforms had affinity for endogenous neuroactive compounds. We used dopamine, norepinephrine, epinephrine, serotonin, histamine, acetylcoline and trace amines as neuroactive compounds. Monoamines such as dopamine, norepinephrine and serotonin and tyramine inhibited CYP2D activities, indicating that these compounds had affinity for CYP2D isoforms. These results suggested that these compounds were candidates for CYP2D substrate. CYP2D6 metabolized these compounds to unidentified metabolites. Tyramine metabolite was idinitified as dopamine using LC/MAS/MAS analysis. This result mean tyramine was hydroxylated to dopamine by CYP2D6. Other CYP isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2E1, and 3A4) did not have this dopamine formation activities. Dopamine formation from tyramine was specific activity of CYP2D isoforms. An affinity of CYP2D6 for tyramine was higher than that of monoamine oxidase, judging from their Km values for tyramine metabolism activities. We prepared liver and brain microsomes from rat and monkey. Western blot analysis showed that CYP2D enzyme proteins existed in these brain microsomes. These brain microsomes had bufuralol 1'-hydroxylation activity, a typical activity for CYP2D isoforms. These brain microsomes also had dopamine formation activities from tyramine. These results suggested that in the brain, CYP2D isoforms formed dopamine from tyramine. Our results suggested that dopamine was formed via not only l-dopa but also tyramine, and CYP2D isoforms had some part in dopamine level by the synthesis from tyramine.
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