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Metabolism of Catecholamines by Catechol‐ O ‐Methyltransferase in Cells Expressing Recombinant Catecholamine Transporters
Author(s) -
Eshleman Amy J.,
Stewart Emilie,
Evenson Anna K.,
Mason John N.,
Blakely Randy D.,
Janowsky Aaron,
Neve Kim A.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.69041459.x
Subject(s) - catechol o methyl transferase , catecholamine , recombinant dna , catechol , metabolism , transporter , chemistry , biochemistry , methyltransferase , biology , endocrinology , gene , methylation , allele
To determine if catechol‐ O ‐methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3 H‐substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L‐M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3 H‐catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41‐0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [ 3 H]serotonin and [ 3 H]MPP + , was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 m M ) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [ 3 H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 m M ) decreased the apparent K m of transport from 0.61 µ M to 0.34 µ M without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.