Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters.
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 3H-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 3H-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, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM 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.
Eshleman
A J
AJ
Medical Research Service, Veterans Affairs Medical Center, and Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland 97201, U.S.A.
Stewart
E
E
Evenson
A K
AK
Mason
J N
JN
Blakely
R D
RD
Janowsky
A
A
Neve
K A
KA
D002352:Carrier Proteins / Q000378:metabolism*; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D065098:Catechol O-Methyltransferase Inhibitors; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000493:pharmacokinetics; D004563:Electrochemistry / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D026901:Membrane Transport Proteins; D009638:Norepinephrine / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D002352:Carrier Proteins; D065098:Catechol O-Methyltransferase Inhibitors; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase; D004298:Dopamine; D009638:Norepinephrine