METABOLISM OF CATECHOLAMINES BY CATECHOL-O-METHYLTRANSFERASE IN CELLSEXPRESSING RECOMBINANT CATECHOLAMINE TRANSPORTERS

To determine if catechol-O-methyltransferase (COMT) metabolizes catech olamines within cell lines used for heterologous expression of plasmal emmal transporters and alters the measured characteristics of H-3-subs trate transport, the uptake of monoamine transporter substrates was as sessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cell s) that had been transfected with the recombinant human transporters, Uptake and cellular retention of H-3-catecholamines was increased by u p to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with p otencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT , [H-3]serotonin and [H-3]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 [H-3]dopamine by C6 cells expressing the dopamine trans porter demonstrated that tropolone (1 mM) decreased the apparent K-m o f transport from 0.61 mu M to 0.34 mu M without significantly altering the maximal velocity of transport, These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter depositio n and thus the apparent kinetic characteristics of transport.