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 K(m) of transport from 0.61 μM to 0.34 μM 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of neurochemistry|
|State||Published - Oct 1997|
- Dopamine transporter
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience