Catechol-O-methyltransferase activity in CHO cells expressing norepinephrine transporter

1 We examined the existence of catecholamine metabolizing enzymes (catechol -O-methyltransferase, COMT, and monoamine oxidase, MAO) in CHO cells transf ected with norepinephrine (NE) transporter (NET) cDNA. 2 NET activity was studied by incubating cells with [H-3]-NE (0.5 mu Ci ml( -1), 20 min) in a Na+ containing medium. Incubation with [H-3]-NE lead to [ H-3] accumulation at 47797 +/- 4864 d.p.m. per well. Specific inhibitors of NET abolished this uptake. 3 During post-uptake incubation, [H-3] leaked rapidly from cells and the ex tracellular phase comprised 89% of total radioactivity within 40 min. Both [H-3] retention and [H-3] 'leakage' were largely unaffected by inhibitors f or MAO. In contrast, COMT inhibitors, U-0521 and Ro 41-0960, dose-dependent ly increased intracellular [H-3]-NE retention with a maximal increase of 4. 5 fold. The EC,, for Ro 41-0960 was 139-times lower than that of U-0521. U- 0521 largely inhibited [H-3] 'leakage' and doubled the apparent V-max for [ H-3]-NE uptake. 4 Addition of U-0521 during uptake incubation increased intracellular NE co ntent by 8 fold. Normetanephrine, the COMT-dependent metabolite of NE, was formed in large quantities during post-uptake incubation. U-0521 significan tly inhibited the formation of NMN with an equal preservation of intracellu lar NE. 5 CHO cells expressing NET possess COMT activity, which is responsible for the metabolism of NE to form lipophilic metabolite normetanephrine. The app arent 'properties' of the NET function expressed in CHO cells changed, afte r inhibition of COMT, in such a way closer to that described in the native neuronal preparations.