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 transfected with norepinephrine (NE) transporter (NET) cDNA. 2. NET activity was studied by incubating cells with [3H]-NE (0. 5 microCi ml-1, 20 min) in a Na+ containing medium. Incubation with [3H]-NE lead to [3H] accumulation at 47797+/-4864 d.p.m. per well. Specific inhibitors of NET abolished this uptake. 3. During post-uptake incubation, [3H] leaked rapidly from cells and the extracellular phase comprised 89% of total radioactivity within 40 min. Both [3H] retention and [3H] 'leakage' were largely unaffected by inhibitors for MAO. In contrast, COMT inhibitors, U-0521 and Ro 41-0960, dose-dependently increased intracellular [3H]-NE retention with a maximal increase of 4.5 fold. The EC50 for Ro 41-0960 was 139-times lower than that of U-0521. U-0521 largely inhibited [3H] 'leakage' and doubled the apparent Vmax for [3H]-NE uptake. 4. Addition of U-0521 during uptake incubation increased intracellular NE content by 8 fold. Normetanephrine, the COMT-dependent metabolite of NE, was formed in large quantities during post-uptake incubation. U-0521 significantly inhibited the formation of NMN with an equal preservation of intracellular NE. 5. CHO cells expressing NET possess COMT activity, which is responsible for the metabolism of NE to form lipophilic metabolite normetanephrine. The apparent 'properties' of the NET function expressed in CHO cells changed, after inhibition of COMT, in such a way closer to that described in the native neuronal preparations.
Percy
E
E
Alfred and Baker Medical Unit, Baker Medical Research Institute, St Kilda Road Central, PO Box 6492, Melbourne 8008, Victoria, Australia.
Kaye
D M
DM
Lambert
G W
GW
Gruskin
S
S
Esler
M D
MD
Du
X J
XJ
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D065098:Catechol O-Methyltransferase Inhibitors; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008995:Monoamine Oxidase / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D027981:Symporters; D014162:Transfection
D002352:Carrier Proteins; D065098:Catechol O-Methyltransferase Inhibitors; D004791:Enzyme Inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; D027981:Symporters; D008995:Monoamine Oxidase; D002394:Catechol O-Methyltransferase; D009638:Norepinephrine