Related Experiment Videos
Catechol-O-methyltransferase activity in CHO cells expressing norepinephrine transporter
E Percy1, D M Kaye, G W Lambert
1Alfred and Baker Medical Unit, Baker Medical Research Institute, St Kilda Road Central, PO Box 6492, Melbourne 8008, Victoria, Australia.
British Journal of Pharmacology
|October 12, 1999
Summary
Chinese Hamster Ovary (CHO) cells expressing the norepinephrine transporter (NET) also have catechol-O-methyltransferase (COMT) activity. Inhibiting COMT enhances norepinephrine retention and alters transporter properties, mimicking native neuronal preparations.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Chinese Hamster Ovary (CHO) cells are widely used for expressing recombinant proteins.
- Understanding the functional expression of neurotransmitter transporters in heterologous systems is crucial for pharmacological research.
- Catecholamine-O-methyltransferase (COMT) and monoamine oxidase (MAO) are key enzymes in catecholamine metabolism.
Purpose of the Study:
- To investigate the presence and activity of catecholamine metabolizing enzymes, specifically COMT and MAO, in CHO cells engineered to express the norepinephrine transporter (NET).
- To characterize the impact of COMT and MAO inhibition on norepinephrine (NE) uptake, retention, and metabolism within these engineered CHO cells.
Main Methods:
- CHO cells were transfected with norepinephrine transporter (NET) cDNA.
- NET activity was assessed by measuring the uptake of radiolabeled [3H]-norepinephrine ([3H]-NE) in the presence and absence of specific transporter inhibitors.
- The effects of COMT inhibitors (U-0521, Ro 41-0960) and MAO inhibitors on [3H]-NE retention and metabolism were evaluated during post-uptake incubation.
Main Results:
- CHO cells expressing NET demonstrated significant uptake of [3H]-NE, which was blocked by NET-specific inhibitors.
- COMT inhibitors dose-dependently increased intracellular [3H]-NE retention and reduced [3H]-NE efflux, while MAO inhibitors had minimal effect.
- COMT inhibition enhanced apparent NET Vmax and altered NE handling, making NET properties in CHO cells more akin to native neuronal preparations.
- Normetanephrine, a COMT-dependent metabolite, was detected, and its formation was inhibited by U-0521, preserving intracellular NE.
Conclusions:
- CHO cells expressing NET possess intrinsic COMT activity responsible for metabolizing NE to normetanephrine.
- Inhibition of COMT activity in these cells significantly alters NE handling and retention.
- COMT inhibition makes the functional properties of NET expressed in CHO cells more representative of native neuronal NET behavior.