Human norepinephrine transporter kinetics using rotating disk electrode voltammetry.

Burnette WB, Bailey MD, Kukoyi S, Blakely RD, Trowbridge CG, Justice JB
Anal Chem. 1996 68 (17): 2932-8

PMID: 8794928 · DOI:10.1021/ac960022x

Rotating disk electrode (RDE) voltammetry is applied to the measurement of the transport of the catecholamine neurotransmitters norepinephrine (4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, NE) and dopamine (3,4-dihydroxyphenethylamine, DA) in suspensions of LLC-NET cells, a line of porcine kidney cells expressing the human norepinephrine transporter (hNET). Initial rate of transport was assessed by following the initial decrease in neurotransmitter after its addition to the cell suspension, as measured by the decrease in oxidation current at +0.45 V vs Ag/AgCl. The initial rate of norepinephrine uptake was saturable, with Vmax and KM of 197 +/- 17 amol min-1 cell-1 and 1.64 +/- 0.46 microM, respectively. The RDE method also allows observation of outward transport (efflux) of the DA or NE previously taken up by the cells. Outward transport was induced by the addition of either d-amphetamine (d-AMPH) or p-tyramine (4-hydroxyphenethylamine, p-TYR), which are also substrates for the NE transporter. The technique was also used to monitor accelerated NE uptake by cells preloaded with p-TYR, a phenomenon distinguishing carriers from channels. Together, these findings document the utility of RDE for the nonisotopic measurement of neurotransmitter influx and efflux from transfected mammalian cells.

MeSH Terms (16)

Animals Carrier Proteins Cells, Cultured Dopamine Dopamine Plasma Membrane Transport Proteins Electrochemistry Humans Kinetics Membrane Glycoproteins Membrane Transport Proteins Nerve Tissue Proteins Norepinephrine Norepinephrine Plasma Membrane Transport Proteins Swine Symporters Tyramine

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