HUMAN NOREPINEPHRINE TRANSPORTER KINETICS USING ROTATING-DISK ELECTRODE VOLTAMMETRY

Rotating disk electrode (RDE) voltamme try is applied to the measureme nt of the transport of the catecholamine neurotransmitters norepinephr ine (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 transporte r(hNET). initial rate of transport was assessed by following the initi al decrease in neurotransmitter after its addition to the cell suspens ion, as measured by the decrease in oxidation current at +0.45 V vs Ag /Cl. The initial rate of norepinephrine uptake was saturable, with V-m ax and R(M) of 197 +/- 17 amol min(-1) cell(-1) and 1.64 +/- 0.46 mu M , respectively. The RDE method also allows observation of outward tran sport (efflux) of the DA or NE previously taken up by the cells, Outwa rd transport was induced by the addition of either d-amphetamine (d-AM PH) or p-tyramine (4-hydroxyphenethylamine, p-TYR), which are also sub strates for the NE transporter, The technique was also used to monitor accelerated NE uptake by cells preloaded with p-TYR, a phenomenon dis tinguishing carriers from channels. Together, these findings document the utility of RDE for the nonisotopic measurement of neurotransmitter influx and efflux from transfected mammalian cells.