Quantitative microdialysis of serotonin and norepinephrine: pharmacological influences on in vivo extraction fraction.

The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction (Ed) of serotonin (5HT) and norepinephrine (NE). Paroxetine administration decreased the Ed of 5HT in the nucleus accumbens from 24 +/- 3 to 18 +/- 0.2% (p < 0.05). Similarly, desipramine infusion reduced the NE Ed from 35 +/- 2 to 26 +/- 1% (p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the Ed of either 5HT or NE. Perfusion with agonists for the 5HT, alpha-adrenergic, D2 and histamine receptors had no effect on the Ed of 5HT. In the same manner, perfusion with the alpha-adrenergic agonists, methoxamine or clonidine, did not affect the Ed of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change Ed of monoamines. These results suggest that, like DA, changes in the Ed for 5HT or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

Human norepinephrine transporter kinetics using rotating disk electrode voltammetry.

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.