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Related Experiment Videos

Human norepinephrine transporter kinetics using rotating disk electrode voltammetry

W B Burnette1, M D Bailey, S Kukoyi

  • 1Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.

Analytical Chemistry
|September 1, 1996
PubMed
Summary

Rotating disk electrode voltammetry effectively measures norepinephrine and dopamine transport in cells. This nonisotopic method quantifies neurotransmitter influx and efflux, aiding transporter studies.

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Area of Science:

  • Electrochemistry
  • Neuroscience
  • Cell Biology

Background:

  • Catecholamine neurotransmitters like norepinephrine (NE) and dopamine (DA) are crucial for neuronal signaling.
  • Understanding their transport mechanisms is vital for neurological research and drug development.
  • The human norepinephrine transporter (hNET) plays a key role in NE reuptake.

Purpose of the Study:

  • To evaluate the utility of rotating disk electrode (RDE) voltammetry for measuring catecholamine transport in mammalian cells.
  • To quantify the kinetics of norepinephrine uptake by cells expressing the human norepinephrine transporter (hNET).
  • To observe and characterize the efflux of neurotransmitters from cells using RDE.

Main Methods:

  • Utilized RDE voltammetry to monitor the oxidation current decrease of NE and DA at +0.45 V vs Ag/AgCl.

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  • Assessed initial transport rates by measuring the rapid decline in extracellular neurotransmitter concentration after addition to LLC-NET cell suspensions.
  • Induced and measured neurotransmitter efflux using RDE by adding substrates like d-amphetamine (d-AMPH) and p-tyramine (p-TYR).

    • Main Results:

    • Demonstrated saturable norepinephrine uptake with Vmax of 197 +/- 17 amol min-1 cell-1 and KM of 1.64 +/- 0.46 microM.
    • Successfully observed and quantified outward transport (efflux) of NE and DA induced by d-AMPH and p-TYR.
    • Showcased accelerated NE uptake in p-TYR preloaded cells, distinguishing transporter-mediated uptake from passive diffusion.

    Conclusions:

    • RDE voltammetry is a valuable nonisotopic technique for quantifying neurotransmitter influx and efflux in transfected mammalian cells.
    • The study provides kinetic parameters for NE transport mediated by hNET.
    • RDE enables detailed characterization of transporter activity, including substrate-induced efflux and carrier-mediated uptake phenomena.