Voltammetric Approaches to Kinetics and Mechanism of the Norepinephrine Transporter

Abstract

Rotating-disk electrode voltammetry (RDEV) has been used to determine kinetic arameters of dopamine (DA) and norepinephrine (NE) at the hNET in LLC-NET cells. As uptake occurs, the decreasing current is recorded for 6 min as a new steady-state is approached. To induce efflux, 6 μ l of unbuffered electrolyte as a control or 1, 10, or 100 μ M of selected substrate is added to the suspension, and the efflux of DA is monitored. At first, there is little l-amphetamine relative to the high concentration of DA on the inside of the cells to compete for outward transport. However, as it accumulates intracellularly, it competes with DA for binding to the empty transporter and DA efflux decreases. At the same time, DA concentration in the medium increases, causing an increase in the inward flux of DA. Amphetamine is known to inhibit the uptake of norepinephrine across the neuronal membrane and participate in the transmitter's release through the transporter. It was hypothesized that amphetamine-induced release was through an exchange diffusion mechanism. Because of the highly lipophilic nature of amphetamine, it is also capable of entering the cell by diffusion, complicating the analysis. The effect of using methylphenidate as an effluxing agent was also examined. Methylphenidate, as with d -amphetamine, has been used to treat attention deficit disorder with hyperactivity. Its mode of action is believed to be similar to that of cocaine in that it inhibits the DA transporter.