Abstract
High-affinity monoamine transporters are targets for prescribed medications and stimulant drugs of abuse. Therefore, assessing monoamine transporter activity for candidate medications and newly-emerging drugs of abuse provides essential information for industry, academia, and public health. Radiotracer binding and uptake inhibition are the gold standard assays for determining drug–transporter interaction profiles. The combined results from such assays yield a unique biochemical fingerprint for each compound. Over time, different assay methods have been developed to assess transporter activity, and the comparability of data across various assay platforms remains largely unclear. Here, we compare the effects of six well-established stimulants in two different cell-based uptake inhibition assays, one method using adherent cells and the other using suspended cells. Furthermore, we compare the data from transfected cell lines derived from different laboratories and data reported from rat synaptosomes. For transporter inhibitors, IC50 values obtained by the two experimental methods were comparable, but using different transfected cell lines yielded disparate results. For transporter substrates, differences between the two cell lines were less pronounced but the drugs displayed different inhibition potencies when evaluated by the two methods. Our study illustrates the inherent limitations when comparing transporter inhibition data from different laboratories and stresses the importance of including appropriate control experiments with reference compounds when investigating new drugs of interest.
Highlights
High-affinity monoamine transporters (MATs) for serotonin (5hydroxytryptamine [5-HT]), dopamine, and norepinephrine (SERT, dopamine transporter (DAT), and norepinephrine transporter (NET), respectively) are transmembrane proteins of the solute carrier 6 family that transport substrate molecules across the plasma membrane bilayer, using ion gradients (Na+, Cl-) as a driving force
We investigated the effects of different assay conditions and different transfected cell lines on kinetic constants and functional activities of six well-established psychostimulants
We compared the inhibitory potency of these substances among cell lines expressing rat MATs and to previously published data from rat synaptosomes
Summary
High-affinity monoamine transporters (MATs) for serotonin (5hydroxytryptamine [5-HT]), dopamine, and norepinephrine (SERT, DAT, and NET, respectively) are transmembrane proteins of the solute carrier 6 family that transport substrate molecules across the plasma membrane bilayer, using ion gradients (Na+, Cl-) as a driving force. These transporters contain 12 transmembrane helical domains with intracellular amino and carboxy termini (Torres et al, 2003). In addition to approved prescription medications, most stimulant drugs of abuse act on MATs, with especially potent actions at DAT and NET Based on their molecular mechanism of action, psychostimulants can be classified as either uptake inhibitors or releasers. Releasers (e.g., amphetamine) bind to transporter proteins but are subsequently transported into neurons where they increase extracellular transmitter concentrations by disrupting intracellular vesicular storage of the transmitter or by reversing the direction of the membrane transporter flux (Cozzi et al, 1998; Scholze et al, 2000; Rothman and Baumann, 2006; Bulling et al, 2012; Eshleman et al, 2013; Rosenauer et al, 2013; Luethi et al, 2018a)
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