Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters

This manuscript outlines a high-throughput 96-well plate assay to measure the vesicular uptake of the false fluorescent neurotransmitter FFN206 by the vesicular monoamine transporter 2 (VMAT2) in near real-time and following pharmacological and environmental toxicant exposure in HEK293 cells.

Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters

This manuscript outlines a high-throughput 96-well plate assay to measure the vesicular uptake of the false fluorescent neurotransmitter FFN206 by the vesicular monoamine transporter 2 (VMAT2) in near real-time and following pharmacological and environmental toxicant exposure in HEK293 cells.

Tenofovir Disoproxil Fumarate Is a New Substrate of ATP-Binding Cassette Subfamily C Member 11

ABSTRACT Tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, after conversion to tenofovir (TFV), is mainly eliminated by glomerular filtration and active tubular secretion. The major adverse effect of tenofovir is nephrotoxicity; however, the exact mechanism remains poorly understood. In this study, the ATP-binding cassette subfamily C member 11 (ABCC11; multidrug resistance protein 8 [MRP8]) transporter, which is abundant in proximal tubular cells, was demonstrated to act as an efflux transporter of tenofovir. Real-time PCR (RT-PCR) and indirect immunofluorescence assays were used to determine MRP8 overexpression in a continuous cell line. Tenofovir accumulations were assessed by cytotoxicity, cellular transport, and vesicular uptake assays. Substrate specificity was confirmed using MK-571, an MRP-specific inhibitor, and methotrexate, which served as a known substrate. Intracellular and intravesicular concentrations of tenofovir were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 50% cytotoxic concentration (CC50) of TDF in MRP8-overexpressing cells was 4.78 times higher than that of parental cells. Transport assays also showed that the intracellular accumulation of tenofovir in MRP8-overexpressing cells was 55 times lower than that in parental cells and was partly reversed by MK-571. Similarly, an “inside-out” vesicular uptake assay, using Sf9 inverted membrane vesicles to allow measuring of accumulation of the substrates into the vesicles, demonstrated a higher intravesicular concentration of tenofovir in MRP8-overexpressing vesicles than in Sf9 insect control vesicles. These effects were effectively reversed by increasing concentrations of the specific inhibitor MK-571. In conclusion, tenofovir is a new substrate of the MRP8 transporter. An alteration in the activity of this efflux pump may increase the intracellular accumulation of tenofovir in proximal renal tubular cells.

Vesicular uptake of N-acetylaspartylglutamate is catalysed by sialin (SLC17A5)

NAAG (N-acetylaspartylglutamate) is an abundant neuropeptide in the vertebrate nervous system. It is released from synaptic terminals in a calcium-dependent manner and has been shown to act as an agonist at the type II metabotropic glutamate receptor mGluR3. It has been proposed that NAAG may also be released from axons. So far, however, it has remained unclear how NAAG is transported into synaptic or other vesicles before it is secreted. In the present study, we demonstrate that uptake of NAAG and the related peptide NAAG2 (N-acetylaspartylglutamylglutamate) into vesicles depends on the sialic acid transporter sialin (SLC17A5). This was demonstrated using cell lines expressing a cell surface variant of sialin and by functional reconstitution of sialin in liposomes. NAAG uptake into sialin-containing proteoliposomes was detectable in the presence of an active H+-ATPase or valinomycin, indicating that transport is driven by membrane potential rather than H+ gradient. We also show that sialin is most probably the major and possibly only vesicular transporter for NAAG and NAAG2, because ATP-dependent transport of both peptides was not detectable in vesicles isolated from sialin-deficient mice.