Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

Abstract

We describe the regulation mechanisms of the Na+-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-dl-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na+-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (Vmax) of Na+-dependent alanine uptake in Caco-2 cells without affecting the Michaelis–Menten constant (Kt). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na+-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na+-dependent neutral amino acid transporter ASCT2, but not the other Na+-dependent neutral amino acid transporters ATB0,+ and B0AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.