Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells.
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 (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). 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 ATB(0,+) and B(0)AT1. 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.
Uchiyama
Tomomi
T
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Yamashina-ku, Japan.
Matsuda
Yasuyuki
Y
Wada
Miyuki
M
Takahashi
Satoru
S
Fujita
Takuya
T
D000409:Alanine / Q000378:metabolism; D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015778:Minor Histocompatibility Antigens; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000378:metabolism / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D026962:Amino Acid Transport System ASC; D015778:Minor Histocompatibility Antigens; D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; C420506:SLC1A5 protein, human; D003609:Dactinomycin; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D003513:Cycloheximide; D012964:Sodium; D006162:Guanylate Cyclase; D000409:Alanine