Comparisons of uptake and cell surface binding among pyridoxal, pyridoxine, and pyridoxamine in RAW264.7 cells.
OBJECTIVE
Vitamin B6 (B6) suppresses the expression of cyclooxygenase-2 stimulated by lipopolysaccharide in mouse macrophage RAW264.7 cells. The greatest effect is recognized for pyridoxal (PL) compared with pyridoxamine (PM), pyridoxine (PN), and pyridoxal 5'-phosphate (PLP). However, it has not been elucidated why PL has the strongest effect. We compared the uptakes and cell surface interactions among PL, PM, PN, and PLP in RAW264.7 cells.


METHODS
Cyclo-oxygenase-2 mRNA expression was evaluated by real-time polymerase chain reaction. Intracellular B6 concentrations were measured by high-performance liquid chromatography. Interactions of B6s with the cell surface were analyzed using a surface plasmon resonance biosensor. B6 uptake speeds were measured using [(3)H]-PN.


RESULTS
The intracellular PLP levels did not change significantly when cells were cultured in medium containing PL, PM, PN, or PLP. Only PL interacted with the cell surface. Although PM and PN were associated with the cell surface, their binding was only recognized during sample loading. After the change to phosphate buffered saline after sample loading, the binding resonances of PM and PN returned to baseline, whereas that of PL did not. Uptake of [(3)H]-PN was inhibited by non-labeled PN, PL, or PLP, but not PM, at 1 microM. The inhibition rate of PL was higher than those of PN and PLP.


CONCLUSIONS
The inhibition of cyclo-oxygenase-2 mRNA expression by PL may be related to the cell surface interaction of PL, rather than the intracellular PLP level. The uptake mechanism for PN and PL may differ from that for PM.
Kanouchi
Hiroaki
H
Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan. kano@agri.kagoshima-u.ac.jp
Shibuya
Mayumi
M
Tsukamoto
Shuntaro
S
Fujimura
Yoshinori
Y
Tachibana
Hirofumi
H
Yamada
Koji
K
Oka
Tatsuzo
T
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry / Q000493:pharmacokinetics*; D051379:Mice; D011730:Pyridoxal / Q000378:metabolism / Q000493:pharmacokinetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025101:Vitamin B 6 / Q000737:chemistry / Q000493:pharmacokinetics*
D016861:Cyclooxygenase Inhibitors; D012333:RNA, Messenger; D011730:Pyridoxal; D025101:Vitamin B 6; D051546:Cyclooxygenase 2