title: Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP.
abstract: Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient.
authors.affiliation: Laboratory of General and Comparative Biochemistry, University of Liège, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Bettendorff
authors.forename: P
authors.initials: P
authors.lastname: Wins
mesh_terms: D000157:Aconitine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D013831:Thiamine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014701:Veratridine / Q000494:pharmacology*
chemical_list: D001496:Batrachotoxins; D015222:Sodium Channels; D013779:Tetrodotoxin; D014701:Veratridine; D000584:Amiloride; D000255:Adenosine Triphosphate; D013831:Thiamine; D000157:Aconitine