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

Nerve cells are particularly sensitive to thiamine deficiency. We stud ied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [C-14]thiamine was taken up through a saturab le high affinity mechanism (K-m = 35 nM). This was blocked by low conc entrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 m u M) and batrachotoxin (IC50 = 0.9 mu M). These effects were not antag onized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the m echanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (K-m = 0.8 mM) , insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibi ted (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intra cellular 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 fr ee thiamine was barely significant. These data suggest a secondary act ive transport of thiamine, the main driving force being thiamine phosp horylation rather than the sodium gradient.