Abstract

We recently showed that thiamine uptake by neuroblastoma cells is mediated by two saturable transport systems: the first with high affinity for thiamine ( K m = 35 nM) is blocked by veratridine; the other, with low affinity is blocked by Ca 2+. The driving force for thiamine uptake is its phosphorylation to thiamine diphosphate (TDP) by thiamine pyrophosphokinase and subsequent binding of this cofactor to apoenzymes. Our results suggest that cells of neuronal origin possess mechanisms regulating the intracellular concentration of thiamine. At low external thiamine, the vitamin is taken up by a high-affinity transporter and pyrophosphorylated in thiamine diphosphate (TDP) : this is the TDP pool of slow turnover. An intraover extracellular concentration gradient of free thiamine is observed at low external concentration of the vitamin. At higher external thiamine concentration, TDP accumulation is limited by the binding capacitiy to the apoenzymes and unbound TDP (i.e. a small pool of fast turnover) is quickly hydrolyzed. Thiamine is slowly released by the cells by at least two different mechanisms. The first, accounting for a maximum of 50% of total thiamine release, is stimulated by external thiamine and is blocked by veratridine, suggesting that it is a self-exchange mechanism catalyzed by the high affinity thiamine transporter. The remaining thiamine efflux is neither sensitive to veratridine nor to Ca 2+ and its mechanism is unknown. About 25% of intracellular thiamine is not released, even after treatment of the cells with digitonin, thus maintaining an apparent gradient. This suggests a binding or sequestration in intracellular compartments. In neuroblastoma cells, the affinity of the transporter for thiamine is the highest reported so far, but the rate of transport is 2–3 orders of magnitude lower than in hepatocytes. In vivo, liver may be a thiamine reservoir which is replenished at high plasma thiamine concentrations, while the brain would remain able to pump thiamine (albeit slowly) and maintain steep gradients even when the external thiamine concentration becomes very low.

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