THIAMINE HOMEOSTASIS IN NEUROBLASTOMA-CELLS

We recently showed that thiamine uptake by neuroblastoma cells is medi ated by two saturable transport systems: the first with high affinity for thiamine (K-m = 15 nM) is blocked by veratridine; the other, with low affinity is blocked by Ca2+. The driving force for thiamine uptake is its phosphorylation to thiamine diphosphate (TDP) by thiamine pyro phosphokinase and subsequent binding of this cofactor to apoenzymes. O ur results suggest that cells of neuronal origin possess mechanisms re gulating the intracellular concentration of thiamine. At low external thiamine, the vitamin is taken up by a high-affinity transporter and p yrophosphorylated in thiamine diphosphate (TDP) : this is the TDP pool of slow turnover. An intra-over extracellular concentration gradient of Free thiamine is observed at low external concentration of the vita min. At higher external thiamine concentration, TDP accumulation is li mited by the binding capacity to the apoenzymes and unbound TDP (i.e. a small pool of fast turnover) is quickly hydrolyzed. Thiamine is slow ly released by the cells by at least two different mechanisms. The fir st, accounting for a maximum of 50% of total thiamine release, is stim ulated by external thiamine and is blocked by veratridine, suggesting that it is a self-exchange mechanism catalyzed by the high affinity th iamine transporter. The remaining thiamine efflux is neither sensitive to veratridine nor to Ca2+ and its mechanism is unknown. About 25% of intracellular thiamine is not released, even after treatment of the c ells with digitonin, thus maintaining an apparent gradient. This sugge sts a binding or sequestration in intracellular compartments. In neuro blastoma cells, the affinity of the transporter for thiamine is the hi ghest reported so far, but the rate of transport is 2-3 orders of magn itude lower than in hepatocytes. In vivo, liver may be a thiamine rese rvoir which is replenished at high plasma thiamine concentrations, whi le the brain would remain able to pump thiamine (albeit slowly) and ma intain steep gradients even when the external thiamine concentration b ecomes very low.