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A new function of tetrahydrobiopterin (BH4) was demonstrated in vitro in which BH4 stimulated serotonergic cells to release serotonin (5HT). RBL2H3 cells were pre-loaded with serotonin at 100 μM for 6 hours. The cells were able to release serotonin in response to either 30 mM KCl or 30 nM A23187 up to 2.26±2.06% (n=15) or 9.75± 1.38% (n=15 ), respectively. On exposure to 6R-L-erythrotetrahydrobiopterin (6RBH4) at 100 μM, the 5HT-Ioaded cells secreted serotonin up to 8.13± 1.43% of the cellular 5HT within 60 min. The effect was a concentration dependent response and the half maximum output was observed at 1 nM or lower. Sepiapterin (> 10 μM) caused minimal release and 6S-L-erythro-tetrahydrobiopterin (6SBH4,≤0 .4 mM) did not cause significant release. The results suggest that the effect of 6RBH4 was highly stereospecific and that it worked extracellularly.

Cellular uptake of sepiapterin and tetrahydrobiopterin was compared quantitatively with RBL2H3 cells in culture. RBL2H3 is a mast cell line and has the ability to synthesize serotonin. Sepiapterin was incorporated and converted into tetrahydrobiopterin in the cell. The incorporation of sepiapterin was about 10 times faster than tetrahydrobiopterin. A low concentration of tetrahydrobiopterin was incorporated in a saturable manner up to about 10 μM, and it was superimposed upon a linearly concentration- dependent process. Overnight treatment with DAHP depleted endogenous tetrahydrobiopterin. The DAHP treated cells took up tetrahydrobiopterin faster than untreated cells.