Ability of RBL2H3 Cells to Lower Environmental Tetrahydrobiopterin concentration

Abstract

Tetrahydrobiopterin works as a redox-cofactor inside cells for phenylalanine hydroxylase (1), tyrosine hydroxylase (2), tryptophan hydroxylase (3, 4), and nitric oxide synthetase (5, 6). Other functions oftetrahydrobiopterin so far proposed are modulation of more complex cellular functions such as mitosis (7, 8), apoptosis (9-11), and exocytotic release of dopamine (1214) and serotonin (15). Within them, the suggested stimulation of monoamine release by BH4, first demonstrated in the rat brain using a microdialysis technique by Dr. Miwa and his colleagues in the early 90s, drew our attention because it was strongly suggested that BH4 works outside the cells (12). Recently, we established an in vitro experimental system to explore the BH4 function to stimulate monoaminergic cells to release monoamine (16). Recent work revealed an outline of this process. 6R-L-erythro-5,6,7,8tetrabydrobiopterin (6RBH4) administered .to culture cells of the RBL2H3 cell line was effective in releasing cellular serotonin at around 10-11 M, while an unnatural diastereomer at the C6 position, 6SBH4, was not effective even at much higher concentrations, Furthermore, 6SBH4 was found to be a strong antagonist to 6RBH4 in stimulating serotonin release. This specificity is a remarkable difference from the case when BH4 was utilized as a redox-cofactor for monooxygenases. As the redox-cofactor of these enzymes, 6SBH4 works relatively well in vitro (17), although it as well as 6RBH4. The other remarkable point was that 6RBH4 administered in the medium worked from outside the cells. These studies were made by measuring serotonin release with RBL2H3 cells, of a mast cell-like neoplastic cell origin, and serotonin-loaded PC-12 cells, of a pheochromocytoma origin, both having ability to release monoamines in response to physiological stimulation. These cells have been employed in many studies to explore generic functions of mast cells and sympathetic neurons. Between these cells, no essential differences were found with respect to the BH4-response. Therefore, these observations suggested that BH4 might work as a signal mediator to regulate cellular functions of a wide variety of cells in tissue. As for the suggested function of BH4 as being physiologically significant, however, at least two questions should be answered: 1) when and where BH4 is released and 2) how the environmental BH4 concentration is kept low in the real tissues enough to lower the threshold level or to raise the signal/noise ratio. Two possible mechanisms were expected: one was a high affinity uptake of environmental BH4 and the other was active breakdown of BH4 by quick uptake of BH4 and oxidation of it. 'This work focused on the latter mechanism. Observations described in this paper were presented at the 12th International Conference on Pteridine and Folate, Mar. 18-25, St. Moritz, Switzerland.