Summary 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.
Article Rapid Turnover of Tryptophan Hydroxylase: The Turnover Rate is Unaffected after Elevation of the Enzyme Amount Induced by Treatment of Cells with Calcium Ionophores and Protease Inhibitors was published on August 1, 1996 in the journal Pteridines (volume 7, issue 3).
Abstract A new function of tetrahydrobiopterin (BH 4 ) was demonstrated in vitro in which BH 4 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 (6RBH 4 ) 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 (6SBH 4 ,≤0 .4 mM) did not cause significant release. The results suggest that the effect of 6RBH 4 was highly stereospecific and that it worked extracellularly.
A 69-year-old woman, who developed acute respiratory distress syndrome (ARDS) after coronary artery bypass grafting, underwent venovenous extracorporeal membrane oxygenation (V-V ECMO) because conventional ventilatory support was ineffective. We used a covalently bonded heparin surface ECMO system, including an artificial lung, a centrifugal pump, cannulas, tubing and connectors, that was maintained with low-dose systemic heparinization, the patient was weaned from ECMO after 186 hours. During ECMO, her platelet count was about half of the initial level and markedly elevated thrombin-antithrombin complex (TAT), plasmin-alpha 2 plasmin inhibitor complex (PIC) and D-dimer were decreased by the use of heparin and protease inhibitors. V-V ECMO seems to be useful even in patients with severe adult respiratory failure and can be performed safely if a heparin covalent circuit is applied.
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.
