Tetrahydrobiopterin transport through plasma membranes and regulation of nitric oxide- and monoamine-dependent signaling.

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Tetrahydrobiopterin (BH4) in the cells is an essential cofactor of nitric oxide synthase (NOS), and of tyrosine hydroxylase and tryptophan hydroxylase, rate-limiting enzymes in catecholamine and serotonin biosynthetic pathways, respectively. BH4 in the extracellular fluid is also known to induce exocytotic release of catecholamines and serotonin, and to enhance the degradation of nitric oxide (NO). Since BH4 is synthesized in the cells, BH4 transport through plasma membranes is one of the most important processes in the regulation of intra- and extracellular BH4 levels In this project, we studied the BH4 transport through plasma membrane using pheochromocytoma PC12 cells and rat basophilic leukemia RBL2H3 cells. Effect of extracellular BH4 on the cell physiology was also examined.1. BH4 outward flow (BH4 secretion) and BH4 inward flow (BH4 uptake) were performed by different transport systems, respectively. The BH4 outward flow was inhibited by reserpine and tetrabenazine, which are kn … More own to be vesicular monoamine transporters, suggesting that the transporter responsible for BH4 outward flow might be a members of a family of toxin-extruding antiporters (TEXANs).2. Outward flow of BH4 from PC12 cells was down-regulated by both cyclic AMP and cyclic GMP, and protein phosphorylation was suggested to be involved in this down-regulation.3. Although PC12 and RBL2H3 cells can take up BH4 in the medium, BH4 thus incorporated was strictly distinguished from the endogenous BH4 by the cells : BH4 of the exogenous origin was quickly oxidized and subsequently excreted to the medium. Therefore, the addition of sepia pterin, an intermediate in the BH4 salvage pathway, into the medium raised cellular BH4 level more efficiently than BH4 addition into the medium.4. Effects of extracellular BH4 on the phosphorylation of ion transporters are now being examined, since we found that Na+,K+-ATPase and Na+-K+-2CI- cotransporter were regulated by the phosphorylation of the protein molecules and since BH4 was expected to induce intracellular Ca2+ elevation which can trigger the protein phosphorylation reactions. Less