Regulation of tetrahydrobiopterin and monoamine transport by cyclic GMP

结果发现，环磷酸鸟苷(CGMP)刺激PC12细胞的囊泡单胺转运活性，cAMP(CAMP)则抑制其活性。我们还发现，cGMP和cAMP都下调了四氢生物蝶呤(BH4)的转运，四氢生物蝶呤是一氧化氮合酶和芳香氨基酸羟基酶的辅助因子，通过各种细胞的质膜。在这项工作中，我们研究了BH4的转运机制以及cGMP对BH4转运和囊泡单胺转运的影响。1.BH4转运：内源性BH4向胞外液的外流和外源BH4向细胞内的内流是由不同的转运系统介导的。BH4的外流存在三条途径：1)转运蛋白途径，属于离子梯度依赖的毒素挤出逆向转运蛋白(德克萨斯)家族。这种类型的BH4转运蛋白被cGMP下调。2)BH4转运途径属于水通道蛋白家族，对渗透压敏感，涉及膜蛋白。因此，该途径受Na-K-ATPase和K、Cl-离子转运体的调节。3)由多药耐药蛋白(MDR)等ABC转运蛋白家族成员介导的途径。另一方面，BH4转运蛋白负责外源性BH4摄取到细胞质中，特异性存在于肠上皮细胞和肾脏中。这种类型的BH4转运活性也在红细胞中被检测到。2.囊泡单胺转运：cGMP以剂量依赖的方式上调囊泡单胺转运。未检测到cGMP对囊泡单胺转运体(VMAT)分子的磷酸化作用。CGMP似乎影响了VMAT分子对囊泡膜的靶向性。研究正在寻找可能参与VMAT的蛋白质，作为cGMP效应的中介，VMAT靶向分泌囊泡膜。

We found that the vesicular monoamine transport activity of neuronal cell line, PC12, was stimulated by cyclic GMP (cGMP) while it was inhibited by cyclic AMP (cAMP). We also found that both cGMP and cAMP down-regulated the transport of tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthase and for aromatic amino acid hydroxylases, through plasma membranes of various cells. In this works, we examined the mechanisms of BH4 transport and the effects of cGMP on the BH4 transport and on the vesicular monoamine transport.1.BH4 transport : Outward flow of endogenous BH4 to extracellular fluid and inward flow of exogenous BH4 into the cells were mediated by different transport systems. Three pathways were found to be present for BH4 outward flow : 1)a pathway by transporter belonged to ion gradient-dependent toxin-extruding antiporter (TEXAN) family. This type of BH4 transporter was down-regulated by cGMP. 2)a pathway which was sensitive to osmotic pressure and involved a membrane protein belonged to aquaporin family for BH4 transport. This pathway was therefore regulated by Na+-K+-ATPase and ion transporters for K+ and Cl- ions. 3)a pathway mediated by a member of ABC transporter family such as multi-drug resistant protein (MDR).On the other hand, BH4 transporter which is responsible for exogenous BH4 uptake into cytoplasm was specifically present in intestinal epithelial cells and in kidney. This type of BH4 transport activity was also detected in erythrocytes.2.Vesicular monoamine transport : Vesicular monoamine transport was up-regulated by cGMP in a dose-dependent manner. Phosphorylation of the vesicular monoamine transporter (VMAT) molecules by cGMP was not detected. cGMP seemed to affect the targeting of VMAT molecules to the vesicular membranes. Studies is in progress to search proteins which might be involved in the VMAT targeting to the secretory vesicle membranes as a mediater of cGMP effect.