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Mechanisms of cyclic AMP-dependent regulation of vesicular monoamine transport and of extracellular dopamine concentration of dopaminergic neurons in central nervous system.

中枢神经系统中多巴胺能神经元的囊泡单胺转运和细胞外多巴胺浓度的环AMP依赖性调节机制。

基本信息

批准号：
07672027
负责人：
NAKANISHI Nobuo
金额：
$ 1.41万
依托单位：
Meikai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

Vesicular monoamine transport (VMT) plays a vital role in regulation of extracellular neurotransmitter levels. We found that cyclic AMP (cAMP) down-regulated VMT in pheochromocytoma PC12 cells. In this project, we investigated the mechanism of this cAMP action on the VMT,and cAMP effect on extracellular dopamine level in central nervous system. Effect of protein kinase C (PKC) on the VMT was also examined.1.We demonstrated that cAMP has inhibitory effect on VMT and thereby greatly increases extracellular dopamine level of PC12 cultures (J.Neurochem.64,600-607, l995). Then we established an assay method for VMT activity with PC12 cells in culture by permeabilizing their plasma membranes with digitonin. This method made it possible to test the effects of endogenous neuromodulators and various drugs on the VMT (J.Biochem.118,291-296, l995).2.Studies with protein kinase inhibitors and protein phosphatase inhibitors, protein phosphorylation process(es) was shown to be involved in the cAMP-dependent inhibition of VMT (Pteridines 6,126-128,1995 ; FEBS Lett.368,411-414,1995).3.PKC activators inhibited the VMT and this was also mediated by protein phosphorylation (submitted for publication).4.Administration of dibutyryl cAMP (dBcAMP) by microdialysis apparatus into striatum of freely moving rats increased extracellular DA concentration. From the comparison of this effect of dBcAMP with those of DA uptake inhibitors, reserpine and nomifensine, the increase in extracellular DA by dBcAMP was thought to be resulted from the inhibition of VMT.5.Preliminary studies with antibody against vesicular monoamine transporter (VMAT) peptide suggested that phosphorylation of the transporter protein might not be involved in the inhibition of VMT by PKA or PKC.This should be further examined with techniques of molecular genetics, e.g., the mutant VMATcDNA which lacks consensus sites for phosphorylation by protein kinases.

囊泡单胺转运(VMT)在细胞外神经递质水平的调节中起着重要作用。我们发现cAMP(CAMP)下调嗜铬细胞瘤PC12细胞VMT的表达。在本项目中，我们研究了cAMP对VMT的作用机制，以及cAMP对中枢神经系统细胞外多巴胺水平的影响。1.我们证明cAMP对VMT有抑制作用，从而显著增加PC12细胞外多巴胺水平(神经化学杂志，64,600-607,1995)。在此基础上，我们建立了一种测定PC12细胞VMT活性的方法，即用洋地黄素使其质膜通透性。这种方法使得检测内源性神经调节剂和各种药物对VMT的影响成为可能(J.BioChem.118,291-296,1995)。2.蛋白激酶抑制剂和蛋白磷酸酶抑制剂的研究表明，蛋白质磷酸化过程(ES)参与了对VMT的cAMP依赖的抑制(蝶类6,126-128,1995；FEBS Lett.368,411-414,1995)。3.PKC激动剂抑制VMT，这也是通过蛋白质磷酸化(提交发表)。4.微透析器向自由活动大鼠纹状体给予二丁酰cAMP(DBcAMP)增加细胞外DA浓度。从dBcAMP与DA摄取抑制剂利血平和诺米芬辛的作用比较来看，dBcAMP增加细胞外DA的作用被认为是由于抑制VMT。5.抗泡单胺转运体(VMAT)多肽抗体的初步研究表明，转运蛋白的磷酸化可能不参与PKA或PKC对VMT的抑制作用，这需要用分子遗传学技术进一步研究，例如突变的VMATcDNA缺乏蛋白激酶磷酸化的共识位点。