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

囊泡单胺转运（VMT）在细胞外神经递质水平的调节中起着至关重要的作用。我们发现，环磷酸腺苷（cAMP）下调VMT在嗜铬细胞瘤PC 12细胞。本课题研究了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。蛋白激酶C（PKC）对VMT的影响也被检测。1.我们证明cAMP对VMT具有抑制作用，从而大大增加了PC 12培养物的细胞外多巴胺水平（J.Neurochem.64，600 -607，1995）。建立了毛地黄皂苷透化PC 12细胞膜测定VMT活性的方法。这种方法使得测试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，蛋白磷酸化过程参与cAMP依赖性VMT抑制（Pteridines 6，126 - 128，1995; FEBS Lett.368，411 - 414，1995）。3. PKC激活剂抑制VMT，这也是由蛋白磷酸化介导的（提交出版）。4.通过微透析装置将二丁酰cAMP（dBcAMP）施用到自由活动大鼠的纹状体中增加了细胞外DA浓度。从dBcAMP与DA摄取抑制剂利血平和诺米芬辛的这种作用的比较来看，dBcAMP引起的细胞外DA含量的增加可能与抑制VMT有关。5.用抗囊泡单胺转运体（VMAT）抗体的初步研究肽的研究表明，转运蛋白的磷酸化可能不参与PKA或PKC对VMT的抑制。分子遗传学技术，例如，缺乏蛋白激酶磷酸化的共有位点的突变VMATcDNA。

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.