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

水疱单胺转运（VMT）在调节细胞外神经递质水平中起着至关重要的作用。我们发现环AMP （cAMP）下调嗜铬细胞瘤PC12细胞的VMT。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。蛋白激酶C （PKC）对VMT的影响也进行了研究。我们证明cAMP对VMT有抑制作用，从而大大增加PC12培养物的细胞外多巴胺水平(J.Neurochem。万,64600 - 607年)。然后，我们建立了一种用洋地黄苷渗透PC12细胞质膜的方法来检测培养PC12细胞的VMT活性。这种方法可以测试内源性神经调节剂和各种药物对VMT的影响（J.Biochem.）。118291 - 296年万)。2。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（es）参与camp依赖性的VMT抑制（Pteridines 6,126-128,1995; FEBS letters .368,411-414,1995）。PKC激活剂抑制VMT，这也是由蛋白磷酸化介导的（提交发表）。通过微透析仪将二丁基cAMP （dBcAMP）注入自由运动大鼠纹状体，使细胞外DA浓度升高。从dBcAMP与DA摄取抑制剂利血平和诺非芬的作用比较，dBcAMP的细胞外DA增加被认为是由抑制VMT.5引起的。针对囊泡单胺转运蛋白（VMAT）肽抗体的初步研究表明，转运蛋白的磷酸化可能与PKA或PKC对VMT的抑制无关。这应该用分子遗传学技术进一步研究，例如，突变体VMATcDNA缺乏被蛋白激酶磷酸化的共识位点。

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.