|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
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.