Studies On The Synergestically Increased Expression Of CCK,PPT, And PDYN By KCI And Forskolin And Their Interactions With PACAP Or BDNF In Neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) was found to be synergistically increased by calcium influx and increased content of cAMP in neurons, and is involved in the regulation of synaptic plasticity and thereby related to the control of emotion and other central nervous system (CNS) function. Brain-derived neurotrophic factor (BDNF) expression is increased by PACAP in neurons, and plays an important role in neuronal survival, differentiation and synaptic plasticity. Proteins encoded by the genes of prodynorphin (PDYN), cholecystokinin (CCK), and preprotachykinin (PPT) are reported to be involved in variety of CNS function, and are likewise related to the development or pathology of affective disorders such as anxiety or depression. It was found that KC1and forskolin, an adenylate cyclase activator, synergistically increased PACAP mRNA expression, and increase BDNF gene expression in a non-synergistical manner. However, it is still unclear whether PACAP and BDNF, as endogenous regulators, interact with PDYN, CCK, and PPT gene. And if there is an interaction, how does it go? Therefore, the present study investigated genes expression induced by calcium influx and increased cAMP content in neurons, as well as interactions of PDYN, CCK, and PPT gene expression with PACAP or BDNF. And further study was done to investigate the underlying mechanism after our discovery of PACAP-induced PDYN expression.Primary cultured rat cortical neurons were employed to investigate mRNA expressiooninduced by KC1and/or forskolin, which result in a calcium influx and increased content of cAMP, by techniques of genechip and quantitative RT-PCR. And further study on the effect and mechanism of PACAP-induced PDYN or BDNF mRNA expression was performed by immuno fluorescence and quantitative RT-PCR. It was found that KC1and forskolin synergistically upregulated mRNA expression of PDYN, CCK, and PPT in neurons. Substance P, the biological active product of PPT, increased the expression of BDNF mRNA via a non-NK1receptor mediated pathway. However, this effect is regulated by NMDA receptor. PDYN mRNA expression and its bioactive protein product, dynorphin A, were significantly increased by the treatment of PACAP, while PDYN mRNA expression was decreased by that of BDNF. a further study on the mechanism of PACAP-induced PDYN mRNA expression was performed. It was found that PACAP increased PDYN mRNA expression in a dose-and time-dependent manner. PACAP had much more of an effect than vasoactive intestinal polypeptide (VIP) on PDYN mRNA expression, suggesting a major role of PAC1receptor in PACAP-induced PDYN mRNA expression. PDYN mRNA expression was increased by forskolin, activator of adenylate cyclase, but not TPA, a potent activator of PKC. Meanwhile, the increase in PDYN mRNA expression induced by PACAP was completely inhibited by pretreatment with H89, an inhibitor of PKA, indicating that PACAP-induced Pdyn mRNA expression is dependent on a PACAP/PAC1/PKA pathway. Furthermore, it was found that PACAP-induced Pdyn mRNA expression was actually enhanced by cycloheximide (CHX), an inhibitor of-protein synthesis, indicating thatt de novo protein synthesis is not necessary for the PACAP-induced PDYN mRNA expression. The PACAP-induced BDNF and c-fos mRNA expression was inhibited by APV whereas the expression of PDYN mRNA induced by PACAP was not affected in the presence of APV, indicating no involvement of NMDA-R activation in the PACAP-induced Pdyn mRNA expression.For the first time we found that depolarization and adenylate cyclase activation synergistically increase PDYN, CCK, and PPT mRNA expression in primary cultured rat cortical neurons; SP induced BDNF expression via a non-NK1receptor mediated, but NMDA receptor mediated pathway; PACAP directly induced PDYN mRNA expression via a PAC1/PKA mediated pathway, indicating that PACAP is an endogenous upstream regulator for inducing PDYN mRNA expression. Considering the distribution and biological activities of PACAP and dynorphins in CNS, it is highly possible that a cascade of PACAP-induced gene expression, including the expression of PDYN, could be involved in regulating not only nociception but also a variety of neuronal functions and diseases in the central nervous system.