| Objective: The cAMP-dependent protein kinase(PKA),protein kinase C(PKC)and Src family kinases(SFKs)play important roles in the regulation of neuroplasticity.In this study,we investigated the actions of PKA,PKC and SFKs in the regulation of neuronal excitability in cultured ARC neurons and explore the mechanism.Methods: 1.Primary cultured ARC neurons were isolated from newborn Sprague-Dawley rats(P0-P1).2.The whole-cell patch clamp recordings were performed to measure the neuronal excitability of ARC neurons.3.The expressions of p PKA,p PKC and p SFKs in ARC tissues were determined by western blot analysis;4.We investigated the translocation of PKC by immunofluorescent staining and Western blot analysis.Results: 1.The electrophysiological data indicated that:(1)The action potential firing frequency of cultured ARC neurons was significantly increased by application of PKA agonists in a dose-dependent manner,which is completely blocked by pretreatment of the PKA inhibitor KT5720.(2)PMA,a PKC agonist,dose-dependently enhanced the excitability of ARC neurons at 1 ?M,5 ?M and 10 ?M.This effect was abolished by the PKC inhibitor GF109203 x or CC(Chelerythrine chloride).(3)Application of the SFKs activator EPQ(p Y)EEIPIA up-regulated the firing activity of ARC neurons.The firing frequency began to increase after 3 minutes of treatment,and the effect was significant after 10 minutes or 15 minutes of EPQ(p Y)EEIPIA application(P < 0.01).Pre-incubation of PP2,a SFKs inhibitor,completely blocked the increased excitability induced by EPQ(p Y)EEIPIA.(4)Application of PP2 significantly abolished the increase of firing frequency induced by PKA or PKC agonists,while its inactive analogue PP3 did not elicit so such effects(P < 0.05,P < 0.01).(5)The effect of SFKs on excitability of ARC neurons could be blocked by the PKC inhibitor GF109203 x,but not the PKA inhibitor KT5720.2.By Western blot analysis,our results showed that:(1)The expression of p PKA and p SFKs at Y416 was significantly increased after PKA activation,while the expression of p PKC(pan)and p PKC?/?? was not affected.Pre-incubation of KT5720 inhibited the increase of p PKA and p SFKs expression induced by the PKA agonists.(2)After treatment of cells with PMA,the expression of p PKC(pan),p PKC?/?? as well as p SFKs(Y416)were increased significantly,while the expression of p PKA remained unchanged.Pretreatment with the PKC inhibitors GF109203 x and CC,the increase of p PKC(pan),p PKC?/?? and p SFKs expression induced by PMA was abolished.(3)Pretreatment of cells with PP2 eliminated the changes of p SFKs induced by PKA activation,while the increase of p PKA expression were not affected.Pre-incubation of PP2 inhibited the increase of p SFKs expression induced by PKC activation,while the expression of p PKC(pan)and p PKC?/?? remained unchanged.3.Immunocytochemistry and western blot analysis showed that PMA induced the translocation of PKC? and PKC?? from cytoplasma to cellular membrane.This effect was abolished by the PKC inhibitor CC pre-incubation,whereas another PKC inhibitor GF109203 x or the SFKs inhibitor PP2 had no such effects.Conclusions: Our present study has demonstrated that activation of PKA,PKC as well as SFKs significantly increased the firing frequency of ARC neurons.PKA may act as an upstream factor of SFKs.Inhibition of SFKs blocked increase of excitability induced by PKA,while PKA inhibitors did not affect the increase of excitability in ARC neurons induced by SFKs activation.PKCs and SFKs may interact reciprocally,and thereby up-regulate the firing activity in hypothalamic ARC neurons.Inhibition of SFKs eliminated the increase of firing frequency and the increased expression of p SFKs induced by PKC activation,but did not affect the p PKCs expression.Application of PKC inhibitors blocked the SFKs-induced increase of neuronal excitability in ARC neurons.The PKC translocation may not attribute to the regulation of neuronal excitability by PKC. |
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