姓名施美琪.ppt

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1、姓名:施美琪 系級:生物科技系四年級 學號:91390373,J Neurosci.2005 Sep 14;25(37):8432-8.,Cannabinoid action depends on phosphorylation of dopamine-and cAMP-regulated phosphoprotein of 32 kDa at the protein kinase A site in striatal projection neurons.Andersson M,Usiello A,Borgkvist A,Pozzi L,Dominguez C,Fienberg AA,Sve

2、nningsson P,Fredholm BB,Borrelli E,Greengard P,Fisone G.,名詞解釋:Dopamine-and cAMP-regulated phosphoryprotein of 32 kDa-由dopamine 及 cAMP 所活化之磷酸蛋白(DARPP-32)Projection neuron-A neuron that links one neuropil to one or more othersDopamine-neurontransmitter,cannabinoid(THC),DARPP-32-,CB1,Introduction,CB1 r

3、eceptor,DARPP-32,Thr34,Thr75,P,Depress of motor activity,ex:catalepsy,P,抑制PKA,抑制 PP-1(protein phosphatase-1),CB1 receptor(cannabinoid receptor 1),存在:basal ganglia(由putamen、caudate nucleus、globus pallidus組成),調控 motor behavior,尾核,被殻,蒼白球,G protein coupled receptor 作用:depress motor activity ex:catalepsy

4、,實驗方向,1.利用不同的藥劑來測試DARPP-32磷酸化的程度2.將Thr34 Ala34及Thr75 Ala75,並測試catalepsy情形3.將Dopamine D2 receptor及Adenosine A2 receptor做基因剔除,比較其缺失對phospho-Thr34-DARPP-32的影響,Dopamine Receptor,dopamine D2 receptor 活化後 adenylate cyclase cAMP PKA DARPP-32磷酸化,Materials&Method,Animal:Wild-type mice&DARPP-32 knock-out mice

5、(DARPP-32+/+)(DARPP-32-/-)Wild-type mice&Dopamine D2 receptor knock-out miceWild-type mice&A2A receptor knock-out miceDrug:CP55,940-CB1 receptor agonistSR141716A-CB1 receptor antagonistAM404-A inhibition of anandamide reuptakeKW6002-A2A receptor antagonist 以上藥劑溶在含8%tween80 的食鹽水中,Measurnment of catal

6、epsy,Determination of phosphorylated DARPP-32 將不同的藥劑施打在老鼠身上 並在不同作用時間內將老鼠的頭剪下來 立即放入液態氮中(6s)將striata分離出來 溶在 750l SDS(1%)煮10min,取3l出來做protein 定量 跑SDS-PAGE(10%,load30g)做western blot,Result,Activation of CB1 receptors stimulates phosphorylation of DARPP-32 at Thr34,DARPP-32,P,Thr75,P,Thr34,CP55,940,在注射0.

7、5mg/kg的CP55,940可達最大的phospo-Thr34-DARPP-32,15min,注射CP55,940 1hr後,phospho-Thr34-DARPP-32最高峰注射CP55,940 2hr後,phospho-Thr34-DARPP-32回到baseline,0.5mg/kg,CB1 receptor antngonist SR141716A,會減弱CP55,940增加phospho-Thr34-DARPP-32反應,SR141716A:5mg/kg 作用15min後,CP55,940:0.5mg/kg,作用45min,Examined the ability of endog

8、enous cannabioids(anandamide)to regulate DARPP-32 phosphorylation-use AM404,AM404,-,注射AM404發現其效果和CP55,940類似-皆會使phospho-Thr34-DARPP-32 增加,30min,10mg/kg,SR141716A也同樣會抑制AM404刺激phospho-Thr34-DARPP-32 的能力,結論:利用CB1 receptor agonist 或是 endogenous cannabinoids,都會造成 phospho-Thr34-DARPP-32 的增加,而SR141716A會抑制ph

9、ospho-Thr34-DARPP-32程度,SR141716A:5mg/kg 作用15min後,AM404:10mg/kg,作用45min,PhosphoThr34-DARPP-32 is involved in CB1 receptor-mediated catalepsy,在DARPP-32 KO mice 上注射CP55,940發現catalepsy現象大大減弱,0.5 mg/kg,將Tr34 Ala34,再注射CP55,940,發現catalepsy明顯減弱,突變,0.5 mg/kg,將Thr75 Ala75,再注射CP55,940,發現和wild-type幾乎無差別,突變,Phos

10、pho-Thr34會導致catalepsy,0.5 mg/kg,Intact dopamine D2 and adenosine A2A receptor transmission are regulated for full-cannabinoid action,dopamine和 D2 receptor 結合 adenylate cyclase cAMP PKA DARPP-32磷酸化,block,D2 KO 後再注射CP55,940Phospho-Thr34-DARPP-32 反而下降,0.5 mg/kg 作用60min,在D2 receptor KO mice 注射CP55,940後,

11、catalepsy現象也同樣降低,0.5 mg/kg,A2A receptor KO mice 中加入CP55,940發現phospho-Thr34-DARPP-32 降低,0.5 mg/kg 作用60min,A2A receptor KO mice中加入CP55,940Catalepsy也同樣下降,0.5 mg/kg 作用60min,A2A receptor antagonist,注射KW6002(3mg/kg)後,phospho-Thr34-DARPP-32 降低,CP55,940:0.5mg/Kg KW6002:3mg/Kg作用60min,與CP55,940比較起來,CP55,940+K

12、W6002的catalepsy現象降低,結論:CB1 receptor活化後,進而產生各種效應,需A2A receptor transmission,CP55,940:0.5mg/Kg KW6002:3mg/Kg,A2A receptor,+,PKA,cAMP,D2 receptor,cAMP,-,PKA,DARPP-32,P,discussion,Q:原本D2 receptor 的存在是抑制phospho-Thr34-DARPP-32,但是將D2 receptor KO後,卻發現phospho-Thr34-DARPP-32降低?,A:Glass and Felder(1997)證明當CB1 receptor及D2 receptor在彼此的agonist皆未達飽和狀態且同時被刺激時,會共同形成complex,CB1 receptor,D2 receptor,CB1/D2 complex,G/s,cAMP,Gi/o,THE END,