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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, Fienber
2、g AA, Svenningsson 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 others Dopamine- neurontransmitter,cannabinoid(THC),DARPP-32-,C
3、B1,Introduction,CB1 receptor,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: depr
4、ess motor activity ex: catalepsy,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: Wi
5、ld-type mice & DARPP-32 knock-out mice (DARPP-32+/+) (DARPP-32-/-) Wild-type mice & Dopamine D2 receptor knock-out mice Wild-type mice & A2A receptor knock-out mice Drug: CP55,940 - CB1 receptor agonist SR141716A - CB1 receptor antagonist AM404 - A inhibition of anandamide reuptake KW6002 - A2A rece
6、ptor antagonist 以上藥劑溶在含8% tween80 的食鹽水中,Measurnment of catalepsy,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 phosphorylat
7、ion of DARPP-32 at Thr34,DARPP-32,P,Thr75,P,Thr34,CP55,940,在注射0.5mg/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/
8、kg 作用15min後,CP55,940:0.5mg/kg,作用45min,Examined the ability of endogenous 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
9、是 endogenous cannabinoids , 都會造成 phospho-Thr34-DARPP-32 的增加,而SR141716A會抑制phospho-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ca
10、talepsy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,Phospho-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CP55,940 Pho
11、spho-Thr34-DARPP-32 反而下降,0.5 mg/kg 作用60min,在D2 receptor KO mice 注射CP55,940後,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CP55,940 Catalepsy也同樣下降,0.5 mg/kg 作用60min,A2A receptor antagonist,注射KW6002(3mg/kg)後, phospho-Th
12、r34-DARPP-32 降低,CP55,940:0.5mg/Kg KW6002:3mg/Kg 作用60min,與CP55,940比較起來, CP55,940+KW6002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,