| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 83 === GH3腦下腺垂體癌細胞株，經acetylcholine (ACh)24小時前處理後，會增強thyrotropin-releasinghormone(TRH)引發prolactin及C-fos基因表現。本篇進一步探討ACh對TRH刺激C-fos基因表現促進作用機轉。
我們發現以TMB-8(細胞內鈣離子流動抑制劑)，thapsigargin(calcium/ATPase抑制劑，可以枯竭細胞內貯藏的鈣離子)，verapamil或nifedipine(specific L-type calcium channel blockeL)，EGTA(可螯合鈣離子)或Co2+(non-specificcalcium channel blocke)，與TRH共同處理細胞，結果ACh對TRH刺激c-fos基因表現仍有促進作用。KC1(增加鈣離子內流)可以刺激c-fos基因表現，且此作用被Ach促進。以高濃度PMA長期處理細胞以去除protein kinase C，發現ACh促進作用仍然存在，而低濃度PMA所刺激的c-fos基因表現，則可以被ACh所促進。進一步以HA1004為Ser/Thr kinase抑制劑處理細胞，發現無法去除ACh的促進作用，而cAMPanalog 8-Br-cAMP刺激c-fos基因表現，可以被ACh所促進。而在transfection實驗中ACh可以促進TRH刺激FC2 plasmid(c-fos promoter-CAT constructplasmid) CAT活性的表現。
所以根據我們的結果，ACh的促進作用，可能發生在TRH刺激c-fos基因表現的下游蛋白，這個蛋白參與calcium、protein kinase C及cAMP對c-fos基因表現的調控。
We have previously showed acetylcholine (ACh) pretreatment potentiated thyrotropin-releasing hormone (TRH) induced c-fos gene expression in GH3 pituitary tumor cells. In the present study, we investigated the mechanisms underlying this potentiation effect.
In the presence of TMB-8 to inhibit the intracellular calcium mobilization or thapsigargin to deplete intracellular calcium pool, the augmentation on TRH-stimulated c-fos mRNA expression by ACh persisted. Moreover, neither L-type channel blocker verapamil or nifedipine nor EGTA or Co2+ was able to inhibit the potentiation effect of ACh. KC1 increased c-fos mRNA expression, and this effect was potentiated by ACh. These results indicate that calcium is possibly not involved in the potentiation of ACh, but calcium stimulates c-fos mRNA expression were potentiated by ACh. Depleting cellular PKC by high dosage phorbol myristic acetate (PMA) have no effect on the potentiation of ACh. However, the PMA stimulated c-fos expression was potentiated by ACh. In addition, 8-Br-cAMP stimulated c-fos mRNA expression was potentiated in the ACh conditioned cells.
The possibility that the augmentation by ACh was exerted at the transcriptional levels was examined with fusion gene construct composed of chloramphenicol-acetyl transferase (CAT) reporter gene driven by c- fos promoter (FC2 plasmid). Transfection of FC2 plasmid into GH3 cells show that TRH stimulated-CAT activity was increased by ACh pretreatment.
Our results suggest that the signaling pathway downstream of calcium, PKC and cAMP appear to be responsible for the potentiation effect that eventually leads to accelerated c-fos gene transcription in the ACh conditioned cells.
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