摘要
目的 时空观察PtdIns(4, 5)P2 在细胞内水解及再合成的动态变化过程,并比较渥曼青霉素 (wortmannin)、氯化锂(LiCl)、U73122和新霉素四种阻断剂对PtdIns(4, 5)P2 代谢过程的影响。方法 利用绿色荧光蛋白 (GFP)与磷脂酶Cδ1 (PLCδ1 )PH区 (PLCδ1PH)的融合体 (PLCδ1PH GFP)作为标记PtdIns(4, 5)P2 的特异性荧光探针,使用激光扫描共聚焦显微镜技术动态观察及测量PtdIns( 4, 5 )P2 位置的变化。结果 在CHO细胞,用ATP刺激内源性表达的P2Y受体,可引起PLCδ1PH GFP荧光转位可恢复性的变化;渥曼青霉素和LiCl不影响PLC激活所介导的PLCδ1PH GFP荧光由胞膜到胞浆的转位变化,但可阻断此转位的恢复;U73122可以阻断PLC激活所介导的PLCδ1PH GFP荧光由胞膜到胞浆的转位变化; 新霉素对PLCδ1PH GFP的转位无影响,但可抑制未表达PLCδ1PH GFP的细胞中PLC激活后水解PtdIns(4,5)P2 的作用。结论 PLCδ1PH GFP可作为一种有价值的荧光探针来标记PtdIns( 4, 5 )P2 的动态变化;渥曼青霉素、LiCl、U73122和新霉素四种阻断剂对PtdIns(4, 5)P2 代谢过程有不同的影响;PLCδ1PH GFP可阻断新霉素发挥其抑制PLC水解PtdIns(4, 5)P2 的作用。
Aim To visualize the dynamics of PtdIns(4,5)P 2 hyd ro lysis and resynthesis, and modulate it by pharmacological agents wortmannin, LiC l, U73122 and neomycin. Methods We used a fusion construct of g reen fluorescent protein(GFP) with the PH domain of phospholipase C δ1(PL C δ1PH)(PLC δ1PH-GFP) known to bind PtdIns(4,5)P 2 specifically , and laser-scanning confocal microscopy to trace PtdIns(4,5)P 2 translocatio n. Results Stimulation of endogenous P 2Y receptors by ATP in CHO cells induced a reversible PLC δ1PH-GFP translocation, indicating Pt dIns(4,5)P 2 hydrolysis through the receptor-mediated phospholipase C (PLC) ac tivation. Wortmannin and LiCl did not affect the translocation of PLC δ1PH -GFP from plasma membrane to cytosol but blocked the recovery after the translo cation. The transient translocation from plasma membrane was blocked by the PLC inhibitor U73122 but was not affected by another PLC inhibitor neomycin. However , in the absence of PLC δ1PH-GFP expression, neomycin inlibited the recep tor-induced PLC hydrolysis of PtdIns(4,5)P 2.Conclusion PLC δ1PH-GFP can be used as a valuable fluorescence probe to visualize the dyn amic change of PtdIns(4,5)P 2 in living cells. Wortmannin, LiCl, U73122 and neo mycin have distinct modulation effects on PtdIns(4,5)P 2 metabolism. PLC δ1 PH,when bound to PtdIns(4,5)P 2,prevents neomycin from inhibiting PLC hydro lyzing PtdIns(4,5)P 2.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2005年第1期27-32,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金 (No30270361 )
科技部重大基础研究前提研究专项基金 (No2003CCA00300 )
国家杰出青年科学基金(No30325038)资助课题
