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.