RESUMEN
Aim: To visualize the dynamics of PtdIns (4,5) P2 hydrolysis and resynthesis, and modulate it by pharmacological agents wortmannin, LiCl, U73122 and neomycin. Methods: We used a fusion construct of green fluorescent protein (GFP) with the PH domain of phospholipase Cδ1 (PLC δ1 PH) (PLCδ1 PH-GFP) known to bind PtdIns(4,5) P2 specifically, and laser-scanning confocal microscopy to trace PtdIns (4,5) P2 translocation. Results: Stimulation of endogenous P2Y receptors by ATP in CHO cells induced a reversible PLCδ1 PH-GFP translocation, indicating PtdIns (4,5) P 2 hydrolysis through the receptor-mediated phospholipase C (PLC) activation. Wortmannin and LiCl did not affect the translocation of PLC δ1 PH-GFP from plasma membrane to cytosol but blocked the recovery after the translocation. 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 PLCδ1 PH-GFP expression, neomycin inlibited the receptor-induced PLC hydrolysis of PtdIns(4,5) P2. Conclusion: PLCδ1 PH-GFP can be used as a valuable fluorescence probe to visualize the dynamic change of PtdIns (4,5) P2 in living cells. Wortmannin, LiCl, U73122 and neomycin have distinct modulation effects on PtdIns(4,5) P2 metabolism. PLC δ1 PH, when bound to PtdIns(4,5) P2, prevents neomycin from inhibiting PLC hydrolyzing PtdIns(4,5) P2.
RESUMEN
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 ?1(PL C ?1PH)(PLC ?1PH-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 ?1PH-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 ?1PH -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 ?1PH-GFP expression, neomycin inlibited the recep tor-induced PLC hydrolysis of PtdIns(4,5)P 2.Conclusion PLC ?1PH-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 ?1 PH,when bound to PtdIns(4,5)P 2,prevents neomycin from inhibiting PLC hydro lyzing PtdIns(4,5)P 2.