1. Regulation Of The Proliferation And Migration By CysLT₁ Receptor In Vascular Endothelial Cells 2. Transfection With 5-lipoxygenase/green Fluorescence Protein For Evaluating Injury-induced 5-lipoxygenase Translocation To The Nuclear Membrane

Objective:Cysteinyl leukotrienes(CysLTs,including LTC₄,LTD₄ and LTE₄)are 5-lipoxygenase(5-LOX)metabolites of arachidonic acid.The effects of CysLTs are mediated by two receptors,cysteinyl receptor 1 and 2(CysLT₁ and CysLT₂) receptors.CysLT₁ is one of the CysLT receptors that have been cloned and characterized.The proliferation and migration of endothelial cells are the important processes of angiogenesis.CysLT₁ receptor is involved in angiogenesis,but the regulatory effect of CysLT₁ receptor on the angiogenesis in vascular endothelial cells is unclear.This study was aimed to determine the effects of CysLT₁ receptor on the proliferation and migration of endothelial cells and the possible mechanisms.Methods:After treatment with LTD₄ for 24 h,48 h and 72 h,the cell proliferation was determined by trypan blue exclusion assay and the 5-bromodeoxyuridine(Brd U) incorporation.After treatment with LTD₄ for 12 h,24 h and 36 h,the migration was detected by wound healing.After treatment with LTD₄ for 5 min,30 min,1 h,4 h and 12 h,the phosphorylation of ERK1/2 was detected by Western blotting.The selective CysLT₁ receptor antagonist montelukast,nonselective antagonist Bay u9773 were pretreated for 30 min to evaluate the effects of CysLT₁ receptor.The ERK1/2 inhibitor U0126 were pretreated for 30 min to observe the role of ERK1/2 in the migration of endothelial cells. Results:LTD₄ had no obvious effect on the proliferation of endothelial cells.LTD₄ (0.1-100 nmol/L)significantly induced the migration of endothelial cells at 12 h after treatment,and LTD₄(10 nmol/L)induced the phosphorylation of ERK1/2 at 0.5 and 1 h after treatment.The effects of LTD₄ on cell migration and the phosphorylation of ERK1/2 were inhibited by the selective CysLT₁ receptor antagonist montelukast and nonselective antagonist Bay u9773(25 nmol/L).The LTD₄-induced migration was also inhibited by ERK1/2 inhibitor U0126(1μmol/L).Conclusion:1.LTD₄ does not obviously affect the proliferation of endothelial cells.2.CysLT₁ receptor mediates the migration of endothelial cells,which is regulated by the phosphorylation of ERK1/2. Objective:To evaluate the changes in 5-lipoxygenase(5-LOX)localization after injuries in the PC12 cells transfected with green fluorescence protein(GFP)/5-LOX with a visualized method.Methods:PC12 cells were stably transfected with pEGFP-C2/5-LOX(GFP/5-LOX) or pEGFP-C2 vectors(control).After oxygen-glucose deprivation(OGD),H₂O₂ or NMDA treatments,GFP/5-LOX localization in the cells were observed under a fluorescence microscope.Wild-type 5-LOX was determined by immunostaining after the treatments.Results:In the GFP/5-LOX-transfected cells,GFP/5-LOX was primarily localized in the nucleus;while in the GFP-transfected cells,GFP was localized in both the cytoplasm and nucleus.After OGD and H₂O₂ treatments,GFP/5-LOX was translocated to the nuclear membrane in 50.6%and 57.7%cells.However,after NMDA treatment or in GFP-transfeeted cells,no translocation was observed. Wild-type 5-LOX was distributed in the nuclei and cytoplasm,and all the 3 treatments induced 5-LOX translocation to the nuclear membrane.Conclusion:1.In the PC12 cells stably transfected with GFP/5-LOX,GFP/5-LOX is primarily distributed in the nuclei. 2.The OGD-,H₂O₂- and NMDA-induced 5-LOX translocation exhibits different properties.