Effect Of Na～+, K～+-ATPase Inhibition On Growth And Death Of Vascular Endothelial Cells And Its Mechanisms

Objective: To study the effect of Na+,K+-ATPase inhibition by ouabain on cell growth, cell death and cell junction in the cultured vascular endothelial cells ECV304 and the levels of the free intracellular calcium and free-radical in ECV304 cells were measured by Laser confocal microscopy.To measure the mRNA level of Na+,K+-ATPaseα1-Subunit,β1-Subunit,VE-cadherin and Snail gene to further investigate the mechanisms of ouabain on ECV304 cells.Methods: (1) Growth inhibition of ouabain on ECV304 cells was analyzed by MTT assay.(2) Microscopic structure changes and ultrastructural feature of cells were observed by light microscopy and transmission electric microcopy.The feature of cell death was studied by Hoechst33342/PI staining, nuclear morphological assessment of apoptotic cells and dead cells were observed by fluorescence microscopy.(3) The levels of the free intracellular calcium and free-radical in ECV304 cells were measured by laser confocal microscope.(4) The DNA ladder was revealed by agarose gel electrophoresis.(5) The mRNA expression of Na+,K+-ATPaseα1,β1-Subunit, VE-cadherin and Snail gene were studied by reverse transcription PCR(RT-PCR).Results: (1) 10μmol/L ouabain treated for 24 hours could stimulate the necrosis of ECV304 cells;The median inhibitive concentration (IC50) of ouabain for 24,48,72 h on ECV304 was 0.624,0.184,0.04μmol/L respectively.Ouabain could inhibite vascular endothelial cells growth in a dose and time-dependme nt manner.(2) The obvious morphological changes of cells treated with ouabain were detected by light microscopy. 5～10μmol/L ouabain treated for 12～24 hours could stimulate the necrosis of ECV304 cells;When treated with 0.1μmol/L ouabain for 24～48 hours,the cells became sphericalin shape,detached,obviously defluxion and final death.Apoptotic cells nuclear chromatin condensation,chromatin margination and the loss of cell-cell contacts were revealed by electricmicrocopy.ECV304 cells treated with 0.1μmol/L ouabain nuclear chromatin condensation and fragmentation shaped bright-pearl structure were detected by fluorescence microscopy. (3) The DNA ladder were revealed by agarose gel electrophoresis.The treatment of ECV304 cells with 0.1μmol/L ouabain for 48 and 60 hours respectively,after 48 hours induced internucleosomal DNA fragmentation in the form of a laddering pattern;While after 60 hours appeared smear pattern.(4) The production of free-radicals and the levels of the free intracellular calcium in ECV304 cells had distinct difference after adminis tration of ouabain in a time-and dose-dependant manner.(5) Na+ ,K+-ATPaseα1-Subunit mRNA expression was significantly up-regulated in ECV304 cells treated with ouabain while theβ1-Subunit expression conversely showed a significant decrease. (6) Snail mRNA expression were poor in normal ECV304 cells while the VE-cadherin mRNA expression were strong.on the contrary,Snail mRNA expression were significantly up-regulated and VE-cadherin expression showed a significant decrease in ECV304 cells treated with ouabain in a dose and time-dependment manner.Conclusion: (1) ouabain has a potent growth-inhibitory effect on ECV304 cells in a dose-and time-dependent manner. (2) The effect of ouabain at the concentration of 10μmol/L could stimulate the necrosis of ECV304 cells;But 0.1μmol/L ouabain could efficiently induce apoptosis of ECV304 cells.(3) The production of free-radicals and the levels of the free intracellular calcium increased after treated with ouabain.(4) It was revealed that the protent level of Na+,K+-ATPaseα1-Subunit and Snail increased and that ofβ1-Subunit and VE-cadherin decreased after treated with ouabain.Those results suggested that ouabain could reduce the cell junctions in vascular endothelial cells and induce ECV304 cells death may be by altering the expression of those genes.