Effects of ouabain on proliferation of human endothelial cells correlate with Na+,K+-ATPase activity and intracellular ratio of Na+ and K+

Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+](i)/[K+](i)). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na+](i)/[K+](i) ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na+](i)/[K+](i) ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of Rb-86(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+](i)/[K+](i) ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na+](i)/[K+](i) ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na+](i)/[K+](i) ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+](i)/[K+](i) ratio.