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
86Rb
+ 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.
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