Stimulation of large-conductance Ca²⁺-activated K⁺ channels by Evans blue in cultured endothelial cells of human umbilical veins

The effect of Evans blue (EB) on large-conductance Ca²⁺-activated K⁺ (BK(Ca)) channels was investigated in cultured endothelial cells of human umbilical veins. In whole-cell configuration, EB (50 μM) reversibly increased the amplitude of K⁺ outward currents (I(K)). When the patch pipettes were filled with 10 mM EGTA, its stimulatory effect on I(K) was unaltered. Further application of EB in the presence of suramin, a blocker of P₂-purinergic receptor, or AOPCP, an inhibitor of 5'-nucleotidase, still increased I(K). However, charybdotoxin (100 nM) suppressed EB-induced increase in I(K). In inside-out configuration, bath application of EB (50 μM) did not change single channel conductance but significantly increased the activity of BK(Ca) channels. The EB-induced increase in the activity of BK(Ca) channels was independent on internal Ca²⁺. EB (50 μM) shifted the activation curve of BK(Ca) channels to less positive membrane potentials by approximately 20 mV. The change in the kinetic behavior of BK(Ca) channels caused by EB in these cells is due to an increase in mean open time and a decrease in mean closed time. These results indicate that EB can stimulate the activity of BK(Ca) channel in endothelial cells. This effect is unrelated to its blockade of P₂-purinergic receptors or inhibition of 5'-nucleotidase. The direct stimulation of these ionic channels by EB may contribute to its effect on capillary permeability.