Interplay between Na⁺/K⁺-ATPase-dependent Src-mediated Ca²⁺ sensitization and BK_Ca channel negative feedback...

The Na⁺/K⁺-ATPase α₂ isoform regulates vascular tone by modulating Na⁺/Ca²⁺ exchange and Src kinase-dependent signaling within membrane microdomains. Inhibition of the Na⁺/K⁺-ATPase by cardiotonic steroids promotes vascular contraction through increased intracellular Ca²⁺ and enhanced Ca²⁺ sensitization of the contractile apparatus. This effect is counterbalanced by a negative feedback mechanism involving large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. Given inconsistent reports regarding ouabain-induced potentiation of agonist-evoked vasoconstriction, we hypothesized that the effects of ouabain depend on the agonist (thromboxane A₂ receptor agonist U46619, noradrenaline, and the α₁-adrenoceptor agonist methoxamine) used. We specifically assessed changes in intracellular Ca²⁺, Ca²⁺ sensitization, and the contribution of BK_Ca-channel-mediated feedback. Rat mesenteric small arteries were studied using isometric myography. Intracellular Ca²⁺ was measured with the ratiometric dye FURA-2/AM, and signaling pathways were analyzed by Western blotting. Micromolar ouabain enhanced U46619-induced contraction but had no statistically significant effect on responses to noradrenaline or methoxamine. BK_Ca channel inhibition with iberiotoxin increased sensitivity to U46619 via elevated intracellular Ca²⁺. Combined ouabain and iberiotoxin treatment increased basal tone and augmented contractile responses to all agonists tested. Notably, ouabain enhanced Ca²⁺ sensitization during U46619 stimulation through Src-dependent MYPT1 phosphorylation. Ouabain also increased Src and MYPT1 phosphorylation during both U46619 and methoxamine stimulation. These findings identify the Na⁺/K⁺-ATPase as a signaling scaffold that promotes Src-mediated Ca²⁺ sensitization. This pro-contractile effect requires a sufficient intracellular Ca²⁺ level, partly regulated by BK_Ca-channel dependent negative feedback.