Open Access

Daniel Løgstrup Nielsen, Josef Ali Khalid, Anna Giné Martínez, Elizaveta V. Melnikova, Benjamin Hørning Risager, Rudolf Schubert, Christian Staehr, Vladimir V. Matchkov

• 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+ (BKCa) 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 BKCa-channel-mediated feedback. Rat mesenteric small arteries were studied using isometric myography. Intracellular Ca2+ was measured with the ratiometric dyeThe 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+ (BKCa) 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 BKCa-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. BKCa 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 BKCa-channel dependent negative feedback.…