The Na,K-ATPase modulates arterial tone by means of ionic gradients, and via the Na,K-ATPase-dependent signal transduction, including the Src kinase signaling pathway. Inhibition of the Na,K-ATPase leads to accumulation of intracellular Na⁺, which modifies the Na,Ca-exchanger activity leading to elevation of intracellular Ca²⁺ and smooth muscle contraction. Src kinase activates upon interaction with the Na,K-ATPase inhibitor, i.e., cardiotonic steroid ouabain, which leads to sensitization of smooth muscle cell contractile machinery and potentiation of arterial contraction. It has been proposed that both Na,K-ATPase-dependent mechanisms potentiate the contraction in response to different pro-contractile stimuli, however, their contribution depends on contractile agonist.

This study aims to analyze the agonist-dependence (methoxamine, U46619, and noradrenaline) of the pro-contractile action of ouabain, and its effect at different activation states in the vascular wall.

Male Wistar Hannover rats were obtained from Janvier-Labs (France) and sacrificed with CO₂ inhalation. Mesenteric arteries (2^nd – 3^rd order branches) were dissected and used in isometric myography, where the contraction was induced by either U46619 (thromboxane A₂ mimetic), methoxamine (alpha1-adrenergic receptor agonist), or noradrenaline (unspecific adrenoreceptor agonist). The arterial segments were compared under control conditions and in the presence of either ouabain, or iberiotoxin (IbTX), the inhibitor of big conductance Ca²⁺-activated potassium channels, or in the combination of these drugs. Intracellular Ca²⁺ was measured with Ca²⁺-sensitive dye, FURA-2/AM simultaneously with contractile responses in myograph. Western blot with arterial segment lysates was performed to assess the phosphorylation of Src kinase and MYPT protein responsible for Ca²⁺ sensitization. The comparison of concentration-response curves was done with two-way ANOVA. Data presented as mean±standard deviation.

Ouabain (10 µM) significantly potentiated the contraction in response to U46619 (pEC50 changes from 6.2±0.5 to 6.8±0.7, n=7, P=0.016). IbTX also potentiated the U46619-induced contraction in comparison with the control (pEC50: 7.5±3, n=7, P<0.0001). Pro-contractile effects of IbTX and ouabain were not additive (pEC50: 7.1±0.7, n=5, P=0.0003). In contrast, ouabain showed no clear potentiation of methoxamine-induced constriction (pEC50: 5.2±0.7 and 5.5±0.3, n=7, P=0.92). However, the pro-contractile effect of IbTX was still seen (pEC50: 5.8±0.3, n=7, P=0.03) but this was not affected by a combination of IbTX and ouabain (pEC50: 5.7±0.2, n=7, P=0.13). Finally, no potentiation of vessel contractility by ouabain was observed in the stimulation with noradrenaline (pEC50: 5.9±0.2 and 6.0±0.1, n=6, P=0.20), neither with IbTX (pEC50: 6.1±0.2, n=6, P=0.28), but this effect was strengthened in the presence of both IbTX and ouabain (pEC50: 6.2±0.2, n=6, P=0.03). Further analyses demonstrated that a major part of ouabain pro-contractile action is not associated with elevation of intracellular Ca²⁺ but with Ca²⁺ sensitization. In contrast, IbTX strongly elevates intracellular Ca²⁺ in smooth muscle cells.

This study demonstrates the agonist-dependent character of the pro-contractile action of ouabain, where agonists whose action is primarily mediated by Ca²⁺-sensitization are most sensitive to inhibition of the Na,K-ATPase. Importantly, this ouabain action can further be potentiated by the elevation of intracellular Ca²⁺ with IbTX. This study suggests that caution should be taken when comparing the pro-contractile ouabain action for different agonists and at different activation states of the vascular wall.