Aims: In the vascular wall of cerebral arteries, micromolar ouabain inhibits the α2 Na,K-ATPase and activates cSrc kinase, which was shown to increase Ca²⁺-sensitivity of the smooth muscle cell contractile machinery. It has been proposed, that this cSrc-dependent Ca²⁺-sensitization depends on the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) by Rho kinase signaling, as the conventional pathway for Ca²⁺-sensitization of vascular smooth muscle cells. However, this Src-Rho-MYPT1 signaling remains to be shown and validated in cerebral arteries. Familial hemiplegic migraine type 2 is associated with mutations in the α2 Na,K-ATPase, including the G301R missense mutation, associated with reduced abundancy of the Na,K-ATPase in cerebrovascular smooth muscle cells, resulting in increased cSrc activation. We aimed to test whether cSrc potentiates Ca²⁺-sensitivity via Rho-kinase-dependent MYPT1 phosphorylation in middle cerebral arteries and to compare this signaling in mice bearing the G301R mutation (ATP1A2^+/G301R) and wild types.

Methods: Middle cerebral artery contractility to thromboxane A₂ analog, U46619, was assessed ex-vivo in isometric myograph. The effects of pre-incubation with Rho kinase inhibitor, Y27632 (3·10^-6M) and ouabain (10^-5M) were studied. Arteries from ATP1A2^+/G301R and wild type mice were compared.
To assess cSrc and MYPT1 phosphorylation and its dependence on Rho kinase, U46619-stimualted middle cerebral arteries from wild type and ATP1A2^+/G301R mice were studied with Western blot upon pre-incubation with Y27632, ouabain or both.

Results: Ouabain potentiated the U46619-induced contraction of middle cerebral arteries from wild type mice (n=5), while Y27632 inhibited it (n=4). In the presence of Y27632, ouabain still potentiated the contraction of wild type arteries (n=5). In contrast, ouabain did not significantly affect the contraction of ATP1A2^+/G301R mice (n=5), neither under control conditions nor after Rho kinase inhibition, while Y27632 still suppressed the contraction (n=3). Western blot data indicates a positive correlation between cSrc kinase and MYPT1 phosphorylation, which is only partially prevented by Rho kinase inhibition (24 mice divided into 6 groups exposed to 4 different interventions).

Conclusion: We suggest, that cSrc kinase increases Ca²⁺-sensitivity in vascular smooth muscle cells via MYPT1 phosphorylation. Our results suggest that this arterial tonus regulating pathway is partially independent of Rho kinase signaling.