Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 and WT showed similar contractile responses. The Atp1a2 MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca change was smaller than in WT. The enhanced constriction of Atp1a2 MCA was associated with increased cSrc activation, stronger sensitization to [Ca] and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 mice had reduced resting blood flow through MCA in comparison with WT mice . FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone.

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We tested here the hypothesis that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance, and synchronized [Ca] transients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2, and by an inhibitor of the Na-K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity whereas pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation.

Aims Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na,K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain. Methods and Results Neurovascular coupling was compared in α2+/G301R and matching wild type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative PCR, Western blot and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found. Conclusions This study suggests that an abnormally high cerebrovascular hyperemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack. Translational Perspective Migraine, especially familial migraine with aura, is strongly associated with severe cerebrovascular events. Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by transient hypoperfusion, which is followed by hyperperfusion of the affected hemisphere. We have previously described the mechanism responsible for FHM2-associated vasospasm of cerebral arteries and brain hypoperfusion. In this study, we characterize the mechanism for an abnormally strong cerebral vasodilator response that follows vasospasm and causes delayed hyperperfusion in a migraine attack. These defects may contribute to the cerebrovascular damage seen in patients with migraine with aura and are suggestive as future therapeutic targets.

Ga-PSMA PET/CT is currently used for detection of prostate cancer including metastases, even at low prostate-specific antigen values. A grown number of reports have shown increased uptake of PSMA in neovessels of nonprostatic malignancies including lung cancer, and recently a case report has demonstrated increased PSMA uptake in colorectal adenocarcinoma. In this case report, we demonstrate increased Ga-PMSA uptake on PET/CT in metastases from previously treated colon adenocarcinoma, and it illustrates the importance of histology of suspicious lesions on Ga-PSMA PET/CT.