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Despite successful recanalization, a significant number of patients with ischemic stroke experience impaired local brain tissue reperfusion with adverse clinical outcome. The cause and mechanism of this multifactorial complication are yet to be understood. At the current moment, major attention is given to dysfunction in blood-brain barrier and capillary blood flow but contribution of exaggerated constriction of cerebral arterioles has also been suggested. In the brain, arterioles significantly contribute to vascular resistance and thus control of perfusion. Accordingly, pathological changes in arteriolar wall function can, therefore, limit sufficient reperfusion in ischemic stroke, but this has not yet received sufficient attention. Although an increased vascular tone after reperfusion has been demonstrated in several studies, the mechanism behind it remains to be characterized. Importantly, the majority of conventional mechanisms controlling vascular contraction failed to explain elevated cerebrovascular tone after reperfusion. We propose here that the Na,K-ATPase-dependent Src kinase activation are the key mechanisms responsible for elevation of cerebrovascular tone after reperfusion. The Na,K-ATPase, which is essential to control intracellular ion homeostasis, also executes numerous signaling functions. Under hypoxic conditions, the Na,K-ATPase is endocytosed from the membrane of vascular smooth muscle cells. This initiates the Src kinase signaling pathway that sensitizes the contractile machinery to intracellular Ca 2+ resulting in hypercontractility of vascular smooth muscle cells and, thus, elevated cerebrovascular tone that can contribute to impaired reperfusion after stroke. This mechanism integrates with cerebral edema that was suggested to underlie impaired reperfusion and is further supported by several studies, which are discussed in this article. However, final demonstration of the molecular mechanism behind Src kinase-associated arteriolar hypercontractility in stroke remains to be done.

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 +/-G301R ) 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 2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 +/-G301R and WT showed similar contractile responses. The Atp1a2 +/-G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca 2+ change was smaller than in WT. The enhanced constriction of Atp1a2 +/-G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca 2+ ] i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 +/-G301R 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 2+ , which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

New Findings: What is the topic of this review? This symposium report discusses the previously unrecognized pro-contractile role of chloride ions in rat arteries at early stages of postnatal development. What advances does it highlight? It highlights the postnatal decline in the contribution of chloride ions to regulation of arterial contractile responses and potential trophic role of sympathetic nerves in these developmental alterations., Abstract: Chloride ions are important for smooth muscle contraction in adult vasculature. Arterial smooth muscle undergoes structural and functional remodelling during early postnatal development, including changes in K + currents, Ca 2+ handling and sensitivity. However, developmental change in the contribution of Cl - to regulation of arterial contraction has not yet been explored. Here, we provide the first evidence that the role of Cl - in α 1 -adrenergic arterial contraction prominently decreases during early postnatal ontogenesis. The trophic influence of sympathetic nerves is a potential mechanism for postnatal decline of the contribution of Cl - to the vascular contraction., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Aims: The Na,K-ATPase is involved in a large number of regulatory activities including cSrc-dependent signalling. Upon inhibition of the Na,K-ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist-induced contraction by ouabain is mediated through Na,K-ATPase-cSrc signalling-dependent sensitization of vascular smooth muscle cells to Ca 2+ ., Methods: Agonist-induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca 2+ ] i in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K-ATPase were transiently downregulated by siRNA transfection in vivo., Results: Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium-dependent. Ouabain sensitized smooth muscle cells to Ca 2+ , and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca 2+ sensitization. Downregulation of the Na,K-ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition., Conclusion: Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca 2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

TMEM16A is essential for Ca 2+ -activated Cl - conductance in vascular smooth muscle. The importance of TMEM16A for agonist-induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca 2+ -activated Cl - conductance, for example modulation of Ca 2+ channel expression. Mice with constitutive, smooth muscle-specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild-types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down-regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild-type mice were not different. Aorta from TG mice showed reduced agonist-induced constriction, while their responses to elevated K + were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild-types. Surprisingly, tail arteries from TG mice constricted less to elevated K + too and were more sensitive to nifedipine-induced relaxation. Consistently, TMEM16A down-regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L-type Ca 2+ channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Aims: We have previously characterized a cGMP-dependent Ca(2+)-activated Cl(-) current in vascular smooth muscle cells (SMCs) and have shown its dependence on bestrophin-3 expression. We hypothesize that this current is important for synchronization of SMCs in the vascular wall. In the present study, we aimed to test this hypothesis by transfecting rat mesenteric small arteries in vivo with siRNA specifically targeting bestrophin-3., Methods and Results: The arteries were tested 3 days after transfection in vitro for isometric force development and for intracellular Ca(2+) in SMCs. Bestrophin-3 expression was significantly reduced compared with arteries transfected with mutated siRNA. mRNA levels for bestrophin-1 and -2 were also significantly reduced by bestrophin-3 down-regulation. This is suggested to be secondary to specific bestrophin-3 down-regulation since siRNAs targeting different exons of the bestrophin-3 gene had identical effects on bestrophin-1 and -2 expression. The transfection affected neither the maximal contractile response nor the sensitivity to norepinephrine and arginine-vasopressin. The amplitude of agonist-induced vasomotion was significantly reduced in arteries down-regulated for bestrophins compared with controls, and asynchronous Ca(2+) waves appeared in the SMCs. The average frequency of vasomotion was not different. 8Br-cGMP restored vasomotion in arteries where the endothelium was removed, but oscillation amplitude was still significantly less in bestrophin-down-regulated arteries. Thus, vasomotion properties were consistent with those previously characterized for rat mesenteric small arteries. Data from our mathematical model are consistent with the experimental results., Conclusion: This study demonstrates the importance of bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(Ca,cGMP))) chloride conductances are important. The effect of anion substitution and Cl(-) channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl(-) (o) replacement with aspartate blocked vasomotion while 36mM SCN(-) (o) (substituted for Cl(-)) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca(2+)](i) disappeared with 36mM SCN(-). DIDS and Zn(2+) blocked I (Cl(Ca,cGMP)) but not I (Cl(Ca)). Vasomotion was not sensitive to DIDS and Zn(2+), and DIDS and Zn(2+) induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn(2+) was sensitive to 36mM SCN(-) (o). The anion substitution data indicate that Cl(-) is crucial for the V (m) and [Ca(2+)](i) oscillations underlying vasomotion. The Cl(-) channel blocker data are consistent with both CaCCs being important.

Vasomotion is a rhythmic variation in microvascular diameter. Although known for more than 150 years, the cellular processes underlying the initiation of vasomotion are not fully understood. In the present study a model of a single cell is extended by coupling a number of cells into a tube. The simulated results point to a permissive role of cGMP in establishing intercellular synchronization. In sufficient concentration, cGMP may activate a cGMP-sensitive calcium-dependent chloride channel, causing a tight spatiotemporal coupling between release of sarcoplasmic reticulum calcium, membrane depolarization, and influx of extracellular calcium. Low [cGMP] is associated only with unsynchronized waves. At intermediate concentrations, cells display either waves or whole cell oscillations, but these remain unsynchronized between cells. Whole cell oscillations are associated with rhythmic variation in membrane potential and flow of current through gap junctions. The amplitude of these oscillations in potential grows with increasing [cGMP], and, past a certain threshold, they become strong enough to entrain all cells in the vascular wall, thereby initiating sustained vasomotion. In this state there is a rhythmic flow of calcium through voltage-sensitive calcium channels into the cytoplasm, making the frequency of established vasomotion sensitive to membrane potential. It is concluded that electrical coupling through gap junctions is likely to be responsible for the rapid synchronization across a large number of cells. Gap-junctional current between cells is due to the appearance of oscillations in the membrane potential that again depends on the entrainment of sarcoplasmic reticulum and plasma membrane within the individual cell.

The present study investigated the influence of media thickness on myogenic tone and intracellular calcium concentration ([Ca(2+)](i)) in rat skeletal muscle small arteries. A ligature was loosely tied around one external iliac artery of 5-wk-old spontaneously hypertensive rats. At 18 wk of age, femoral artery blood pressure was 102 +/- 11 mmHg (n = 15) on the ligated side and 164 +/- 6 mmHg (n = 15) on the contralateral side. Small arteries feeding the gracilis muscle had a reduced media cross-sectional area and a reduced media-to-lumen ratio on the ligated side, where also the range of myogenic constriction was shifted to lower pressures. However, when expressed as a function of wall stress, diameter responses were nearly identical. [Ca(2+)](i) was higher in vessels from the ligated hindlimb at pressures above 10 mmHg, but vasoconstriction was not accompanied by changes in [Ca(2+)](i). Thus the myogenic constriction here seems due primarily to changes in intracellular calcium sensitivity, which are determined mainly by the force per cross-sectional area of the wall and therefore altered by changes in vascular structure.

Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline perfusion. An original mathematical model was applied to evaluate the mechanism of changes in vascular tone regulation. A 3-week suspension resulted in decreased responses to sympathetic nerve stimulation, as well as to exogenous noradrenaline, the latter effect being registered when the pressure level was close to normal. Mathematical simulation indicated that long-term suspension induces both structural and functional changes in the vascular bed of the hind limbs, one of which is a disorder in the myogenic mechanisms of vascular tone regulation. In suspended rats, suppression of the myogenic response can be one of the reasons for decreased vessel reactivity to constrictor stimuli and, consequently, for disturbances in blood flow regulation in skeletal muscles of the hind limbs.