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Large-conductance, calcium-activated potassium channels (BK channels) and the Na/K-ATPase are expressed universally in vascular smooth muscle. The Na/K-ATPase may act via changes in the intracellular Ca 2+ concentration mediated by the Na/Ca exchanger (NCX) and via Src kinase. Both pathways are known to regulate BK channels. Whether BK channels functionally interact in vascular smooth muscle cells with the Na/K-ATPase remains to be elucidated. Thus, this study addressed the hypothesis that BK channels limit ouabain-induced vasocontraction. Rat mesenteric arteries were studied using isometric myography, FURA-2 fluorimetry and proximity ligation assay. The BK channel blocker iberiotoxin potentiated methoxamine-induced contractions. The cardiotonic steroid, ouabain (10 -5  M), induced a contractile effect of IBTX at basal tension prior to methoxamine administration and enhanced the pro-contractile effect of IBTX on methoxamine-induced contractions. These facilitating effects of ouabain were prevented by the inhibition of either NCX or Src kinase. Furthermore, inhibition of NCX or Src kinase reduced the BK channel-mediated negative feedback regulation of arterial contraction. The effects of NCX and Src kinase inhibition were independent of each other. Co-localization of the Na/K-ATPase and the BK channel was evident. Our data suggest that BK channels limit ouabain-induced vasocontraction by a dual mechanism involving the NCX and Src kinase signaling. The data propose that the NCX and the Src kinase pathways, mediating the ouabain-induced activation of the BK channel, act in an independent manner., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

In the vascular wall, the Na,K-ATPase plays an important role in the control of arterial tone. Through cSrc signaling, it contributes to the modulation of Ca2+ sensitivity in vascular smooth muscle cells. This review focuses on the potential implication of Na,K-ATPase-dependent intracellular signaling pathways in severe vascular disorders; ischemic stroke, familial migraine, and arterial hypertension. We propose similarity in the detrimental Na,K-ATPase-dependent signaling seen in these pathological conditions. The review includes a retrospective proteomics analysis investigating temporal changes after ischemic stroke. The analysis revealed that the expression of Na,K-ATPase α isoforms is down-regulated in the days and weeks following reperfusion, while downstream Na,K-ATPase-dependent cSrc kinase is up-regulated. These results are important since previous studies have linked the Na,K-ATPase-dependent cSrc signaling to futile recanalization and vasospasm after stroke. The review also explores a link between the Na,K-ATPase and migraine with aura, as reduced expression or pharmacological inhibition of the Na,K-ATPase leads to cSrc kinase signaling up-regulation and cerebral hypoperfusion. The review discusses the role of an endogenous cardiotonic steroid-like compound, ouabain, which binds to the Na,K-ATPase and initiates the intracellular cSrc signaling, in the pathophysiology of arterial hypertension. Currently, our understanding of the precise control mechanisms governing the Na,K-ATPase/cSrc kinase regulation in the vascular wall is limited. Understanding the role of vascular Na,K-ATPase signaling is essential for developing targeted treatments for cerebrovascular disorders and hypertension, as the Na,K-ATPase is implicated in the pathogenesis of these conditions and may contribute to their comorbidity., (© 2023 The Author(s).)

The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output and myocardial perfusion without affecting blood pressure in humans, but the cardiovascular sites of action remain obscure. Here, we test the hypothesis in rats that 3-OHB acts directly on the heart to increase cardiac contractility and directly on blood vessels to lower systemic vascular resistance. We investigate effects of 3-OHB on (a) in vivo hemodynamics using echocardiography and invasive blood pressure measurements, (b) isolated perfused hearts in Langendorff systems, and (c) isolated arteries and veins in isometric myographs. We compare Na-3-OHB to equimolar NaCl added to physiological buffers or injection solutions. At plasma concentrations of 2-4 mM in vivo, 3-OHB increases cardiac output (by 28.3±7.8%), stroke volume (by 22.4±6.0%), left ventricular ejection fraction (by 13.3±4.6%), and arterial dP/dt max (by 31.9±11.2%) and lowers systemic vascular resistance (by 30.6±11.2%) without substantially affecting heart rate or blood pressure. Applied to isolated perfused hearts at 3-10 mM, 3-OHB increases left ventricular developed pressure by up to 26.3±7.4 mmHg and coronary perfusion by up to 20.2±9.5%. Beginning at 1-3 mM, 3-OHB relaxes isolated coronary (EC 50 =12.4 mM), cerebral, femoral, mesenteric, and renal arteries as well as brachial, femoral, and mesenteric veins by up to 60% of pre-contraction within the pathophysiological concentration range. Of the two enantiomers that constitute racemic 3-OHB, D-3-OHB dominates endogenously; but tested separately, the enantiomers induce similar vasorelaxation. We conclude that increased cardiac contractility and generalized systemic vasorelaxation can explain the elevated cardiac output during 3-OHB administration. These actions strengthen the therapeutic rationale for 3-OHB in heart failure management., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of K ATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of K ATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation., (© 2023. The Author(s).)

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor β. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P <0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P =0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Two α-isoforms of the Na + ,K + -ATPase (α 1 and α 2 ) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α 2 -isoform (G301R; α 2 +/G301R mice) have decreased expression of cardiac α 2 -isoform but elevated expression of the α 1 -isoform. We aimed to investigate the contribution of the α 2 -isoform function to the cardiac phenotype of α 2 +/G301R hearts. We hypothesized that α 2 +/G301R hearts exhibit greater contractility due to reduced expression of cardiac α 2 -isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α 2 +/G301R hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α 2 +/G301R hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α 2 +/G301R hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α 2 +/G301R hearts, which was associated with increased systolic work.

Heterozygous mice (α 2 +/G301R mice) for the migraine-associated mutation (G301R) in the Na + ,K + -ATPase α 2 -isoform have decreased expression of cardiovascular α 2 -isoform. The α 2 +/G301R mice exhibit a pro-contractile vascular phenotype associated with decreased left ventricular ejection fraction. However, the integrated functional cardiovascular consequences of this phenotype remain to be addressed in vivo. We hypothesized that the vascular response to α 2 -isoform-specific inhibition of the Na + ,K + -ATPase by ouabain is augmented in α 2 +/G301R mice leading to reduced cardiac efficiency. Thus, we aimed to assess the functional contribution of the α 2 -isoform to in vivo cardiovascular function of wild-type (WT) and α 2 +/G301R mice. Blood pressure, stroke volume, heart rate, total peripheral resistance, arterial dP/dt, and systolic time intervals were assessed in anesthetized WT and α 2 +/G301R mice. To address rate-dependent cardiac changes, cardiovascular variables were compared before and after intraperitoneal injection of ouabain (1.5 mg/kg) or vehicle during atrial pacing. The α 2 +/G301R mice showed an enhanced ouabain-induced increase in total peripheral resistance associated with reduced efficiency of systolic development compared to WT. When the hearts were paced, ouabain reduced stroke volume in α 2 +/G301R mice. In conclusion, the ouabain-induced vascular response was augmented in α 2 +/G301R mice with consequent suppression of cardiac function.

In the last years, the field of inheritable ventricular arrhythmia disease modelling has changed significantly with a push towards the use of novel cellular cardiomyocyte based models. However, there is a growing need for new in vivo models to study the disease pathology at the tissue and organ level. Zebrafish provide an excellent opportunity for in vivo modelling of inheritable ventricular arrhythmia syndromes due to the remarkable similarity between their cardiac electrophysiology and that of humans. Additionally, many state-of-the-art methods in gene editing and electrophysiological phenotyping are available for zebrafish research. In this review, we give a comprehensive overview of the published zebrafish genetic models for primary electrical disorders and arrhythmogenic cardiomyopathy. We summarise and discuss the strengths and weaknesses of the different technical approaches for the generation of genetically modified zebrafish disease models, as well as the electrophysiological approaches in zebrafish phenotyping. By providing this detailed overview, we aim to draw attention to the potential of the zebrafish model for studying arrhythmia syndromes at the organ level and as a platform for personalised medicine and drug testing., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Several local Ca 2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca 2+ transients near the plasma membrane, and suggested the importance of this signaling for regulation of intercellular coupling and smooth muscle cell contraction. The mechanism behind these Na,K-ATPase-dependent "Ca 2+ flashes" remains to be elucidated. In addition to its conventional ion transport function, the Na,K-ATPase is proposed to contribute to intracellular pathways, including Src kinase activation. The microtubule network is important for intracellular signaling, but its role in the Na,K-ATPase-Src kinase interaction is not known. We hypothesized the microtubule network was responsible for maintaining the Na,K-ATPase-Src kinase interaction, which enables Ca 2+ flashes. Methods: We characterized Ca 2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca 2+ -sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca 2+ . The Na,K-ATPase α2 isoform was knocked down with siRNA and the microtubule network was disrupted with nocodazole. An involvement of the Src signaling was tested pharmacologically and with Western blot. Protein interactions were validated with proximity ligation assays. Results: The Ca 2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca 2+ flashes, as did inhibition of tyrosine phosphorylation with genistein and PP2, and the inhibitor of the Na,K-ATPase-dependent Src activation, pNaKtide. Ouabain-induced Ca 2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca 2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca 2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rognant, Kravtsova, Bouzinova, Melnikova, Krivoi, Pierre, Aalkjaer, Jepps and Matchkov.)

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats ( n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Background: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR., Methods: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox )., Results: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D 3 ), and parathyroid hormone levels. Renal tubular α -Klotho protein expression was increased in KO mice but vascular α -Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice., Conclusions: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis., (Copyright © 2022 by the American Society of Nephrology.)

Objective: Investigating the cardiovascular system is challenging due to its complex regulation by humoral and neuronal factors. Despite this complexity, many existing research methods are limited to the assessment of a few parameters leading to an incomplete characterization of cardiovascular function. Thus, we aim to establish a murine in vivo model for integrated assessment of the cardiovascular system under conditions of controlled heart rate. Utilizing this model, we assessed blood pressure, cardiac output, stroke volume, total peripheral resistance, and electrocardiogram (ECG)., Hypothesis: We hypothesize that (i) our in vivo model can be utilized to investigate cardiac and vascular responses to pharmacological intervention with the α 1 -agonist phenylephrine, and (ii) we can study cardiovascular function during artificial pacing of the heart, modulating cardiac function without a direct vascular effect., Methods: We included 12 mice that were randomly assigned to either vehicle or phenylephrine intervention through intraperitoneal administration. Mice were anesthetized with isoflurane and intubated endotracheally for mechanical ventilation. We measured blood pressure via a solid-state catheter in the aortic arch, blood flow via a probe on the ascending aorta, and ECG from needle electrodes on the extremities. Right atrium was electrically paced at a frequency ranging from 10 to 11.3 Hz before and after either vehicle or phenylephrine administration., Results: Phenylephrine significantly increased blood pressure, stroke volume, and total peripheral resistance compared to the vehicle group. Moreover, heart rate was significantly decreased following phenylephrine administration. Pacing significantly decreased stroke volume and cardiac output both prior to and after drug administration. However, phenylephrine-induced changes in blood pressure and total peripheral resistance were maintained with increasing pacing frequencies compared to the vehicle group. Total peripheral resistance was not significantly altered with increasing pacing frequencies suggesting that the effect of phenylephrine is primarily of vascular origin., Conclusion: In conclusion, this in vivo murine model is capable of distinguishing between changes in peripheral vascular and cardiac functions. This study underlines the primary effect of phenylephrine on vascular function with secondary changes to cardiac function. Hence, this in vivo model is useful for the integrated assessment of the cardiovascular system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rajanathan, Pedersen, Thomsen, Botker and Matchkov.)

Neurovascular coupling ensures rapid and precise delivery of O 2 and nutrients to active brain regions. Chronic stress is known to disturb neurovascular signaling with grave effects on brain integrity. We hypothesized that stress-induced neurovascular disturbances depend on stress susceptibility. Wistar male rats were exposed to 8 weeks of chronic mild stress. Stressed rats with anhedonia-like behavior and with preserved hedonic state were identified from voluntary sucrose consumption. In brain slices from nonstressed, anhedonic, and hedonic rats, neurons and astrocytes showed similar intracellular Ca 2+ responses to neuronal excitation. Parenchymal arterioles in brain slices from nonstressed, anhedonic, and hedonic rats showed vasodilation in response to neuronal excitation. This vasodilation was dependent on inward rectifying K + channel (K ir 2) activation. In hedonic rats, this vasodilation was transient and followed by vasoconstriction insensitive to K ir 2 channel inhibition with 100 µM BaCl 2 . Isolated arteries from hedonic rats showed increased contractility. Elevation of bath K + relaxed isolated middle cerebral arteries in a concentration-dependent and K ir 2-dependent manner. The vasorelaxation to 20-24 mM K + was reduced in arteries from hedonic rats. The expression of voltage-gated K + channels, K v 7.4, was reduced in the cerebral arteries from hedonic rats, whereas the expression of arterial inward-rectifying K + channels, K ir 2.1 was similar to that of nonstressed and anhedonic rats. We propose that preserved hedonic state is associated with increased arterial contractility caused by reduced hyperpolarizing contribution of K v 7.4 channels leading to biphasic cerebrovascular responses to neuronal excitation. These findings reveal a novel potential coping mechanism associated with altered neurovascular signaling.

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In ex vivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

Background: The electroneutral Na + /HCO 3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking., Methods: Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH 4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH 4 + ] was measured in renal biopsies, NH 4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice., Results: Basolateral Na + /HCO 3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na + /HCO 3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH 4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH 4 + gradient, reduced the capacity for urinary NH 4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO 3 - ] from their initial decline., Conclusions: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO 3 - uptake and transepithelial NH 4 + reabsorption in mTALs, renal medullary NH 4 + accumulation, urinary NH 4 + excretion, and early recovery of arterial blood pH and standard [HCO 3 - ]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H + released during NH 4 + reabsorption across mTALs., (Copyright © 2021 by the American Society of Nephrology.)

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

Introduction: The average age of the diving population is rising, and the risk of atherosclerosis and cardiovascular disease in divers are accordingly increasing. It is an open question whether this risk is altered by diving per se . In this study, we examined the effect of 7-weeks simulated diving on endothelial function and mitochondrial respiration in atherosclerosis-prone rats., Methods: Twenty-four male ApoE knockout (KO) rats (9-weeks-old) were fed a Western diet for 8 weeks before 12 rats were exposed to simulated heliox dry-diving in a pressure chamber (600 kPa for 60 min, decompression of 50 kPa/min). The rats were dived twice-weekly for 7 weeks, resulting in a total of 14 dives. The remaining 12 non-diving rats served as controls. Endothelial function of the pulmonary and mesenteric arteries was examined in vitro using an isometric myograph. Mitochondrial respiration in cardiac muscle tissues was measured using high-resolution respirometry., Results and Conclusion: Both ApoE KO diving and non-diving rats showed changes in endothelial function at the end of the intervention, but the extent of these changes was larger in the diving group. Altered nitric oxide signaling was primarily involved in these changes. Mitochondrial respiration was unaltered. In this pro-atherosclerotic rat model of cardiovascular changes, extensive diving appeared to aggravate endothelial dysfunction rather than promote adaptation to oxidative stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Berenji Ardestani, Matchkov, Hansen, Jespersen, Pedersen and Eftedal.)

Membrane transporters and their functional contribution in vasculature change during early postnatal development. Here we tested the hypothesis that the contribution of Cl - channels to arterial contraction declines during early postnatal development and this decline is associated with the trophic sympathetic influence. Endothelium-denuded saphenous arteries from 1- to 2-week-old and 2- to 3-month-old male rats were used. Arterial contraction was assessed in the isometric myograph, in some experiments combined with measurements of membrane potential. mRNA and protein levels were determined by qPCR and Western blot. Sympathectomy was performed by treatment with guanethidine from the first postnatal day until 8-9-week age. Cl - substitution in the solution as well as Cl - -channel blockers (MONNA, DIDS) had larger suppressive effect on the methoxamine-induced arterial contraction and methoxamine-induced depolarization of smooth muscle cells in 1- to 2-week-old compared to 2- to 3-month-old rats. Vasculature of younger group demonstrated elevated expression levels of TMEM16A and bestrophin 3. Chronic sympathectomy increased Cl - contribution to arterial contraction in 2-month-old rats that was associated with an increased TMEM16A expression level. Our study demonstrates that contribution of Cl - channels to agonist-induced arterial contraction and depolarization decreases during postnatal development. This postnatal decline is associated with sympathetic nerves development.

A Ca 2+ -activated Cl - channel protein, ANO1, is expressed in vascular smooth muscle cells where Cl - current is thought to potentiate contraction by contributing to membrane depolarization. However, there is an inconsistency between previous knockout and knockdown studies on ANO1's role in small arteries. In this study, we assessed cardiovascular function of heterozygous mice with global deletion of exon 7 in the ANO1 gene. We found decreased expression of ANO1 in aorta, saphenous and tail arteries from heterozygous ANO1 knockout mice in comparison with wild type. Accordingly, ANO1 knockdown reduced the Ca 2+ -activated Cl - current in smooth muscle cells. Consistent with conventional hypothesis, the contractility of aorta from ANO1 heterozygous mice was reduced. Surprisingly, we found an enhanced contractility of tail and saphenous arteries from ANO1 heterozygous mice when stimulated with noradrenaline, vasopressin, and K + -induced depolarization. This difference was endothelium-independent. The increased contractility of ANO1 downregulated small arteries was due to increased Ca 2+ influx. The expression of L-type Ca 2+ channels was not affected but expression of the plasma membrane Ca 2+ ATPase 1 and the Piezo1 channel was increased. Expressional analysis of tail arteries further suggested changes of ANO1 knockdown smooth muscle cells toward a pro-contractile phenotype. We did not find any difference between genotypes in blood pressure, heart rate, pressor response, and vasorelaxation in vivo. Our findings in tail and saphenous arteries contrast with the conventional hypothesis and suggest additional roles for ANO1 as a multifunctional protein in the vascular wall that regulates Ca 2+ homeostasis and smooth muscle cell phenotype., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

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 mice) 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 hyperaemic 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 polymerase chain reaction, 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. hyperaemic 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 hyperaemic 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., Conclusion: This study suggests that an abnormally high cerebrovascular hyperaemic 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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear. We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort. We show that endothelial cells express the putative HCO 3 - -sensor receptor-type tyrosine-protein phosphatase RPTPγ, which enhances endothelial intracellular Ca 2+ -responses in resistance arteries and facilitates endothelium-dependent vasorelaxation only when CO 2 /HCO 3 - is present. Consistent with waning RPTPγ-dependent vasorelaxation at low [HCO 3 - ], RPTPγ limits increases in cerebral perfusion during neuronal activity and augments decreases in cerebral perfusion during hyperventilation. RPTPγ does not influence resting blood pressure but amplifies hyperventilation-induced blood pressure elevations. Loss-of-function variants in PTPRG , encoding RPTPγ, are associated with increased risk of cerebral infarction, heart attack, and reduced cardiac ejection fraction. We conclude that PTPRG is an ischemia susceptibility locus; and RPTPγ-dependent sensing of HCO 3 - adjusts endothelium-mediated vasorelaxation, microvascular perfusion, and blood pressure during acid-base disturbances and altered tissue metabolism., Competing Interests: KH, CS, PR, CH, SK, MN, VM, EB No competing interests declared, (© 2020, Hansen et al.)

Endothelial dysfunction is recognized as a major contributor to atherosclerosis and has been suggested to be evident far before plaque formation. Endothelial dysfunction in small resistance arteries has been suggested to initiate long before changes in conduit arteries. In this study, we address early changes in endothelial function of atherosclerosis prone rats. Male ApoE knockout (KO) rats (11- to 13-weeks-old) were subjected to either a Western or standard diet. The diet intervention continued for a period of 20-24 weeks. Endothelial function of pulmonary and mesenteric arteries was examined in vitro using an isometric myograph. We found that Western diet decreased the contribution of cyclooxygenase (COX) to control the vascular tone of both pulmonary and mesenteric arteries. These changes were associated with early stage atherosclerosis and elevated level of plasma total cholesterol, LDL and triglyceride in ApoE KO rats. Chondroid-transformed smooth muscle cells, calcifications, macrophages accumulation and foam cells were also observed in the aortic arch from ApoE KO rats fed Western diet. The ApoE KO rats are a new model to study endothelial dysfunction during the earlier stages of atherosclerosis and could help us improve preclinical drug development.

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Aim: Cardiovascular complications, including cardiac arrhythmias, result in high morbidity and mortality in patients with type-2 diabetes mellitus (T2DM). Clinical and experimental data suggest electrophysiological impairment of the natural pacemaker of the diabetic heart. The present study examined sinoatrial node (SAN) arrhythmias in a mouse model of T2DM and physiologically probed their underlying cause., Methods: Electrocardiograms were obtained from conscious diabetic db/db and lean control db/+ mice. In vivo SAN function was probed through pharmacological autonomic modulation with isoprenaline, atropine and carbachol. Blood pressure stability and heart rate variability (HRV) were evaluated. Intrinsic SAN function was evaluated through ex vivo imaging of spontaneous Ca 2+ transients in isolated SAN preparations., Results: While lean control mice showed constant RR intervals during isoprenaline challenge, the diabetic mice experienced SAN arrhythmias with large RR fluctuations in a dose-dependent manner. These arrhythmias were completely abolished by atropine pre-treatment, while carbachol pretreatment significantly increased SAN arrhythmia frequency in the diabetic mice. Blood pressure and HRV were comparable in db/db and db/+ mice, suggesting that neither augmented baroreceptor feedback nor autonomic neuropathy is a likely arrhythmia mechanism. Cycle length response to isoprenaline was comparable in isolated SAN preparations from db/db and db/+ mice; however, Ca 2+ spark frequency was significantly increased in db/db mice compared to db/+ at baseline and after isoprenaline., Conclusion: Our results demonstrate a dysfunction of cardiac pacemaking in an animal model of T2DM upon challenge with a β-adrenergic agonist. Ex vivo, higher Ca 2+ spark frequency is present in diabetic mice, which may be directly linked to in vivo arrhythmias., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Na,K-ATPase is a membrane transporter that is critically important for skeletal muscle function. Mdx and Bla/J mice are the experimental models of Duchenne muscular dystrophy and dysferlinopathy that are known to differ in the molecular mechanism of the pathology. This study examines the function of α1- and α2-Na,K-ATPase isozymes in respiratory diaphragm and postural soleus muscles from mdx and Bla/J mice compared with control С57Bl/6 mice. In diaphragm muscles, the motor endplate structure was severely disturbed (manifested by defragmentation) in mdx mice only. The endplate membrane of both Bla/J and mdx mice was depolarized due to specific loss of the α2-Na,K-ATPase electrogenic activity and its decreased membrane abundance. Total FXYD1 subunit (modulates Na,K-ATPase activity) abundance was decreased in both mouse models. However, the α2-Na,K-ATPase protein content as well as mRNA expression were specifically and significantly reduced only in mdx mice. The endplate membrane cholesterol redistribution was most pronounced in mdx mice. Soleus muscles from Bla/J and mdx mice demonstrated reduction of the α2-Na,K-ATPase membrane abundance and mRNA expression similar to the diaphragm muscles. In contrast to diaphragm, the α2-Na,K-ATPase protein content was altered in both Bla/J and mdx mice; membrane cholesterol re-distribution was not observed. Thus, the α2-Na,K-ATPase is altered in both Bla/J and mdx mouse models of chronic muscle pathology. However, despite some similarities, the α2-Na,K-ATPase and cholesterol abnormalities are more pronounced in mdx mice.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1-10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the 2 Na,K-ATPase isozyme and without changes in 1 and 2 Na,K-ATPase protein content. Ouabain (10-20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the 1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the 2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment., Competing Interests: The authors declare no conflict of interest.

Introduction: The number of divers is rising every year, including an increasing number of aging persons with impaired endothelial function and concomitant atherosclerosis. While diving is an independent modulator of endothelial function, little is known about how diving affects already impaired endothelium. In this study, we questioned whether diving exposure leads to further damage of an already impaired endothelium., Methods: A total of 5 male and 5 female ApoE knockout (KO) rats were exposed to simulated diving to an absolute pressure of 600 kPa in heliox gas (80% helium, 20% oxygen) for 1 h in a dry pressure chamber. 10 ApoE KO rats (5 males, 5 females) and 8 male Sprague-Dawley rats served as controls. Endothelial function was examined in vitro by isometric myography of pulmonary and mesenteric arteries. Lipid peroxidation in blood plasma, heart and lung tissue was used as measures of oxidative stress. Expression and phosphorylation of endothelial NO synthase were quantified by Western blot., Results and Conclusion: A single simulated dive was found to induce endothelial dysfunction in the pulmonary arteries of ApoE KO rats, and this was more profound in male than female rats. Endothelial dysfunction in males was associated with changing in production or bioavailability of NO; while in female pulmonary arteries an imbalance in prostanoid signaling was observed. No effect of diving was found on mesenteric arteries from rats of either sex. Our findings suggest that changes in endothelial dysfunction were specific for pulmonary circulation. In future, human translation of these findings may suggest caution for divers who are elderly or have prior reduced endothelial function., (Copyright © 2019 Berenji Ardestani, Matchkov, Eftedal and Pedersen.)

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.)

New Findings: What is the topic of this review? In this review, we consider the role of the Na + ,K + -ATPase in cerebrovascular function and how it might be changed in familial hemiplegic migraine type 2 (FHM2). The primary focus is involvement of the Na + ,K + -ATPase isoforms in regulation of cerebrovascular tone. What advances does it highlight? In this review, we discuss three overall distinct mechanisms whereby the Na + ,K + -ATPase might be capable of regulating cerebrovascular tone. Furthermore, we discuss how changes in the Na + ,K + -ATPase in cerebral arteries might affect brain perfusion and thereby be involved in the pathology of FHM2., Abstract: Familial hemiplegic migraine type 2 (FHM2) has been characterized by biphasic changes in cerebral blood flow during a migraine attack, with initial hypoperfusion followed by abnormal hyperperfusion of the affected hemisphere. We suggested that FHM2-associated loss-of-function mutation(s) in the Na + ,K + -ATPase α2 isoform might be responsible for these biphasic changes in several ways. We found that reduced expression of the α2 isoform leads to sensitization of the contractile machinery to [Ca 2+ ] i via Src kinase-dependent signal transduction. This change in sensitivity might be the underlying mechanism for both abnormally potentiated vasoconstriction and exaggerated vasorelaxation. Moreover, the functional significance of the Na + ,K + -ATPase α2 isoform in astrocytes provides for the possibility of elevated extracellular potassium signalling from astrocytic endfeet to the vascular wall in neurovascular coupling., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

The third author's name should read: V. V. Matchkov.

Key Points: The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large β1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors., Abstract: Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n = 72) were anaesthetized with ketamine (3 mg kg -1 ) and xylazine (0.75 mg kg -1 ) or pentobarbital (60 mg kg -1 ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1 μm U46619 in the presence of 140 μm suramin and 1 μm prazosin. The vasodilatation was inhibited by 1 μm tetrodotoxin and 5 μm guanethidine, although not by the 1 μm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3 μm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1 μm inhibited the vasodilatation, whereas 0.1 μm of the β 2 -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K + ] to 20 mm caused vasodilatation but was converted to vasoconstriction in the presence of 1 μm BIBN4096bs, and constriction to 30 mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic β 1 -adrenoceptor-mediated dilatation., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The Na,K-ATPase is an enzyme essential for ion homeostasis in all cells. Over the last decades, it has been well-established that in addition to the transport of Na + /K + over the cell membrane, the Na,K-ATPase acts as a receptor transducing humoral signals intracellularly. It has been suggested that ouabain-like compounds serve as endogenous modulators of this Na,K-ATPase signal transduction. The molecular mechanisms underlying Na,K-ATPase signaling are complicated and suggest the confluence of divergent biological pathways. This review discusses recent updates on the Na,K-ATPase signaling pathways characterized or suggested in vascular smooth muscle cells. The conventional view on this signaling is based on a microdomain structure where the Na,K-ATPase controls the Na,Ca-exchanger activity via modulation of intracellular Na + in the spatially restricted submembrane space. This, in turn, affects intracellular Ca 2+ and Ca 2+ load in the sarcoplasmic reticulum leading to modulation of contractility as well as gene expression. An ion-transport-independent signal transduction from the Na,K-ATPase is based on molecular interactions. This was primarily characterized in other cell types but recently also demonstrated in vascular smooth muscles. The downstream signaling from the Na,K-ATPase includes Src and phosphatidylinositol-4,5-bisphosphate 3 kinase signaling pathways and generation of reactive oxygen species. Moreover, in vascular smooth muscle cells the interaction between the Na,K-ATPase and proteins responsible for Ca 2+ homeostasis, e.g., phospholipase C and inositol triphosphate receptors, contributes to an integration of the signaling pathways. Recent update on the Na,K-ATPase dependent intracellular signaling and the significance for physiological functions and pathophysiological changes are discussed in this review., (© 2019 Elsevier Inc. All rights reserved.)

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.)

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150⁻350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca 2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca 2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca 2+ . This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca 2+ .

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).)

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pH i , short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pH i and decrease in [Cl - ] i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

Key Points: Substantial information on rat mesenteric small artery physiology and pharmacology based on in vitro experiments is available. Little is known about the relevance of this for artery function in vivo. We here present an intravital model where rat mesenteric small artery diameters are studied under isolated and controlled conditions in situ with simultaneous measurement of blood flow. The responses of the isolated arteries vary with the anaesthetic used, and they are quantitatively but not qualitatively different from the responses seen in vitro., Abstract: Functional characteristics of rat mesenteric small arteries (internal diameter ∼150-200 μm) have been extensively studied in vitro using isometric and isobaric myographs. In vivo, precapillary arterioles (internal diameter < 50 μm) have been studied, but only a few studies have investigated the function of mesenteric small arteries. We here present a novel approach for intravital studies of rat mesenteric small artery segments (∼5 mm long) isolated in a chamber. The agonist-induced changes in arterial diameter and blood flow were studied using video imaging and laser speckle analysis in rats anaesthetized by isoflurane, pentobarbital, ketamine-xylazine, or by a combination of fentanyl, fluanison and midazolam (rodent mixture). The arteries had spontaneous tone. Noradrenaline added to the chamber constricted the artery in the chamber but not the downstream arteries in the intestinal wall. The constriction was smaller when rats were anaesthetized by rodent mixture in comparison with other anaesthetics, where responses were qualitatively similar to those reported in vitro. The contraction was associated with reduction of blood flow, but no flow reduction was seen in the downstream arteries in the intestinal wall. The magnitude of different endothelium-dependent relaxation pathways was dependent on the anaesthesia. Vasomotion was present under all forms of anaesthesia with characteristics similar to in vitro. We have established an intravital method for studying the tone and flow in rat mesenteric arteries. The reactivity of the arteries was qualitatively similar to the responses previously obtained under in vitro conditions, but the choice of anaesthetic affects the magnitude of responses., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6-12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca 2+ level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca 2+ concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca 2+ dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse., (Copyright © 2017 the American Physiological Society.)

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 2+ ] i 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 2+ 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., (Copyright © 2017 the American Physiological Society.)

Cl(-) and HCO3 (-) are the most prevalent membrane-permeable anions in the intra- and extracellular spaces of the vascular wall. Outwardly directed electrochemical gradients for Cl(-) and HCO3 (-) permit anion channel opening to depolarize vascular smooth muscle and endothelial cells. Transporters and channels for Cl(-) and HCO3 (-) also modify vascular contractility and structure independently of membrane potential. Transport of HCO3 (-) regulates intracellular pH and thereby modifies the activity of enzymes, ion channels, and receptors. There is also evidence that Cl(-) and HCO3 (-) transport proteins affect gene expression and protein trafficking. Considering the extensive implications of Cl(-) and HCO3 (-) in the vascular wall, it is critical to understand how these ions are transported under physiological conditions and how disturbances in their transport can contribute to disease development. Recently, sensing mechanisms for Cl(-) and HCO3 (-) have been identified in the vascular wall where they modify ion transport and vasomotor function, for instance, during metabolic disturbances. This review discusses current evidence that transport (e.g., via NKCC1, NBCn1, Ca(2+)-activated Cl(-) channels, volume-regulated anion channels, and CFTR) and sensing (e.g., via WNK and RPTPγ) of Cl(-) and HCO3 (-) influence cardiovascular health and disease., (©2016 Int. Union Physiol. Sci./Am. Physiol. Soc.)

Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations, and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions, and protein kinase signaling pathways. In addition to its "classical" function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids (CTS) triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function.

The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the α1 and α2 subunit isoforms of Na,K-ATPase. The α2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6-12 h) HS on the functioning of the Na,K-ATPase α1 and α2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase α2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with α2 Na,K-ATPase. The loss of α2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolarized resting membrane potential. The decreased α2 Na,K-ATPase activity is caused by a decrease in enzyme activity rather than by altered protein and mRNA content, localization in the sarcolemma, or functional interaction with the nicotinic acetylcholine receptors. The loss of extrajunctional α2 Na,K-ATPase activity depends strongly on muscle use, and even the increased protein and mRNA content as well as enhanced α2 Na,K-ATPase abundance at this membrane region after 12 h of HS cannot counteract this sustained inhibition. In contrast, additional factors may regulate the subset of junctional α2 Na,K-ATPase pool that is able to recover during HS. Notably, acute, low-intensity muscle workload restores functioning of both α2 Na,K-ATPase pools. These results demonstrate that the α2 Na,K-ATPase in rat skeletal muscle is dynamically and acutely regulated by muscle use and provide the first evidence that the junctional and extrajunctional pools of the α2 Na,K-ATPase are regulated differently., (© 2016 Kravtsova et al.)