Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

BACKGROUND: Na+,HCO3--cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. METHODS: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3--cotransport with human breast cancer metastasis. RESULTS: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3--cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3--cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. CONCLUSIONS: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth.

Stress adaptation in rats associate with reduced expression of cerebrovascular Kv7.4 channels and biphasic neurovascular responses.

Neurovascular coupling ensures rapid and precise delivery of O2 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 Ca2+ 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 (Kir2) activation. In hedonic rats, this vasodilation was transient and followed by vasoconstriction insensitive to Kir2 channel inhibition with 100 µM BaCl2. Isolated arteries from hedonic rats showed increased contractility. Elevation of bath K+ relaxed isolated middle cerebral arteries in a concentration-dependent and Kir2-dependent manner. The vasorelaxation to 20-24 mM K+ was reduced in arteries from hedonic rats. The expression of voltage-gated K+ channels, Kv7.4, was reduced in the cerebral arteries from hedonic rats, whereas the expression of arterial inward-rectifying K+ channels, Kir2.1 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 Kv7.4 channels leading to biphasic cerebrovascular responses to neuronal excitation. These findings reveal a novel potential coping mechanism associated with altered neurovascular signaling.

Isoform-specific Na,K-ATPase and membrane cholesterol remodeling in motor endplates in distinct mouse models of myodystrophy.

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.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

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.

The Na,K-ATPase in vascular smooth muscle cells.

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 Ca2+ and Ca2+ 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 Ca2+ 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.

Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase-dependent connexin43 phosphorylation.

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 [Ca2+]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 Ca2+ 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.

Neuron and neuroblast numbers and cytogenesis in the dentate gyrus of aged APPswe/PS1dE9 transgenic mice: Effect of long-term treatment with paroxetine.

Altered neurogenesis may influence hippocampal functions such as learning and memory in Alzheimer's disease. Selective serotonin reuptake inhibitors enhance neurogenesis and have been reported to reduce cerebral amyloidosis in both humans and transgenic mice. We have used stereology to assess the longitudinal changes in the number of doublecortin-expressing neuroblasts and number of granular neurons in the dentate gyrus of APPswe/PS1dE9 transgenic mice. Furthermore, we investigated the effect of long-term paroxetine treatment on the number of neuroblasts and granular neurons, hippocampal amyloidosis, and spontaneous alternation behaviour, a measure of spatial working memory, in transgenic mice. We observed no difference in granular neurons between transgenic and wild type mice up till 18months of age, and no differences with age in wild type mice. The number of neuroblasts and the performance in the spontaneous alternation task was reduced in aged transgenic mice. Paroxetine treatment from 9 to 18months of age reduced hippocampal amyloidosis without affecting the number of neuroblasts or granular neurons. These findings suggest that the amyloidosis affects the differentiation of neuroblasts and spatial working memory, independent of changes in total granular neurons. Furthermore, while long-term paroxetine treatment may be able to reduce hippocampal amyloidosis, it appears to have no effect on total number of granular neurons or spatial working memory.

The importance of n-6/n-3 fatty acids ratio in the major depressive disorder.

This review aims to clarify the relation between the ratio of omega-6 to omega-3 fatty acids and the development of depression. It is explained how these fatty acids are involved in the production of eicosanoids and how these fatty acids can affect the membrane fluidity, by their incorporation into membrane phospholipids. In addition, it is described how omega-3 derivatives are shown to regulate gene transcription. In view of the pathophysiology of depression, the mechanisms of how an altered ratio of omega-6 to omega-3 could be involved in depression are discussed. Possible mechanisms could include an increased production of pro-inflammatory cytokines, which can activate the HPA axis and a changed membrane fluidity, which potentially affects membrane bound enzymes, ion channels, receptor activity and neurotransmitter binding. In view of clinical trials, it is also discussed whether omega-3 supplementation could have a beneficial effect in the treatment of depressive patient. There are strong indications that an increased ratio of membrane omega-6 to omega-3 is involved in the pathogenesis of depression and so far, omega-3 supplementation has shown positive effects in clinical trials.

Chronic selective serotonin reuptake inhibition modulates endothelial dysfunction and oxidative state in rat chronic mild stress model of depression.

Major depression is known to be associated with cardiovascular abnormalities, and oxidative stress has been suggested to play a role. We tested the hypothesis that antidepressant treatment reduces oxidative stress and endothelial dysfunctions in the chronic mild stress (CMS) model of depression in rats. Rats with >30% reduction in sucrose intake after 4 wk of CMS were defined in the study as CMS-susceptible and compared with unstressed controls. Sixteen CMS-susceptible and eight unstressed rats were treated during weeks 5 to 8 of the CMS protocol with escitalopram. Escitalopram-treated rats with >20% recovery in the sucrose consumption during the last 2 wk of treatment were defined as escitalopram responders. Rats that did not reach these criteria were defined as escitalopram nonresponders. In the open field test, escitalopram responders demonstrated anxiolytic effect of treatment. In mesenteric small arteries, escitalopram affected neither NO nor cyclooxygenase-1 (COX-1)-mediated vasodilation. Escitalopram potentiated endothelium-dependent hyperpolarization-like response, which was suppressed in the vehicle-treated CMS-susceptible rats and reduced COX-2-dependent relaxation, which was elevated in the vehicle-treated CMS-susceptible rats. Escitalopram did not affect blood pressure and heart rate, which were elevated in the vehicle-treated CMS-susceptible rats. Oxidative stress markers were changed in association with CMS in liver, heart, and brain. Escitalopram normalized oxidative stress markers in the majority of tissues. This study demonstrates that the antidepressant effect of escitalopram is associated with partial improvement of endothelial function in small arteries affecting COX-2 and endothelium-dependent hyperpolarization-like pathways.

Role of peripheral vascular resistance for the association between major depression and cardiovascular disease.

Major depression and cardiovascular diseases are 2 of the most prevalent health problems in Western society, and an association between them is generally accepted. Although the specific mechanism behind this comorbidity remains to be elucidated, it is clear that it has a complex multifactorial character including a number of neuronal, humoral, immune, and circulatory pathways. Depression-associated cardiovascular abnormalities associate with cardiac dysfunctions and with changes in peripheral resistance. Although cardiac dysfunction in association with depression has been studied in detail, little attention was given to structural and functional changes in resistance arteries responsible for blood pressure control and tissue perfusion. This review discusses recent achievements in studies of depression-associated abnormalities in resistance arteries in humans and animal experimental models. The changes in arterial structure, contractile and relaxing functions associated with depression symptoms are discussed, and the role of these abnormalities for the pathology of major depression and cardiovascular diseases are suggested.

Association between endothelial dysfunction and depression-like symptoms in chronic mild stress model of depression.

OBJECTIVE: Cardiovascular diseases have high comorbidity with major depression. Endothelial dysfunction may explain the adverse cardiovascular outcome in depression; therefore, we analyzed it in vitro. In the chronic mild stress model, some rats develop depression-like symptoms (including "anhedonia"), whereas others are stress resilient. METHODS: After 8 weeks of chronic mild stress, anhedonic rats reduced their sucrose intake by 55% (7%), whereas resilient rats did not. Acetylcholine-induced endothelium-dependent relaxation of norepinephrine-preconstricted mesenteric arteries was analyzed in nonstressed, anhedonic, and resilient rat groups. RESULTS: Small resistance arteries from anhedonic rats were less sensitive to acetylcholine than those of the nonstressed and resilient groups (p = .029). Pathways of endothelium-dependent relaxation were altered in arteries from anhedonic rats. Nitric oxide (NO)-dependent relaxation and endothelial NO synthase expression were increased in arteries from anhedonic rats (0.235 [0.039] arbitrary units and 155.7% [8.15%]) compared with the nonstressed (0.135 [0.012] arbitrary units and 100.0% [8.08%]) and resilient (0.152 [0.018] arbitrary units and 108.1% [11.65%]) groups (p < .001 and p = .002, respectively). Inhibition of cyclooxygenase (COX) activity revealed increased COX-2-dependent relaxation in the anhedonic group. In contrast, endothelial NO synthase- and COX-independent relaxation to acetylcholine (endothelium-dependent hyperpolarization-like response) was reduced in anhedonic rats (p < .001). This was associated with decreased transcription of intermediate-conductance Ca-activated K channels. CONCLUSIONS: Our findings demonstrate that depression-like symptoms are associated with reduced endothelium-dependent relaxation due to suppressed endothelium-dependent hyperpolarization-like relaxation despite up-regulation of the NO and COX-2-dependent pathways in rat mesenteric arteries. These changes could affect peripheral resistance and organ perfusion in major depression.

Hypercontractile Cardiac Phenotype in Mice with Migraine-Associated Mutation in the Na+,K+-ATPase α2-Isoform.

Two α-isoforms of the Na+,K+-ATPase (α1 and α2) 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 α2-isoform (G301R; α2+/G301R mice) have decreased expression of cardiac α2-isoform but elevated expression of the α1-isoform. We aimed to investigate the contribution of the α2-isoform function to the cardiac phenotype of α2+/G301R hearts. We hypothesized that α2+/G301R hearts exhibit greater contractility due to reduced expression of cardiac α2-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 α2+/G301R hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α2+/G301R hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α2+/G301R hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α2+/G301R hearts, which was associated with increased systolic work.

Treatment with the vascular disrupting agent combretastatin is associated with impaired AQP2 trafficking and increased urine output.

Combretastatin A-4 disodium phosphate (CA4P) is a vascular disrupting agent known to mediate its effects primarily on tumor blood vessels. CA4P has previously been shown to induce a significant increase in mean arterial blood pressure and in hemoglobin concentration in mice. In the present study, we examined whether this is associated with a general leakage of water into certain tissues or with changes in renal water handling. Munich-Wistar rats received either CA4P (30 mg/kg body wt) or saline intraperitoneally as a bolus injection. One hour later, hemoglobin concentration and mean blood pressure increased significantly. MRI showed no significant changes in tissue water content following CA4P administration. However, urine output and salt excretion increased 1 h after CA4P treatment, without changes in urinary and medullary osmolality. Aquaporin 2 (AQP2) mRNA levels in kidney inner medulla did not change 1 h after CA4P treatment, but semiquantitative confocal laser-scanning microscopy analysis demonstrated a decrease in phosphorylated AQP2 (pS256-AQP2) apical distribution within the collecting ducts of CA4P-treated rats compared with the characteristic apical localization in control rats. Furthermore, we demonstrated that CA4P cause disruption of microtubules and a weaker apical labeling of pS256-AQP2 in collecting duct principal cells within 1 h. In conclusion, our data indicate that water escapes from the vascular system after CA4P treatment, and it may take place primarily through a renal mechanism. The CA4P-mediated increase in urine output seems to be a local effect in the collecting ducts due to reduced AQP2 trafficking to the apical plasma membrane.

Chronic mild stress-induced depression-like symptoms in rats and abnormalities in catecholamine uptake in small arteries.

OBJECTIVE: Major depression and cardiovascular diseases have a strong comorbidity; however, the reason for this is unknown. In the chronic mild stress (CMS) model of depression, only a fraction of rats develop a major feature of depression-anhedonia-like behavior, whereas other rats are stress resilient. Previous studies suggested that CMS rats also have increased total peripheral vascular resistance. METHODS: On the basis of CMS-induced changes of sucrose intake, a reliable measure for anhedonia, rats were divided into "resilient" and "anhedonic" groups. An interaction between hedonic status and vascular function was studied after 4 and 8 weeks of CMS exposure in vitro in wire myograph on saphenous arteries and mesenteric small arteries (MSAs) from these rats. RESULTS: When comparing the different experimental rat groups, arterial sensitivities to noradrenaline (NA) were similar under control conditions, but in the presence of the neuronal reuptake inhibitor cocaine, arteries from anhedonic rats were more sensitive to NA. No change in perivascular innervation was found, but elevated expression of neuronal NA transporter was detected. Inhibition of extraneuronal uptake with corticosterone (1 µM) suggests that this transport is diminished in MSAs after CMS. The corticosterone-sensitive transporter organic cation cotransporter 2 was shown to be reduced in MSAs after CMS. No CMS-induced changes in the corticosterone-sensitive transport were found in saphenous arteries. CONCLUSIONS: Our results indicate that CMS-induced depression-like symptoms in rats are associated with changes in catecholamine uptake pathways in the vascular wall, which potentially modulates the effect of sympathetic innervation of resistance arteries.

Low maternal care exacerbates adult stress susceptibility in the chronic mild stress rat model of depression.

In the present study we report the finding that the quality of maternal care, in early life, increased the susceptibility to stress exposure in adulthood, when rats were exposed to the chronic mild stress paradigm. Our results indicate that high, as opposed to low maternal care, predisposed rats to a differential stress-coping ability. Thus rats fostered by low maternal care dams became more prone to adopt a stress-susceptible phenotype developing an anhedonic-like condition. Moreover, low maternal care offspring had lower weight gain and lower locomotion, with no additive effect of stress. Subchronic exposure to chronic mild stress induced an increase in faecal corticosterone metabolites, which was only significant in rats from low maternal care dams. Examination of glucocorticoid receptor exon 17 promoter methylation in unchallenged adult, maternally characterized rats, showed an insignificant tendency towards higher total cytosine methylation in rats from low maternal care dams. Assessment of methylation in the resilient versus anhedonic-like rat phenotypes, revealed only minor differences. Thus, maternal care status seems to be a strong predictor or trait marker for the behavioural phenotype.

The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells.

Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ 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 "Ca2+ 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 Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. 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 Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ 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 Ca2+ 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 Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ 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.

Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells.

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca(2+)-activated Cl(-) currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca(2+)-activated Cl(-) current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca(2+)-activated Cl(-) current and to have characteristics distinct from those previously known for Ca(2+)-activated Cl(-) currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca(2+)-activated Cl(-) current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca(2+)-activated Cl(-) current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca(2+)-activated Cl(-) current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca(2+)-activated Cl(-) current, whereas the magnitude of the classic Ca(2+)-activated Cl(-) current was not affected. The majority of previous suggestions that bestrophins are a new Cl(-) channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca(2+)-activated Cl(-) current in smooth muscles in the intact vascular wall.

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

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.

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.

Evaluation of Divers' Neuropsychometric Effectiveness and High-Pressure Neurological Syndrome via Computerized Test Battery Package and Questionnaires in Operational Setting.

Introduction: When divers are compressed to water depths deeper than 150 meter sea water (msw), symptoms of high-pressure neurological syndrome (HPNS) might appear due to rapid increase in pressure on the central nervous system during compression. The aim of this study was to first operate a new computerized tool, designed to monitor divers' wellbeing and cognitive function, and to record the results. The second aim was to evaluate the feasibility and validity of the Physiopad software and HPNS questionnaires as a new tool for monitoring divers wellbeing in an operational setting, including sensible visualization and presentation of results. Methods: The Physiopad was operated onboard Deep Arctic (TechnipFMC Diving Support Vessel). The diving work was performed between 180 and 207 msw. The data from 46 divers were collected from the HPNS questionnaires, Hand dynamometry test, Critical Flicker Fusion Frequency test (CFFF), Adaptive Visual Analog Scale (AVAS), Simple Math Process (MathProc test), Perceptual Vigilance Task (PVT), and Time Estimation Task (time-wall). Result: Diver's subjective evaluation revealed different symptoms, possibly also HPNS related, which lasted from 1 to 5 days in storage, with the common duration being 1 day. The results from Physiopad battery testing showed no signs of significant neurological alteration. Conclusion: The present study showed that there was no association between subjective measurements of HPNS and neuropsychometric test results. We also confirmed the feasibility of using the computerized test battery to monitor saturation divers at work. The HPNS battery and Physiopad software could be an important tool for monitoring diver's health in the future. This tool was not used during the Bahr Essalam project to operationally evaluate any HPNS effect on divers as data analysis was performed post-project.