Concentrations Of Ouabain Research Articles

Ouabain-mediated downregulation of ALKBH5 and IGF2BP2 inhibits the malignant progression of DLBCL.

m6A modification is a crucial epigenetic regulatory mechanism in diffuse large B-cell lymphoma (DLBCL). Low-dose cardiotonic drugs have been shown to induce apoptosis in DLBCL cells through epigenetic modulation. However, the involvement of the cardiotonic drug ouabain in the malignant progression of DLBCL remains unclear. Our study revealed that ouabain indeed contributes to the malignant progression of DLBCL through m6A modification. Through qPCR analysis, we observed a negative correlation between ouabain concentration and the expression levels of the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 in DLBCL cells. Furthermore, high expression levels of ALKBH5 and IGF2BP2 were identified in both the GEO database and DLBCL patient tissue samples. Notably, elevated ALKBH5 and IGF2BP2 promoted cell proliferation both in vitro and in vivo. Inhibition of their expression rendered DLBCL cells more sensitive to ouabain treatment, resulting in significant suppression of cell proliferation, G1/S phase cell cycle arrest, and increased apoptosis. In summary, our results clarify that the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 are involved in the malignant progression of DLBCL, and that the cardiotonic drug ouabain can inhibit the proliferation of DLBCL cells by inhibiting the expression of ALKBH5 and IGF2BP2, which provides new insights into the targeted treatment of DLBCL.

Evaluation of Ouabain's Tissue Distribution in C57/Black Mice Following Intraperitoneal Injection, Using Chromatography and Mass Spectrometry.

Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC-MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: Cmax = 882.88 ± 21.82 ng/g; Tmax = 0.08 ± 0.01 h; T1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: Cmax = 145.24 ± 44.03 ng/g (undetectable at 60 min); Tmax = 0.08 ± 0.02 h; T1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: Cmax = 1072.3 ± 260.8 ng/g; Tmax = 0.35 ± 0.19 h; T1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: Cmax = 2558.0 ± 382.4 ng/g; Tmax = 0.35 ± 0.13 h; T1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS.

Plant secondary metabolite has dose‐dependent effects on bumblebees

The presence of secondary metabolites in flower nectar can mediate interactions between plants, pollinators, herbivores, and microbes. Milkweeds range in concentrations of cardenolides in flower nectar from ~ 1 to 100 ng μl–1. Using three different behavioral assays with bumblebees Bombus impatiens, we examined the impacts of the commercially available cardenolide ouabain at the range of concentrations at which cardenolides naturally occur in milkweeds. We show that after four days of exposure bees in consumption assays drank more of a nectar solution with a low ouabain concentration of 10 ng μl–1 than the control sucrose nectar, and over the course of the experiment bees consumed less of the 100 ng μl–1 ouabain solution than the control. Bee activity levels in Petri dish arena assays were not impacted by ouabain consumption, even at the highest concentrations; however, in free‐flying choice assays, bees preferentially visited artificial flowers containing 10 ng μl–1 ouabain more than flowers with sucrose control, or flowers with 100 ng μl–1 of ouabain. We therefore conclude that cardenolides may provide plants advantages to pollination at the low end of the naturally occurring range of concentrations for cardenolides, but may be costly to plants at the high end of the range. This research highlights that secondary metabolites in nectar may be under selection to be maintained at low concentrations, and species with high concentrations of secondary metabolites may be under conflicting selection pressures to maintain high circulating levels to deter herbivores at the cost of reduced pollination.

Ouabain at nanomolar concentrations is cytotoxic for biliary tract cancer cells.

Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.

#4434 IMPACT OF GENETIC FACTORS FOR SALT-SENSITIVE HYPERTENSION AND RENAL DAMAGE ON HOSPITALIZED COVID-19 PATIENTS

Abstract Background and Aims The coronavirus SARS-CoV-2 infects patients by binding human angiotensin-converting enzyme 2. Also, the ATP1A1 subunit of the plasma membrane Na-K-pump is critical in supporting the entry of SARS-CoV-2 into cells. Targeting of the ouabain binding site on the Na-K-pump by gene silencing or low concentrations of ouabain blocks viral infection in murine model. A recent finding demonstrated a more effective antiviral activity of digoxin and ouabain against SARS-CoV-2 infection in vitro than previously approved antiviral agents such as chloroquine and remdesivir. Moreover, cardiotonic steroids can promote renal inflammation and oxidative stress through the Na-K-pump α-1 and Src kinase signaling complex in both renal epithelial and immune cells. Arterial hypertension is one of the most common comorbidities associated with COVID-19, especially in patients with severe clinical involvement and at risk of death. The aim of this study is to evaluate the genetic aspects of pathways related to hypertension and renal failure, and to Na-K-pump activity such as endogenous ouabain and RAAS system, in order to partly dissect the wide clinical spectrum of the disease in hospitalized patients infected by SARS-CoV-2, with various degrees of symptoms. Method We investigated the relationship between three outcomes and genetic determinants in the COVID-BioB study (ClincalTrials.gov NCT04318366), a characterization of an Italian cohort of about 500 patients, a SARS-CoV-2 positive population recruited during the first wave of pandemic at San Raffaele Scientific Institute with biological samples and clinical assessment data available in an internal biobank. Targeted DNA genotyping was performed by custom arrays on TaqMan OpenArray system (ThermoFisher) for single nucleotide polymorphisms (SNPs). The genetic variants were selected as candidate for salt-sensitive hypertension and renal failure. Associations with genetic markers and outcomes were carried out with logistic regression analysis for outcome absence/presence comparison. Results COVID-19 patients were all hospitalized, with mean age 67.4±13.5 (30.2% female), pneumonia 96.9%, hypertension 51.8%, coronary arterial disease 26.8%, emergency department AKI stage 1- 11.1%, AKI stage 2- 0.4%, CKD stage&amp;gt;3 11.5%, Chronic Obstructive Pulmonary Disease 8.2%, creatinine 1.19±0.70, all at emergency department admission, in-hospital AKI 37.8%, Intensive Care unit admission 18.3%, and in-hospital death 20.6%. The main outcomes for the analysis of SNPs were in-hospital death, AKI and onset of proteinuria. The main findings for the SNP analysis concerned different genetic markers, each specific for different outcomes. A SNP in renin gene (REN), rs10900555, was associated with the in-hospital death (OR 3.84 [95%CI 1.15;12.85], P = .029). PRKG1 gene coding for the protein kinase cGMP-Dependent reported association with AKI for two different SNPs (rs1904694, OR 2.78 [95%CI 1.63;4.72], P = .0002; rs7905063, OR 2.71 [95%CI 1.72;4.23], P = .00002), with the same risk alleles previously linked to salt-sensitive hypertension. TT genotype (at risk for salt-sensitivity) in uromodulin gene (UMOD, SNP rs4293393) increases the risk of proteinuria development (OR 1.86 [95%CI 1.02;3.40], P = .044). Conclusion This genetic analysis, firstly reported on the COVID-BioB cohort, showed an intriguing relation between some polymorphisms previously associated with salt-sensitive hypertension and worst outcome or renal damage, during COVID-19. This genetic stratification may help to identify patients at risk AKI, and (directly or not) for death and renal damage (proteinuria) during COVID-19. Moreover, it may explain, at least in part, the debated relationship between hypertension and severity of COVID-19.

Markedly divergent effects of Ouabain on a Temozolomide-resistant (T98G) vs. a Temozolomide-sensitive (LN229) Glioblastoma cell line

BackgroundGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance. Radiotherapy with Temozolomide (TMZ) is until today the golden standard adjuvant therapy, however, the optimal treatment of recurrent glioblastoma remains controversial. Ouabain belongs to the Cardiotonic Steroids (CTS) the natural ligands of the Na/K-ATPase (NKA). It is established that the NKA represents a signal transducer with either stimulating or inhibiting cell growth, apoptosis, migration and angiogenesis. Over the last decade evidence grew that CTS have anti-tumor properties especially in GBM.AimProceeding from recent studies we wanted to further demonstrate a divergent effect of Ouabain on a TMZ-resistant (T98G) as compared to a TMZ-sensitive (LN229) GBM cell line.MethodsWe analyzed the effect of Ouabain on cell migration and plasma cell membrane potential (PCMP) in the LN229 and T98G GBM cell line as well as underlying mechanisms (Bcl-2 and p-Akt/pan-Akt expression). Moreover, we analyzed the anti-angiogenic effect of Ouabain on human umbilical vein endothelial cells (HUVECs).ResultsT98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10–7 M) resp. (IC50 = 2.72 × 10–7 M) with a strong inverse correlation (R2 = 0.95). In contrast, LN229 cells did not respond to Ouabain in these assays at all. Similarly, only T98G but not LN229 cells revealed Bcl-2 down-regulation at nanomolar Ouabain concentrations. This unique response to Ouabain is associated with a down-regulation of pan-Akt in T98G cells 24 h after Ouabain (1.0 × 10–6 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10–8 M) was demonstrated which correlated strongly with the anti-migratory effect (R2 = 0.85).ConclusionThe TMZ-resistant T98G cell line as compared to the TMZ-sensitive LN229 cell line shows a high sensitivity towards Ouabain. We consider it as a promising new compound especially in recurrent GBM to overcome the resistance to TMZ and irradiation.

Effects of Ouabain in Ehrlich Tumor Development in vitro and in vivo

Ouabain (OUA) is a cardiotonic steroid with an immunomodulatory and anti-inflammatory role in different experimental models. Currently, the potential antineoplastic effect of OUA has been studied, however, research is needed to better understand OUA role during tumor development. Therefore, our aim was to investigate the OUA effects on Ehrlich tumor (ET) development in vitro and in vivo. To evaluate the cytotoxic effects of OUA on ET in vitro the cells were incubated with different concentrations of OUA during 24h and 48h and our results showed that only the [1000 ?M] decreased the number and viability of ET cells in the two analyzed times. To study the OUA effects on ET in vivo, Swiss mice were pretreated with 0.56 mg/kg of OUA intraperitoneally (i.p.) for three consecutive days. To develop ET in the solid form, one hour after the last day of pretreatment, ET cells were inoculated subcutaneously into the footpad and the animals were monitored for 13 days. To develop the ascitic form, ET cells were inoculated (i.p.) and the animals were monitored for 3 days. OUA was able to reduce the thickness and weight of the tumor paw, in addition to reduce the weight of the popliteal lymph node. In the ascitic tumor, OUA reduced the number of neutrophils and macrophages and increased the lymphocytes in the peritoneum. Thus, we demonstrated that OUA affects ET development both in vitro and in vivo. Our results suggest a new perspective in the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain and brings new concepts about the pathophysiological role of this substance.

Generating Widespread and Scalable Retinal Lesions in Adult Zebrafish by Intraocular Injection of Ouabain.

Zebrafish regenerate functional retinal neurons after injury. Regeneration takes place following photic, chemical, mechanical, surgical, or cryogenic lesions, as well as after lesions that selectively target specific neuronal cell populations. An advantage of chemical retinal lesion for studying the process of regeneration is that the lesion is topographically widespread. This results in the loss of visual function as well as a regenerative response that engages nearly all stem cells (Müller glia). Such lesions can therefore be used to further our understanding of the process and mechanisms underlying re-establishment of neuronal wiring patterns, retinal function, and visually mediated behaviors. Widespread chemical lesions also permit the quantitative analysis of gene expression throughout the retina during the period of initial damage and over the duration of regeneration, as well as the study of growth and targeting of axons of regenerated retinal ganglion cells. The neurotoxic Na+/K+ ATPase inhibitor ouabain specifically offers a further advantage over other types of chemical lesions in that it is scalable; the extent of damage can be targeted to include only inner retinal neurons, or all retinal neurons, simply by adjusting the intraocular concentration of ouabain that is used. Here we describe the procedure through which these "selective" vs. "extensive" retinal lesions can be generated.

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.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle

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.

Antiviral activity of ouabain against a Brazilian Zika virus strain

Zika virus (ZIKV) is an emerging arbovirus associated with neurological disorders. Currently, no specific vaccines or antivirals are available to treat the ZIKV infection. Ouabain, a cardiotonic steroid known as Na+/K+-ATPase inhibitor, has been previously described as an immunomodulatory substance by our group. Here, we evaluated for the first time the antiviral activity of this promising substance against a Brazilian ZIKV strain. Vero cells were treated with different concentrations of ouabain before and after the infection with ZIKV. The antiviral effect was evaluated by the TCID50 method and RT-qPCR. Ouabain presented a dose-dependent inhibitory effect against ZIKV, mainly when added post infection. The reduction of infectious virus was accompanied by a decrease in ZIKV RNA levels, suggesting that the mechanism of ZIKV inhibition by ouabain occurred at the replication step. In addition, our in silico data demonstrated a conformational stability and favorable binding free energy of ouabain in the biding sites of the NS5-RdRp and NS3-helicase proteins, which could be related to its mechanism of action. Taken together, these data demonstrate the antiviral activity of ouabain against a Brazilian ZIKV strain and evidence the potential of cardiotonic steroids as promising antiviral agents.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Heparin-induced and caffeine or ouabain supplemented capacitation of frozen-thawed yak (Bos grunniens) spermatozoa.

Our goal was to investigate heparin-induced capacitation of frozen-thawed yak sperm and to assess the effects of caffeine or ouabain supplementation with heparin on sperm capacitation. Sperm were incubated with varying heparin concentrations, namely 0, 12.5, 25, 50 and 100μg/ml, for 0, 15, 30 and 60min. In every treatment, sperm capacitation was assessed using microscopic examination of the sperm acrosomal status and western blot analysis of the levels of tyrosine phosphorylation (Tyr-P). Based on our results, the optimal condition for frozen-thawed yak sperm capacitation was a 30-min exposure to 50-μg/ml heparin. Next, we incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of caffeine supplementation, namely 0, 2.5, 5 and 10mM for 30min. Interestingly, caffeine significantly increased yak sperm acrosome reaction (AR) and Tyr-P (p<.05). The optimal caffeine concentration was 5mM, followed by 2.5 and 10mM, with the lowest AR and Tyr-P found in sperm cells that did not receive any caffeine. To examine the effects of ouabain on sperm capacitation, we next incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of ouabain, namely 0, 25, 50 and 100µM for 30min. We demonstrated that ouabain supplementation did not alter yak sperm AR or Tyr-P in sperm cells, relative to the control (p>.05). In summary, our findings suggested that caffeine acts synergistically with heparin to increase yak sperm capacitation, but ouabain does not synergize with heparin to promote yak sperm capacitation.

Bimodal modulation of short-term motor memory via dynamic sodium pumps in a vertebrate spinal cord

Dynamic neuronal Na+/K+ pumps normally only respond to intense action potential firing owing to their low affinity for intracellular Na+. Recruitment of these Na+ pumps produces a post-activity ultraslow afterhyperpolarization (usAHP) up to ∼10mV in amplitude and ∼60s in duration, which influences neuronal properties and future network output. In spinal motor networks, the usAHP underlies short-term motor memory (STMM), reducing the intensity and duration of locomotor network output in a manner dependent on the interval between locomotor bouts. In contrast to tonically active Na+ pumps that help set and maintain the resting membrane potential, dynamic Na+ pumps are selectively antagonized by low concentrations of ouabain, which, we show, blocks both the usAHP and STMM. We examined whether dynamic Na+ pumps and STMM can be influenced by neuromodulators, focusing on 5-HT and nitric oxide. Bath-applied 5-HT alone had no significant effect on the usAHP or STMM. However, this is due to the simultaneous activation of two distinct 5-HT receptor subtypes (5-HT7 and 5-HT2a) that have opposing facilitatory and suppressive influences, respectively, on these two features of the locomotor system. Nitric oxide modulation exerts a potent inhibitory effect that can completely block the usAHP and erase STMM. Using selective blockers of 5-HT7 and 5-HT2a receptors and a nitric oxide scavenger, PTIO, we further provide evidence that the two modulators constitute an endogenous control system that determines how the spinal network self-regulates the intensity of locomotor output in light of recent past experience.

Sleep deprivation is associated with increased circulating levels of endogenous ouabain: Potential role in bipolar disorder

Endogenously produced cardiac glycosides, like endogenous ouabain (EO), are putative hormones that have been implicated in the pathophysiology of bipolar disorder. Individuals with bipolar disorder appear to be unable to sufficiently upregulate production of EO in situations of increased need. This study was performed to determine the effect of sleep deprivation on the circulating levels of EO. Plasma EO concentrations were measured by ouabain-radioimmunoassay in heterozygote Na,K-ATPase a2 knockout (KO) mice, which have been used as an animal model of mania, and wildtype siblings at baseline and after sleep fragmentation utilizing the moving bar method. a2 KO animals had elevated endogenous ouabain concentrations compared to wild type controls (0.82 ± SD 0.22 nM vs 0.26 ± 0.02, P = 0.03). Sleep fragmentation increased ouabain concentrations in wild type mice (0.53 ± 0.08 nM sleep fragmentation vs 0.26 ± 0.02 nM baseline, P = 0.04), but not in a2 KO mice (0.60 ± 0.07 nM sleep fragmentation vs 0.82 ± 0.22 nM baseline, P > 0.05). These studies demonstrate that sleep disturbance can increase EO in control mice but animals that exhibit some manic behaviors are unable to increase EO production.

Empagliflozin Ameliorates Ouabain-Induced Na+ and Ca2+ Dysregulations in Ventricular Myocytes in an Na+-Dependent Manner.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a novel class of glucose-lowering agents that have improved clinical outcomes in patients with heart failure; however, their therapeutic mechanisms remain elusive. Although contradictory results have been reported, it has been proposed that improving Na+ homeostasis may be the underlying mechanism of action of SGLT2 inhibitors in heart failure treatment. This study explored whether empagliflozin ameliorates Na+ and Ca2+ handling disorders induced by ouabain in an Na+-dependent manner. Isolated ventricular myocytes of mice were incubated with ouabain to establish a cellular model of Na+ overload. Effects of empagliflozin on Na+ and Ca2+ handling were evaluated using an ionOptix system and a confocal microscope. Distinct cytosolic Na+ levels were established by incubating different ouabain concentrations (10, 50, and 100μmol/L). In the absence of ouabain, 1μmol/L empagliflozin had a negligible impact on Na+ and Ca2+ handling in ventricular myocytes. Ouabain (50μmol/L) significantly enhanced cytosolic Na+ levels and dysregulated Ca2+ handling, including an increased Ca2+ transient amplitude, elevated Ca2+ content in the sarcoplasmic reticulum, and enhanced spontaneous Ca2+ release normalized by treatment with 1μmol/L empagliflozin within 10min. All Na+ and Ca2+ handling abnormalities induced by ouabain were reversed by 1μmol/L empagliflozin. The efficacy of empagliflozin was more potent at higher cytosolic Na+ levels. Pretreatment with the Na+/H+ exchanger (NHE) inhibitor (1μmol/L cariporide) abolished the effects of empagliflozin. Empagliflozin ameliorates ouabain-induced Na+ and Ca2+ handling disorders in a cytosolic Na+-dependent manner, potentially by inhibiting the NHE.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

Possible Antinociceptive Mechanisms Triggered by Nanomolar Ouabain Concentrations in Primary Sensory Neurons

Possible Antinociceptive Mechanisms Triggered by Nanomolar Ouabain Concentrations in Primary Sensory Neurons

Cardiac glycosides cause cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis.

Cardiac glycosides inhibit Na+ /K+ -ATPase and are used to treat heart failure and arrhythmias. They can induce inflammasome activation and pyroptosis in macrophages, suggesting cytotoxicity, which remains to be elucidated in human tissues. To determine the cell-type specificity of this cytotoxicity, we used human monocyte-derived macrophages and non-adherent peripheral blood cells from healthy donors, plus omental white adipose tissue, stromal vascular fraction-derived pre-adipocytes and adipocytes from obese patients undergoing bariatric surgery. All these cells/tissues were treated with nanomolar concentrations of ouabain (50, 100, 500 nM) to investigate the level of cytotoxicity and the mechanisms leading to cell death. In white adipose tissue, we investigated ouabain-mediated cytotoxicity by measuring insulin sensitivity, adipose tissue function and extracellular matrix deposition ex vivo. Ouabain induced cell death through pyroptosis and apoptosis, and was more effective in monocyte-derived macrophages compared to non-adherent peripheral blood mononuclear cell populations. This cytotoxicity is dependent on K+ flux, as ouabain causes intracellular depletion of K+ and accumulation of Na+ and Ca2+ . Consistently, the cell death caused by these ion imbalances can be rescued by addition of potassium chloride to human monocyte-derived macrophages. Remarkably, when white adipose tissue explants from obese patients are cultured with nanomolar concentrations of ouabain, this causes depletion of macrophages, down-regulation of type VI collagen levels and amelioration of insulin sensitivity ex vivo. The use of nanomolar concentration of cardiac glycosides could be an attractive therapeutic treatment for metabolic syndrome, characterized by pathogenic infiltration and activation of macrophages. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.