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BioRxiv : the Preprint Server For... May 2024Thrombosis is a major cause of myocardial infarction and ischemic stroke. The sodium/potassium ATPase (NKA), comprising α and β subunits, is crucial in maintaining...
BACKGROUND
Thrombosis is a major cause of myocardial infarction and ischemic stroke. The sodium/potassium ATPase (NKA), comprising α and β subunits, is crucial in maintaining intracellular sodium and potassium gradients. However, the role of NKA in platelet function and thrombosis remains unclear.
METHODS
We utilized wild-type (WT, α ) and NKA α1 heterozygous (α ) mice, aged 8 to 16 weeks, of both sexes. An intravital microscopy-based, FeCl -induced carotid artery injury thrombosis model was employed for in vivo thrombosis assessment. Platelet transfusion assays were used to evaluate platelet NKA α1 function on thrombosis. Human platelets isolated from healthy donors and heart failure patients treated with/without digoxin were used for platelet function and signaling assay. Complementary molecular approaches were used for mechanistic studies.
RESULTS
NKA α1 haplodeficiency significantly reduced its expression on platelets without affecting sodium homeostasis. It significantly inhibited 7.5% FeCl -induced thrombosis in male but not female mice without disturbing hemostasis. Transfusion of α , but not α , platelets to thrombocytopenic WT mice substantially prolonged thrombosis. Treating WT mice with low-dose ouabain or marinobufagenin, both binding NKA α1 and inhibiting its ion-transporting function, markedly inhibited thrombosis in vivo. NKA α1 formed complexes with leucine-glycine-leucine (LGL)-containing platelet receptors, including P2Y12, PAR4, and thromboxane A2 receptor. This binding was significantly attenuated by LGL>SFT mutation or LGL peptide. Haplodeficiency of NKA α1 in mice or ouabain treatment of human platelets notably inhibited ADP-induced platelet aggregation. While not affecting 10% FeCl -induced thrombosis, NKA α1 haplodeficiency significantly prolonged thrombosis time in mice treated with an ineffective dose of clopidogrel.
CONCLUSION
NKA α1 plays an essential role in enhancing platelet activation through binding to LGL-containing platelet GPCRs. NKA α1 haplodeficiency or inhibition with low-dose ouabain and marinobufagenin significantly inhibited thrombosis and sensitized clopidogrel's anti-thrombotic effect. Targeting NKA α1 emerges as a promising antiplatelet and antithrombotic therapeutic strategy.
PubMed: 38798556
DOI: 10.1101/2024.05.13.593923 -
Cancer & Metabolism May 2024Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine...
BACKGROUND
Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na/K-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells.
METHODS
Live whole-cell measurements of intracellular sodium [Na] were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by H NMR to monitor conversion of [6,6-H]D-glucose to [H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na] was titrated using isotonic buffers with varying [Na] and [K] and introducing an artificial Na plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[F]fluoroglucose (F-FDG) positron emission tomography (PET).
RESULTS
Intracellular [Na] was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na] and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na] in MDA-MB-231 and 4T1 cells and a Na-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected.
CONCLUSIONS
Glycolytic flux correlates with Na-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na]-NKA nexus' in the mechanistic basis of the Warburg effect.
PubMed: 38783368
DOI: 10.1186/s40170-024-00343-5 -
Nature Communications May 2024Elevated intracellular sodium Na adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic...
Elevated intracellular sodium Na adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Na decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH, which are all reversed on lowering Na to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Na elevation. Elevated Na plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.
Topics: Animals; Rats; Mitochondria, Heart; Sodium; Male; Myocardium; Hypoxia-Inducible Factor 1, alpha Subunit; Heart Failure; Adenosine Triphosphate; Citric Acid Cycle; Rats, Sprague-Dawley; Organophosphorus Compounds; Sodium-Calcium Exchanger; Ubiquinone; Sodium-Potassium-Exchanging ATPase; Oxidation-Reduction; Succinic Acid
PubMed: 38769288
DOI: 10.1038/s41467-024-48474-z -
Biology Open May 2024Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside...
Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.
Topics: Wnt Signaling Pathway; Pinocytosis; Colonic Neoplasms; Sodium-Potassium-Exchanging ATPase; Animals; Humans; Ouabain; Cell Line, Tumor; Xenopus
PubMed: 38713004
DOI: 10.1242/bio.060269 -
International Journal of Molecular... Apr 2024Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous...
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: C = 882.88 ± 21.82 ng/g; T = 0.08 ± 0.01 h; T = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: C = 145.24 ± 44.03 ng/g (undetectable at 60 min); T = 0.08 ± 0.02 h; T = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: C = 1072.3 ± 260.8 ng/g; T = 0.35 ± 0.19 h; T = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: C = 2558.0 ± 382.4 ng/g; T = 0.35 ± 0.13 h; T = 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.
Topics: Animals; Ouabain; Tissue Distribution; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Male; Blood-Brain Barrier; Brain; Mass Spectrometry; Kidney; Liver; Chromatography, High Pressure Liquid; Myocardium; Cardiotonic Agents
PubMed: 38673903
DOI: 10.3390/ijms25084318 -
Scientific Reports Apr 2024Lysophosphoglycerides (LPLs) have been reported to accumulate in myocardium and serve as a cause of arrhythmias in acute myocardial ischemia. However, in this study we...
Lysophosphoglycerides (LPLs) have been reported to accumulate in myocardium and serve as a cause of arrhythmias in acute myocardial ischemia. However, in this study we found that LPLs level in the ventricular myocardium was decreased by the onset of acute myocardial ischemia in vivo in rats. Decreasing of LPLs level in left ventricular myocardium, but not right, was observed within 26 min of left myocardial ischemia, regardless of whether arrhythmias were triggered. Lower LPLs level in the ventricular myocardium was also observed in aconitine-simulated ventricular fibrillation (P < 0.0001) and ouabain-simulated III atrioventricular block (P < 0.0001). Shot-lasting electric shock, e.g., ≤ 40 s, decreased LPLs level, while long-lasting, e.g., 5 min, increased it (fold change = 2.27, P = 0.0008). LPLs accumulation was observed in long-lasting myocardial ischemia, e.g., 4 h (fold change = 1.20, P = 0.0012), when caspase3 activity was elevated (P = 0.0012), indicating increased cell death, but not coincided with higher frequent arrhythmias. In postmortem human ventricular myocardium, differences of LPLs level in left ventricular myocardium was not observed among coronary artery disease- and other heart diseases-caused sudden death and non-heart disease caused death. LPLs level manifested a remarkable increasing from postmortem 12 h on in rats, thus abolishing the potential for serving as biomarkers of sudden cardiac death. Token together, in this study we found that LPLs in ventricular myocardium were initially decreased by the onset of ischemia, LPLs accumulation do not confer arrhythmogenesis during acute myocardial ischemia. It is necessary to reassess the roles of LPLs in myocardial infarction.
Topics: Animals; Myocardial Ischemia; Rats; Male; Heart Ventricles; Arrhythmias, Cardiac; Humans; Myocardium; Ventricular Fibrillation; Aconitine; Disease Models, Animal; Ouabain
PubMed: 38670979
DOI: 10.1038/s41598-024-57047-5 -
Disease Models & Mechanisms May 2024Dystonia is thought to arise from abnormalities in the motor loop of the basal ganglia; however, there is an ongoing debate regarding cerebellar involvement. We adopted...
Dystonia is thought to arise from abnormalities in the motor loop of the basal ganglia; however, there is an ongoing debate regarding cerebellar involvement. We adopted an established cerebellar dystonia mouse model by injecting ouabain to examine the contribution of the cerebellum. Initially, we examined whether the entopeduncular nucleus (EPN), substantia nigra pars reticulata (SNr), globus pallidus externus (GPe) and striatal neurons were activated in the model. Next, we examined whether administration of a dopamine D1 receptor agonist and dopamine D2 receptor antagonist or selective ablation of striatal parvalbumin (PV, encoded by Pvalb)-expressing interneurons could modulate the involuntary movements of the mice. The cerebellar dystonia mice had a higher number of cells positive for c-fos (encoded by Fos) in the EPN, SNr and GPe, as well as a higher positive ratio of c-fos in striatal PV interneurons, than those in control mice. Furthermore, systemic administration of combined D1 receptor agonist and D2 receptor antagonist and selective ablation of striatal PV interneurons relieved the involuntary movements of the mice. Abnormalities in the motor loop of the basal ganglia could be crucially involved in cerebellar dystonia, and modulating PV interneurons might provide a novel treatment strategy.
Topics: Animals; Interneurons; Parvalbumins; Disease Models, Animal; Proto-Oncogene Proteins c-fos; Dystonia; Corpus Striatum; Receptors, Dopamine D2; Receptors, Dopamine D1; Cerebellum; Ouabain; Mice, Inbred C57BL; Mice; Male
PubMed: 38616770
DOI: 10.1242/dmm.050338 -
Anais Da Academia Brasileira de Ciencias 2024Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur,...
Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur, leading to acute kidney injury (AKI) due to exacerbated reactive O2 species (ROS) production. However, the consequences of ROS exposure on ionic transport and the regulator local renin-angiotensin-aldosterone system (RAAS) still need to be elucidated at a molecular level. This study evaluated how ROS acutely influences Na+-transporting ATPases and the renal RAAS. Adult male Wistar rats received paraquat (20 mg/kg; ip). After 24 h, we observed body weight loss and elevation of urinary flow and serum creatinine. In the renal cortex, paraquat increased ROS levels, NADPH oxidase and (Na++K+)ATPase activities, angiotensin II-type 1 receptors, tumor necrosis factor-α (TNF-α), and interleukin-6. In the medulla, paraquat increased ROS levels and NADPH oxidase activity but inhibited (Na++K+)ATPase. Paraquat induced opposite effects on the ouabain-resistant Na+-ATPase in the cortex (decrease) and medulla (increase). These alterations, except for increased serum creatinine and renal levels of TNF-α and interleukin-6, were prevented by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol; 1 mmol/L in drinking water), a stable antioxidant. In summary, after paraquat poisoning, ROS production culminated with impaired medullary function, urinary fluid loss, and disruption of Na+-transporting ATPases and angiotensin II signaling.
Topics: Rats; Animals; Male; Renin-Angiotensin System; Reactive Oxygen Species; Paraquat; Angiotensin II; Creatinine; Interleukin-6; Tumor Necrosis Factor-alpha; Rats, Wistar; Kidney; Adenosine Triphosphatases; Sodium; NADPH Oxidases
PubMed: 38597493
DOI: 10.1590/0001-3765202420230971 -
Leukemia Research May 2024Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies...
Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34 cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid's ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.
Topics: Humans; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Models, Theoretical; Cytarabine
PubMed: 38579483
DOI: 10.1016/j.leukres.2024.107485 -
The Journal of Physiological Sciences :... Apr 2024Cardiac glycosides, known as inhibitors of Na,K-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death....
Cardiac glycosides, known as inhibitors of Na,K-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na,K-ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells.
Topics: Humans; Cardiac Glycosides; Glycosides; Sodium-Potassium-Exchanging ATPase; Ouabain; Thyroid Neoplasms; Neoplasm Proteins
PubMed: 38561668
DOI: 10.1186/s12576-024-00914-7