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PNAS Nexus Mar 2023For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions....
For its size, the brain is the most metabolically active organ in the body. Most of its energy demand is used to maintain stable homeostatic physiological conditions. Altered homeostasis and active states are hallmarks of many diseases and disorders. Yet there is currently no direct and reliable method to assess homeostasis and absolute basal activity of cells in the tissue noninvasively without exogenous tracers or contrast agents. We propose a novel low-field, high-gradient diffusion exchange nuclear magnetic resonance (NMR) method capable of directly measuring cellular metabolic activity via the rate constant for water exchange across cell membranes. Exchange rates are s under normal conditions in viable ex vivo neonatal mouse spinal cords. High repeatability across samples suggest that values are absolute and intrinsic to the tissue. Using temperature and drug (ouabain) perturbations, we find that the majority of water exchange is metabolically active and coupled to active transport by the sodium-potassium pump. We show that this water exchange rate is sensitive primarily to tissue homeostasis and provides distinct functional information. In contrast, the apparent diffusion coefficient (ADC) measured with submillisecond diffusion times is sensitive primarily to tissue microstructure but not activity. Water exchange appears independently regulated from microstructural and oxygenation changes reported by ADC and relaxation measurements in an oxygen-glucose deprivation model of stroke; exchange rates remain stable for 30-40 min before dropping to levels similar to the effect of ouabain and never completely recovering when oxygen and glucose are restored.
PubMed: 36970182
DOI: 10.1093/pnasnexus/pgad056 -
Drugs - Real World Outcomes Jun 2023Cardiac glycosides such as digoxin, digitoxin and ouabain are still used around the world to treat patients with chronic heart failure with reduced ejection fraction...
BACKGROUND
Cardiac glycosides such as digoxin, digitoxin and ouabain are still used around the world to treat patients with chronic heart failure with reduced ejection fraction (HFrEF) and/or atrial fibrillation (AF). However, in the US, only digoxin is licensed for treating these illnesses, and the use of digoxin for this group of patients is increasingly being replaced in the US by a new standard of care with groups of more expensive drugs. However, ouabain and digitoxin, and less potently digoxin, have also recently been reported to inhibit SARS-CoV-2 virus penetration into human lung cells, thus blocking COVID-19 infection. COVID-19 is known to be a more aggressive disease in patients with cardiac comorbidities, including heart failure.
OBJECTIVE
We therefore considered the possibility that digoxin might provide at least a measure of relief from COVID-19 in digoxin-treated heart failure patients. To this end, we hypothesized that treatment with digoxin rather than standard of care might equivalently protect heart failure patients with regard to diagnosis of COVID-19, hospitalization and death.
METHODS
To test this hypothesis, we conducted a cross-sectional study by using the US Military Health System (MHS) Data Repository to identify all MHS TRICARE Prime and Plus beneficiaries aged 18-64 years with a heart failure (HF) diagnosis during the period April 2020 to August 2021. In the MHS, all patients receive equal, optimal care without regard to rank or ethnicity. Analyses included descriptive statistics on patient demographics and clinical characteristics, and logistic regressions to determine likelihood of digoxin use.
RESULTS
We identified 14,044 beneficiaries with heart failure in the MHS during the study period. Of these, 496 were treated with digoxin. However, we found that both digoxin-treated and standard-of-care groups were equivalently protected from COVID-19. We also noted that younger active duty service members and their dependents with HF were less likely to receive digoxin compared with older, retired beneficiaries with more comorbidities.
CONCLUSION
The hypothesis of equivalent protection by digoxin treatment of HF patients in terms of susceptibility to COVID-19 infection appears to be supported by the data.
PubMed: 36933173
DOI: 10.1007/s40801-023-00360-8 -
Circulation Research Apr 2023Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na) reabsorption via Src (Src family kinase)...
BACKGROUND
Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na transport similar to ouabain-a cardiotonic steroid.
METHODS
Urine Na excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA.
RESULTS
Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated Src and Erk 1/2 (extracellular signal-regulated protein kinase 1/2); these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N-PET-8-biotin-11-cGMP (2 μM); 8-N-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N-6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA.
CONCLUSIONS
cGMP can bind to NKA and thereby mediate natriuresis.
Topics: Animals; Female; Rats; Adenosine Triphosphatases; Biotin; Cyclic GMP; Natriuresis; Ouabain; Potassium; Rats, Sprague-Dawley; Sodium; Sodium-Potassium-Exchanging ATPase
PubMed: 36919600
DOI: 10.1161/CIRCRESAHA.122.321693 -
Anesthesiology Jun 2023Maintenance of ion homeostasis is essential for normal brain function. Inhalational anesthetics are known to act on various receptors, but their effects on ion...
BACKGROUND
Maintenance of ion homeostasis is essential for normal brain function. Inhalational anesthetics are known to act on various receptors, but their effects on ion homeostatic systems, such as sodium/potassium-adenosine triphosphatase (Na+/K+-ATPase), remain largely unexplored. Based on reports demonstrating global network activity and wakefulness modulation by interstitial ions, the hypothesis was that deep isoflurane anesthesia affects ion homeostasis and the key mechanism for clearing extracellular potassium, Na+/K+-ATPase.
METHODS
Using ion-selective microelectrodes, this study assessed isoflurane-induced extracellular ion dynamics in cortical slices of male and female Wistar rats in the absence of synaptic activity, in the presence of two-pore-domain potassium channel antagonists, during seizures, and during spreading depolarizations. The specific isoflurane effects on Na+/K+-ATPase function were measured using a coupled enzyme assay and studied the relevance of the findings in vivo and in silico.
RESULTS
Isoflurane concentrations clinically relevant for burst suppression anesthesia increased baseline extracellular potassium (mean ± SD, 3.0 ± 0.0 vs. 3.9 ± 0.5 mM; P < 0.001; n = 39) and lowered extracellular sodium (153.4 ± 0.8 vs. 145.2 ± 6.0 mM; P < 0.001; n = 28). Similar changes in extracellular potassium and extracellular sodium and a substantial drop in extracellular calcium (1.5 ± 0.0 vs. 1.2 ± 0.1 mM; P = 0.001; n = 16) during inhibition of synaptic activity and two-pore-domain potassium suggested a different underlying mechanism. After seizure-like events and spreading depolarization, isoflurane greatly slowed extracellular potassium clearance (63.4 ± 18.2 vs. 196.2 ± 82.4 s; P < 0.001; n = 14). Na+/K+-ATPase activity was markedly reduced after isoflurane exposure (greater than 25%), affecting specifically the α2/3 activity fraction. In vivo, isoflurane-induced burst suppression resulted in impaired extracellular potassium clearance and interstitial potassium accumulation. A computational biophysical model reproduced the observed effects on extracellular potassium and displayed intensified bursting when Na+/K+-ATPase activity was reduced by 35%. Finally, Na+/K+-ATPase inhibition with ouabain induced burst-like activity during light anesthesia in vivo.
CONCLUSIONS
The results demonstrate cortical ion homeostasis perturbation and specific Na+/K+-ATPase impairment during deep isoflurane anesthesia. Slowed potassium clearance and extracellular accumulation might modulate cortical excitability during burst suppression generation, while prolonged Na+/K+-ATPase impairment could contribute to neuronal dysfunction after deep anesthesia.
Topics: Rats; Animals; Male; Female; Isoflurane; Rats, Wistar; Homeostasis; Brain; Seizures; Potassium; Sodium; Adenosine Triphosphatases
PubMed: 36893015
DOI: 10.1097/ALN.0000000000004553 -
Molecular Genetics and Genomics : MGG May 2023Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to...
Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to explore the effects of ouabain on proliferation and metabolism towards psoriatic keratinocytes. In human HaCaT keratinocytes, ouabain potently decreased viability, promoted apoptosis and caused G2/M cycle arrest. Metabolomics analysis indicated that ouabain markedly impaired glutathione metabolism. The solute carrier family 7 member 11 (SLC7A11) is an amino acid transporter highly specific to cysteine, which is critical for glutathione synthesis. Ouabain downregulated SLC7A11, reduced cysteine uptake and subsequently inhibited glutathione synthesis, probably through inhibiting Akt/mTOR/beclin axis that regulate protein activity of SLC7A11. The impaired glutathione synthesis and oxidative stress caused by ouabain may contribute to its cytotoxicity towards psoriatic keratinocytes. Our results provide experimental evidence supporting further study of ouabain as a potential anti-psoriatic agent.
Topics: Humans; Ouabain; Cysteine; Keratinocytes; Antineoplastic Agents; Apoptosis; Glutathione; Psoriasis; Cell Proliferation
PubMed: 36856826
DOI: 10.1007/s00438-023-02001-9 -
Discover Oncology Feb 2023Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance....
BACKGROUND
Glioblastoma 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.
AIM
Proceeding 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.
METHODS
We 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).
RESULTS
T98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10 M) resp. (IC50 = 2.72 × 10 M) with a strong inverse correlation (R = 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 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10 M) was demonstrated which correlated strongly with the anti-migratory effect (R = 0.85).
CONCLUSION
The 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.
PubMed: 36840822
DOI: 10.1007/s12672-023-00633-2 -
International Journal of Molecular... Feb 2023Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic...
Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.
Topics: Animals; CD13 Antigens; Chlorocebus aethiops; Porcine epidemic diarrhea virus; Receptors, Virus; RNA, Double-Stranded; RNA, Small Interfering; Swine; Swine Diseases; Vero Cells; Virus Attachment; Coronavirus Infections; Sodium-Potassium-Exchanging ATPase; Host-Pathogen Interactions
PubMed: 36835408
DOI: 10.3390/ijms24044000 -
Biomedicines Jan 2023Heterozygous mice (α mice) for the migraine-associated mutation (G301R) in the Na,K-ATPase α-isoform have decreased expression of cardiovascular α-isoform. The α...
Heterozygous mice (α mice) for the migraine-associated mutation (G301R) in the Na,K-ATPase α-isoform have decreased expression of cardiovascular α-isoform. The α 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-isoform-specific inhibition of the Na,K-ATPase by ouabain is augmented in α mice leading to reduced cardiac efficiency. Thus, we aimed to assess the functional contribution of the α-isoform to in vivo cardiovascular function of wild-type (WT) and α mice. Blood pressure, stroke volume, heart rate, total peripheral resistance, arterial dP/dt, and systolic time intervals were assessed in anesthetized WT and α 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 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 mice. In conclusion, the ouabain-induced vascular response was augmented in α mice with consequent suppression of cardiac function.
PubMed: 36830881
DOI: 10.3390/biomedicines11020344 -
Biomedicines Jan 2023This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell...
This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K uptake and severe K depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.
PubMed: 36830836
DOI: 10.3390/biomedicines11020301 -
Science Advances Feb 2023Dysregulated endocrine hormones (EHs) contribute to tumorigenesis, but how EHs affect the tumor immune microenvironment (TIM) and the immunotherapy of non-small cell...
Dysregulated endocrine hormones (EHs) contribute to tumorigenesis, but how EHs affect the tumor immune microenvironment (TIM) and the immunotherapy of non-small cell lung cancer (NSCLC) is still unclear. Here, endogenous ouabain (EO), an adrenergic hormone, is elevated in patients with NSCLC and closely related to tumor pathological stage, metastasis, and survival. EO promotes the suppression of TIM in vivo by modulating the expression of immune checkpoint proteins, in which programmed cell death protein ligand 1 (PD-L1) plays a major role. EO increases PD-L1 transcription; however, the EO receptor Na- and K-dependent adenosine triphosphatase (Na, K-ATPase) α1 interacts with PD-L1 to trigger the endocytic degradation of PD-L1. This seemingly contradictory result led us to discover the mechanism whereby EO cooperates with Na, K-ATPase α1 to finely control PD-L1 expression and dampen tumoral immunity. In conclusion, the Na, K-ATPase α1/EO signaling facilitates immune escape in lung cancer, and manipulation of this signaling shows great promise in improving immunotherapy for lung adenocarcinoma.
Topics: Humans; Adenocarcinoma of Lung; Adenosine Triphosphatases; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Ligands; Lung; Lung Neoplasms; Tumor Microenvironment; Sodium-Potassium-Exchanging ATPase
PubMed: 36763655
DOI: 10.1126/sciadv.ade5393