-
American Journal of Physiology. Cell... May 2021With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion...
With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na/K-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule WNK activity. In vitro, potassium increased the melting temperature of WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of and mammalian WNK kinases through direct effects of potassium ion on the kinase.
Topics: Animals; Animals, Genetically Modified; Binding Sites; Cell Line; Chlorides; Drosophila Proteins; Drosophila melanogaster; Hydrogen-Ion Concentration; Malpighian Tubules; Mutation; Phosphorylation; Potassium; Protein Serine-Threonine Kinases; Protein Stability; Substrate Specificity
PubMed: 33439774
DOI: 10.1152/ajpcell.00456.2020 -
American Journal of Physiology. Cell... Apr 2024Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the... (Review)
Review
Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na/Ca exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and β subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.
Topics: Humans; Rats; Animals; Ouabain; Sodium-Potassium-Exchanging ATPase; Ligands; Digoxin; Cardiotonic Agents; Hypertension; Heart Failure; Enzyme Inhibitors; Calcium Signaling; Binding Sites
PubMed: 38223926
DOI: 10.1152/ajpcell.00273.2023 -
The Journal of Clinical Investigation Jun 2020Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by...
Although autophagy is generally protective, uncontrolled or excessive activation of autophagy can be detrimental. However, it is often difficult to distinguish death by autophagy from death with autophagy, and whether autophagy contributes to death in cardiomyocytes (CMs) is still controversial. Excessive activation of autophagy induces a morphologically and biochemically defined form of cell death termed autosis. Whether autosis is involved in tissue injury induced under pathologically relevant conditions is poorly understood. In the present study, myocardial ischemia/reperfusion (I/R) induced autosis in CMs, as evidenced by cell death with numerous vacuoles and perinuclear spaces, and depleted intracellular membranes. Autosis was observed frequently after 6 hours of reperfusion, accompanied by upregulation of Rubicon, attenuation of autophagic flux, and marked accumulation of autophagosomes. Genetic downregulation of Rubicon inhibited autosis and reduced I/R injury, whereas stimulation of autosis during the late phase of I/R with Tat-Beclin 1 exacerbated injury. Suppression of autosis by ouabain, a cardiac glycoside, in humanized Na+,K+-ATPase-knockin mice reduced I/R injury. Taken together, these results demonstrate that autosis is significantly involved in I/R injury in the heart and triggered by dysregulated accumulation of autophagosomes due to upregulation of Rubicon.
Topics: Animals; Autophagosomes; Autophagy; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Myocardial Reperfusion Injury; Myocardium; Up-Regulation
PubMed: 32364533
DOI: 10.1172/JCI132366 -
Inflammopharmacology Dec 2021Ouabain is a cardiac steroid hormone with immunomodulatory effects. It inhibits neutrophils migration induced by different stimuli, but little is known about the...
Ouabain is a cardiac steroid hormone with immunomodulatory effects. It inhibits neutrophils migration induced by different stimuli, but little is known about the mechanisms involved in this effect. Thus, the aim of this study was to evaluate the ouabain effect on chemotactic signaling pathways in neutrophils. For that, mice neutrophils were isolated from bone marrow, treated with ouabain (1, 10, and 100 nM) for 2 h, submitted to transwell chemotaxis assay and flow cytometry analysis of Akt, ERK, JNK, and p38 phosphorylation induced by zymosan. Ouabain treatment (1, 10 and, 100 nM) reduces neutrophil chemotaxis induced by chemotactic peptide fMLP, but this substance did not inhibit Akt, ERK, and JNK activation induced by zymosan. However, ouabain (1 and 10 nM) reduced p38 phosphorylation in zymosan-stimulated neutrophils. These results suggest that ouabain may interfere in neutrophil migration through p38 MAPK inhibition.
Topics: Animals; Cell Movement; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Female; Flow Cytometry; Mice; Neutrophils; Ouabain; Phosphorylation; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 34792671
DOI: 10.1007/s10787-021-00882-z -
Genes & Diseases May 2021With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques,... (Review)
Review
With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na,K-ATPase is ∼1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na,K-ATPase, decrease the [Na]/[K]-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical Na /K -dependent and Na /K -independent cell responses to CTS are discussed.
PubMed: 33997173
DOI: 10.1016/j.gendis.2020.01.008 -
Nature Communications Oct 2019Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions....
Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.
Topics: A549 Cells; Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Breast Neoplasms; Cardiac Glycosides; Cell Line, Tumor; Cell Membrane; Cellular Senescence; Chondrocytes; Digoxin; Female; Fibroblasts; Humans; Hydrogen-Ion Concentration; Mice; Osteoarthritis; Ouabain; Proscillaridin; Pulmonary Fibrosis; Xenograft Model Antitumor Assays
PubMed: 31636264
DOI: 10.1038/s41467-019-12888-x -
Cell Calcium Mar 2020Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic... (Review)
Review
Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic steroids have been used clinically to treat heart failure and more recently atrial fibrillation. Due to their ability to bind and inhibit the ubiquitous transport enzyme sodium potassium pump, thus regulating intracellular Na concentration in every living cell, they are also an essential tool for research into the sodium potassium pump structure and function. Exogenous CTS have been clearly demonstrated to affect cardiovascular system through modulation of vagal tone, cardiac contraction (via ionic changes) and altered natriuresis. Reports of a number of endogenous CTS, since the 1980s, have intensified research into their physiologic and pathophysiologic roles and opened up novel therapeutic targets. Substantive evidence pointing to the role of endogenous ouabain and marinobufagenin, the two most prominent CTS, in development of cardiovascular disease has accumulated. Nevertheless, their presence, structure, biosynthesis pathways and even mechanism of action remain unclear or controversial. In this review the current state-of-the-art, the controversies and the remaining questions surrounding the role of endogenous cardiotonic steroids in health and disease are discussed.
Topics: Animals; Cardiac Glycosides; Cardiovascular Diseases; Humans; Models, Biological; Signal Transduction; Sodium-Potassium-Exchanging ATPase
PubMed: 31896530
DOI: 10.1016/j.ceca.2019.102156 -
Cancers Dec 2022Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the...
Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the lethality remains high. Therefore, there is an urgent need for more effective ATC treatments. Cardiotonic steroids, such as ouabain, have been shown to have therapeutic potential in cancer treatment. Thus, we aimed to evaluate ouabain's effects in human anaplastic thyroid cells. For this, 8505C cells were cultured in the presence or absence of ouabain. Viability, cell death, cell cycle, colony formation and migratory ability were evaluated in ouabain-treated and control 8505C cells. The expression of differentiation and epithelial-to-mesenchymal transition (EMT) markers, as well as IL-6, TGFb1 and their respective receptors were also quantified in these same cells. Our results showed that ouabain in vitro decreased the number of viable 8505C cells, possibly due to an inhibition of proliferation. A reduction in migration was also observed in ouabain-treated 8505C cells. In contrast, decreased mRNA levels of PAX8 and TTF1 differentiation markers and increased levels of the N-cadherin EMT marker, as well as IL-6 and TGFb1, were found in ouabain-treated 8505C cells. In short, ouabain may have anti-proliferative and anti-migratory effect on 8505C cells, but maintains an aggressive and undifferentiated profile.
PubMed: 36551653
DOI: 10.3390/cancers14246168 -
Experimental Eye Research Nov 2021Adult zebrafish are capable of functional retinal regeneration following damage. A goal of vision science is to stimulate or permit a similar process in mammals to treat... (Review)
Review
Adult zebrafish are capable of functional retinal regeneration following damage. A goal of vision science is to stimulate or permit a similar process in mammals to treat human retinal disease and trauma. Ideally such a process would reconstitute the stereotyped, two-dimensional topographic patterns and regional specializations of specific cell types, functionally important for representation of the visual field. An example in humans is the cone-rich fovea, essential for high-acuity color vision. Stereotyped, global topographic patterns of specific retinal cell types are also found in zebrafish, particularly for cone types expressing the tandemly-replicated lws (long wavelength-sensitive) and rh2 (middle wavelength-sensitive) opsins. Here we examine whether regionally specialized patterns of LWS1 and LWS2 cones are restored in regenerated retinas in zebrafish. Adult transgenic zebrafish carrying fluorescent reporters for lws1 and lws2 were subjected to retinal lesions that destroy all neurons but spare glia, via intraocular injection of the neurotoxin ouabain. Regenerated and contralateral control retinas were mounted whole or sectioned, and imaged. Overall spatial patterns of lws1 vs. lws2 opsin-expressing cones in regenerated retinas were remarkably similar to those of control retinas, with LWS1 cones in ventral/peripheral regions, and LWS2 cones in dorsal/central regions. However, LWS2 cones occupied a smaller fraction of regenerated retina, and several cones co-expressed the lws1 and lws2 reporters in regenerated retinas. Local patterns of regenerated LWS1 cones showed modest reductions in regularity. These results suggest that some of the regional patterning information, or the source of such signals, for LWS cone subtypes may be retained by undamaged cell types (Müller glia or RPE) and re-deployed during regeneration.
Topics: Animals; Animals, Genetically Modified; Models, Animal; Regeneration; Retina; Zebrafish
PubMed: 34653519
DOI: 10.1016/j.exer.2021.108789 -
Phytomedicine : International Journal... Apr 2023Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively...
BACKGROUND
Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate.
PURPOSE
To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA).
STUDY DESIGN
The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels.
METHODS
Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA).
RESULTS
Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence.
CONCLUSION
Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.
Topics: Rats; Humans; Animals; Guinea Pigs; Anti-Arrhythmia Agents; Matrines; Ouabain; Aconitine; Arrhythmias, Cardiac; Ion Channels; Myocytes, Cardiac; Isoproterenol; Potassium; Action Potentials
PubMed: 36738478
DOI: 10.1016/j.phymed.2023.154688