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The Journal of Biological Chemistry Mar 2022Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune...
Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na/K-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2',3'-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.
Topics: A549 Cells; Cardiac Glycosides; Cell Line, Tumor; Digoxin; Humans; Immune Checkpoint Proteins; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kynurenine; Neoplasms; Ouabain; STAT1 Transcription Factor; Sodium-Potassium-Exchanging ATPase
PubMed: 35150740
DOI: 10.1016/j.jbc.2022.101707 -
International Journal of Molecular... Dec 2020Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is...
Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.
Topics: Apoptosis; Cardiotonic Agents; Cell Communication; Cell Proliferation; Gap Junctions; Humans; Neoplasms; Ouabain; Signal Transduction; Tumor Cells, Cultured
PubMed: 33396341
DOI: 10.3390/ijms22010358 -
The Journal of Physiology Feb 19681. Changes in membrane conductance and potential of sodium-loaded frog muscle fibres were found when the external recovery solution was changed: from cold to warm, to...
1. Changes in membrane conductance and potential of sodium-loaded frog muscle fibres were found when the external recovery solution was changed: from cold to warm, to warm plus ouabain, to cold plus ouabain. Comparisons of these measurements for different external solutions were made by leaving the electrodes implanted in the same fibre during all solution changes. (The recovery solutions contained 10 mM-K and 82 mM-Cl.)2. The membrane potential became more negative on warming, less negative when ouabain was added, and still less negative when the ouabain-containing recovery solution was cooled. The membrane conductance increased on warming, increased further on addition of ouabain, and decreased when the ouabain-containing recovery solution was cooled.3. The increase of conductance which occurred on warming decreased with increasing periods in cold recovery. The increase of conductance which occurred on addition of ouabain decreased if the ouabain was added to the recovery solutions of muscles which were more fully recovered.4. The ouabain-sensitivity of the membrane conductance may be dependent upon the sodium-pump rate, or the extent of recovery of the sodium-loaded muscle fibre in the potassium- and chloride-containing recovery solutions.5. It is suggested that if the potassium conductance of the membrane increases with decreasing sodium-pump rates, then during the initial part of the recovery period a non-electrogenic mechanism must be producing a substantial part of the early net potassium influx.
Topics: Animals; Anura; Biological Transport, Active; Chlorides; Cold Temperature; Electric Conductivity; Hindlimb; Hot Temperature; In Vitro Techniques; Ions; Membrane Potentials; Muscles; Ouabain; Potassium; Sodium
PubMed: 5639364
DOI: 10.1113/jphysiol.1968.sp008421 -
BMC Complementary and Alternative... Jun 2019Staphylococcus aureus is a notorious pathogen which often causes nosocomial and community attained infections. These infections steadily increased after evolving the...
BACKGROUND
Staphylococcus aureus is a notorious pathogen which often causes nosocomial and community attained infections. These infections steadily increased after evolving the resistance due to indecorous practice of antibiotics and now become a serious health issue. Ouabain is a Na/K-ATPase inhibitor that leads to increase the heart contraction in patients with congestive heart failure.
METHODS
In the present study, in vitro antimicrobial effect of ouabain together with aminoglycosides was determined against clinical and non-clinical S. aureus strains. Using checkerboard, Gentamycin uptake and biofilm assays, we analysed he interactions of ouabain with aminoglycosides.
RESULTS
Ouabain induced the staphylocidal potency of aminoglycosides by remarkably reducing the MIC of gentamycin (GEN) by 16 (0.25 μg/mL), 8 folds (0.5 μg/mL) amikacin (AMK); and 16 folds (1.0 μg/mL) with kanamycin (KAN), compared to their individual doses. OBN severely reduced cell viability within 60 min with GEN (1 μg/mL), KAN (2 μg/mL) and 90 min with AMK (1 μg/mL). This bactericidal effect was enhanced due to GEN uptake potentiated by 66% which led to increase the cell permeability as revealed by leakage of bacterial ATP and nitrocefin assay. The biofilm adherence disrupted by 80 and 50% at 5 mg/mL and 1.5 mg/mL OBN and 50 and 90% biofilm formation was inhibited at 5 mg/mL (MBIC) and 10 mg/mL (MBIC), respectively. Moreover, OBN with GEN further induced biofilm inhibition by 67 ± 5% at pH 7.0.
CONCLUSIONS
Taken together, we established that OBN synergizes the antimicrobial activity of aminoglycosides that induces cell killing due to intracellular accumulation of GEN by disturbing cell homeostasis. It may be proven an effective approach for the treatment of staphylococcal infections.
Topics: Aminoglycosides; Anti-Bacterial Agents; Biofilms; Drug Synergism; Microbial Sensitivity Tests; Ouabain; Staphylococcus aureus
PubMed: 31170971
DOI: 10.1186/s12906-019-2532-6 -
The Journal of Physiology May 1985To investigate the mechanism by which ouabain causes acute increase in peripheral resistance, the effect of ouabain on vessel tone, 22Na efflux and membrane potential...
To investigate the mechanism by which ouabain causes acute increase in peripheral resistance, the effect of ouabain on vessel tone, 22Na efflux and membrane potential has been compared with the binding of [3H]ouabain in rat isolated mesenteric resistance vessels (lumen diameter approximately 200 microns). Ouabain at concentrations between 1 X 10(-8) and 1 X 10(-3) M had no effect on the tone of resting vessels but caused potentiation of the tone of vessels which were submaximally contracted with noradrenaline. In the entire concentration range, these acute potentiating effects of ouabain on vessel tone correlated well with an acute inhibitory effect on 22Na efflux and with acute [3H]ouabain binding, consistent with the vasoconstrictor effect of ouabain being associated with inhibition of the Na-K pump. At all concentrations, in both resting and activated vessels, ouabain exposure caused an acute concentration-dependent depolarization of the membrane. At low ouabain concentrations (1 X 10(-6) and 1 X 10(-5) M) the potentiating effect on vessel tone and the inhibitory effect on 22Na efflux were transient, so that after 10 min no effect of ouabain was seen. This was consistent with these ouabain concentrations failing to cause a measurable increase in the intracellular Na concentration ([Na]i) after longer term (up to 2 h) exposure. The recovery of 22Na efflux was due to a recovery of the ouabain-sensitive part of the 22Na efflux, suggesting that the transitory nature of these effects is due to stimulation of uninhibited Na-K pumps. The transient effects of low ouabain concentrations contrasted with binding experiments where, with 1 X 10(-6) M-ouabain, the binding first peaked after 30 min with only a small decline thereafter. With higher concentrations of ouabain (1 X 10(-4) and 1 X 10(-3) M), the potentiating effect was also transitory, but 22Na efflux was permanently inhibited and the [Na]i rose. The possibility of there being two populations of ouabain binding sites having different affinities, was supported by the observation of shoulders in the ouabain concentration-effect characteristics obtained with respect to the mechanical potentiation of noradrenaline responses, the 22Na efflux and the acute ouabain binding. In all cases a shoulder was seen between 1 X 10(-6) and 1 X 10(-5) M-ouabain. The results are consistent with the vasoconstrictor effect of ouabain being due to depolarization of the membrane as a consequence of inhibition of the Na-K pump.
Topics: Animals; In Vitro Techniques; Ion Channels; Male; Membrane Potentials; Mesenteric Arteries; Muscle Tonus; Muscle, Smooth, Vascular; Ouabain; Rats; Rats, Inbred WKY; Sodium; Time Factors; Vascular Resistance; Vasoconstriction
PubMed: 2410600
DOI: 10.1113/jphysiol.1985.sp015672 -
The Journal of Physiology Mar 19801. Renin secretion of rat renal cortical slices was measured as a function of extracellular K and ouabain concentrations in the incubation medium.2. A sigmoid...
1. Renin secretion of rat renal cortical slices was measured as a function of extracellular K and ouabain concentrations in the incubation medium.2. A sigmoid relationship was found between renin secretion and log K concentration over the range 1.0-4.0 mM. Secretion was maximal at about 2.25 mM-K and half-maximal at about 1.43 mM-K.3. In media containing 4.0 mM-K, ouabain at 10(-8), 10(-7), and 10(-6)M did not affect renin secretion. Higher concentrations of ouabain inhibited secretion. A sigmoid relationship was found between% inhibition of secretion and log ouabain concentration (10(-6)-10(-3)M). Inhibition was half-maximal at 2.3 x 10(-5)M and complete at 10(-3)M-ouabain.4. Lowering extracellular K concentration from 4.0 to 2.25 mM shifted the dose-effect curve of ouabain to the left. At 2.25 mM-K, inhibition of renin secretion was half-maximal at 10(-5)M-ouabain.5. The inhibitory effect of 2 x 10(-5)M-ouabain (twice the dose for 50% inhibition) in media containing 2.25 mM-K was nearly identical to the combined effect of lowering K to 1.43 mM (the concentration required for 50% inhibition) and adding 10(-5)M-ouabain. This observation suggests that ouabain and low extracellular K act at a common site, presumably on Na, K-ATPase activity, to inhibit renin secretion.6. Neither 10(-3)M-ouabain nor K-free medium inhibited renin secretion when the concentration of free Ca in the medium was lowered to < 10(-8)M. Therefore it is proposed that as a result of Na, K-ATPase inhibition, (a) intracellular Na increases, (b) intracellular Ca increases via Na-Ca exchange, provided that extracellular Ca exceeds 10(-8)M, and that (c) Ca accumulation, in some unknown manner, inhibits renin secretion from rat renal cortical slices.
Topics: Animals; Calcium; Depression, Chemical; Dose-Response Relationship, Drug; In Vitro Techniques; Kidney Cortex; Male; Ouabain; Potassium; Rats; Renin; Secretory Rate
PubMed: 6991669
DOI: 10.1113/jphysiol.1980.sp013154 -
International Journal of Molecular... Oct 2022Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown...
Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na/K ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and β-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.
Topics: Animals; Ouabain; Potassium Channels, Voltage-Gated; Potassium; Potassium Channels; Sodium-Potassium-Exchanging ATPase; Epithelial Cells; Mammals
PubMed: 36362049
DOI: 10.3390/ijms232113257 -
International Journal of Molecular... Sep 2022The 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...
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 ( = 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.
Topics: Animals; Corticosterone; Diaphragm; Interleukin-6; Isoenzymes; Ligands; Male; Muscle, Skeletal; Ouabain; Rats; Rats, Wistar; Saline Solution; Sodium; Sodium-Potassium-Exchanging ATPase
PubMed: 36142836
DOI: 10.3390/ijms231810921 -
American Journal of Physiology. Heart... Mar 2012Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular... (Review)
Review
Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.
Topics: Animals; Cardiotonic Agents; Female; Humans; Hypertension; Hypothalamus; Male; Mice; Ouabain; Pregnancy; Rats; Sodium Chloride, Dietary; Sodium-Potassium-Exchanging ATPase; Sympathetic Nervous System
PubMed: 22058154
DOI: 10.1152/ajpheart.00899.2011 -
International Journal of Molecular... Oct 2016Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone...
Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, α1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/kg/day rostafuroxin for four weeks. In group 1, 2-3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO.
Topics: Androstanols; Animals; Kidney; Kidney Diseases; Nephrectomy; Ouabain; Rats; Rats, Sprague-Dawley; Reperfusion Injury
PubMed: 27754425
DOI: 10.3390/ijms17101728