of ATP, GTP, CTP, 0.03 μM UTP, and 100 μCi of [α-P]UTP (DuPont NEN, 3,000 Ci/mmol) for 15 min at 30°C. The ferret heart contains nearly equal amounts of two isoforms of the sodium pump, one of which is highly sensitive to Time courses of the effects of different ouabain concentrations on myocyte viability. This review focuses on ouabain-mediated activation of cell signaling pathways and secondary messengers that conversely may modulate Na(+),K(+)-ATPase function. Total RNA was isolated and subjected to Northern blot analysis as described under “Experimental Procedures.” B, combined data from several different experiments. than 95% myocytes as assessed by immunofluorescence staining with a myosin heavy chain antibody(11). Effect ofouabain on Na,K-ATPase andelectrolyte transport in the dog ileum in vivo British Society of Gastroenterology (Read et al., 1978). (14) and counted in 0.04% trypan blue using a hemocytometer. Akagawa K., Hara N., and Tsukada Y. myocyte growth. The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na,K-ATPase. 8 and Fig. Incubations were done in the presence of 0.5 mM ouabain and in its absence for 45 min as in Fig. 5), and no synergistic effects of serum and ouabain are apparent (see “Results”). 2The results of these experiments showed that (a) ouabain concentrations of 0.5 mM or higher were required to note an increase in c-fos mRNA and (b) the maximal level of c-fos mRNA, about 5 times that of control, was obtained after 15 min of exposure to ouabain and rapidly declined to control level Therefore, the decrease in the concentration gradient of sodium into ⦠In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+) ⦠probe. 2001 Mar;47(2):383-90. medium, followed by 24 h in serum-free medium) showed nearly identical results (Fig. The reduced contractile response of cells grown in 1 mM extracellular potassium and ouabain (but not isoproterenol) supports the view that elevated intracellular sodium due to Na,K-ATPase inhibition mediates the positive inotropic response to low extracellular potassium and ouabain, probably via augmented transsarcolemmal sodium-calcium exchange. In the heart, partial inhibition of Na + /K +-ATPase by ouabain and related cardiac glycosides causes a modest increase in intracellular Na +, which in turn leads to significant increases in intracellular Ca 2+ through the Na + /Ca 2+ exchanger and in myocardial contractility through the effects of Ca 2+ on contractile proteins . Since induction of c-fos by ouabain involves PKC (Fig. more Fos and Jun proteins than control nuclei, as determined by immunostaining procedures that we have used before to show mM Ca was also added. (B) Effect of glycosylation on CTS binding to the Na +,K +-ATPase. This suggests a role of intracellular Ca in the pathways of the effects of ouabain, phenylephrine, and PMA on c-fos. Time course of ouabain-induced c-fos expression. HeLa cells), and in mouse fibroblasts, required ouabain concentrations that cause complete inhibition of Na/K-ATPase. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). of 5 experiments (p < 0.05). Ouabain is a steroid that binds to the Na (+)/K (+) ATPase, inducing the activation of several intracellular signaling pathways. Interaction between cardiotonic steroids and Na,K-ATPase. Ca concentration was established by calibration procedures and calculations described before (16, 23). Cardiotonic steroids constitute a broad class of specific Na +,K +-ATPase inhibitors, including drugs of clinical importance with multiple physiological effects.The existence of several endogenous cardiotonic steroids suggests ⦠PubMed CrossRef Google Scholar. It is clear, therefore, that the slow induction of the genes by the toxic concentrations Since partial inhibition of the sodium pump by ouabain is known to cause a subsequent increase in functional pump sites Assays of intracellular Ca were done in separate experiments as described under “Experimental Procedures.” Values are means ± S.E. Signal-transducing function of Na1-K1-ATPase is essential for ouabainâs effect on [Ca21] i in rat cardiac myocytes JIANG TIAN,1 XIAOHUA GONG,2 AND ZIJIAN XIE1 1Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614; and 2Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037 Received 24 March 2001; accepted in ï¬nal form 6 July 2001 Cell Mol Biol (Noisy-le-grand). In fish with initially high gill Na+-K+-ATPase activity dry wt) but had no significant effect on the dissociation con- (15) and labeled by T4 DNA kinase using [-P]ATP. in Fig. Exposure of neonatal rat cardiac myocytes to ouabain concentrations that caused partial inhibition of Na/K-ATPase but no loss of viability, increased c-fos and c-jun mRNAs and the transcription factor AP-1. Until the nature of these previously unrecognized effects of 1 C, a prolonged incubation with up to 100 n m ouabain had little effect on cell viability. Control myocytes and those treated with 0.1 μM PMA for 24 h were exposed to 0.1 mM or 0.5 mM ouabain for 45 min and Physiol. The intensities In these contexts, we do not have sufficient data to compare ouabain with other hypertrophic stimuli. The recently published crystal structure of the Na +,K -ATPase phosphoenzyme (E2P) in complex with the widely studied CTS ouabain (4, 5) showed that the high-affinity CTS-binding site is constituted by the transmembrane helices αM1â6 of the cata- (Worthington). The stimulatory effect on ouabain-sensitive H+,K+-ATPase and on cortical Na+,K+-ATPase was also abolished by brefeldin A which inhibits the insertion of pro-teins into the plasma membranes, whereas the inhibitory effect on medullary Na+,K+-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of Na +,K +-ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na + and K + across the membrane of animal cells. inhibition of the cardiac Na/K-ATPase, causing a small increase in intracellular Na, which in turn affects the sarcolemmal Na/Ca exchanger, leading to an increase in intracellular Ca and in the force of contraction(2, 3, 4). 9 and “Results”). experiments were done at 37°C after 24 h of additional incubation under serum-free conditions. More recently, Golomb et al. Involvement of reactive oxygen species in a feed-forward mechanism of Na/K-ATPase-mediated signaling transduction. The increased mRNAs were proportional to the extent of inhibition of Na/K-ATPase and the resulting rise in steady state intracellular Ca concentration. 10), and SRE is known to be stimulated by PKC(38, 40), it seems that ouabain induction of c-fos may be, at least in part, through SRE. 5720. The animals were fasted for 24 hours before surgery. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. Ouabain and related cardiac glycosides are highly specific inhibitors of Na/K-ATPase. 1. 3 . on beating or on the contractile force. 8). Biomed Res Int. c-fos mRNA was measured and normalized to those of GAPDH. other cell lines showed that increased expressions of c-fos and c-jun required several hours of exposure to ouabain concentrations that cause near complete inhibition of Na/K-ATPase. Na + /K +-ATPase activity and ouabain binding of the human enzymes measured under similar conditions suggest that at 100 n m ouabain <50% of the pumps are blocked (e.g. 2010;72:395-412. doi: 10.1146/annurev-physiol-021909-135725. ↵* This work was supported by National Institutes of Health Grant HL-36573 awarded by NHLBI, United States Public Health Service, Examination of this literature shows differences between ouabain effects and those of any other stimulus. Dose-response curve of ouabain effects on c-fos and c-jun expressions in myocytes. 8). In our experiments, Neonatal ventricular myocytes were prepared and cultured as described before(11, 12). 5). 2013 Nov 22;288(47):34249-58. doi: 10.1074/jbc.M113.461020. Effect of ouabain on total transcriptional activity of myocytes. However, recently it has been discovered that circulating endogenous cardiotonic steroids bind to Na(+),K(+)-ATPase, activate cell signaling pathways, and regulate diverse cellular functions. In cardiac myocytes, however, ouabain-induced regulation of c-fos is independent of serum (Fig. Kidneys were removed on day 4, decapsulated, and the cortex was carefully separated from the medulla. Na +,K -ATPase isoform selectivity, with up to fourfold prefer-ence for α2/ 3 over 1(1). Nuclear protein (3-5 μg) was used in the binding reaction; the reaction mixture (20 μl) contained 50 mM NaCl, 1 mM The inhibitory effect observed after 0.5 h infusion was impaired in the hyperleptinaemic group. 2015 Sep 14;589(19 Pt B):2668-74. doi: 10.1016/j.febslet.2015.08.011. In experiments kinase C. Exposure to ouabain for 24-48 h also increased total transcriptional activity and protein content of myocytes. American Society for Biochemistry and Molecular Biology. Clearly, based on available evidence, there seem to be cardiac-specific regulatory effects of ouabain on expressions of Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. It remains to be seen if ouabain's effect on growth can be distinguished from its effects on contractility. Copyright © 2012 Elsevier Inc. All rights reserved. A ouabain-like factor was later found in the adrenal cortex, pituitary and hypothalamus [61,62]. Purified [P]RNA was counted as an index of total transcriptional activity. Finally, depletion of either cholesterol Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na+/K+-ATPase signaling complex. Once ouabain binds to this enzyme, the enzyme ceases to function, leading to an increase of intracellular sodium. 2020 May 11;61(5):1. doi: 10.1167/iovs.61.5.1. Electrophoretic mobility shift assay for AP-1 binding activity was done as described previously(11). COVID-19 is an emerging, rapidly evolving situation. The effects of the solvents deuterated water (2 H 2 O) and dimethyl sulfoxide (Me 2 SO) on [3 H]ouabain binding to (Na +,K +)-ATPase under different ligand conditions were examined.These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg 2+ +ATP+Na +).In contrast, both solvents stimulated type II (i.e., Mg 2+ +P i-, or Mn 2+-dependent) binding of the drug. FEBS Lett. The relative amount of RNA in each sample was normalized to that of 2 are worthy of note at this point. the hypertrophied failing heart with a hypertrophic stimulus. These cultures contained more Effects of the exposure of a myocyte preparation to different ouabain concentrations for 45 min on c-fos and c-jun mRNAs are shown in Fig. A single cell was isolated from the surrounding area using a pinhole Effects of 0.5 mM ouabain, 0.1 mM phenylephrine, and 0.1 μM PMA after 45 min of incubation in this Ca-free medium were determined. These data were used as a guide for the remaining experiments on myocytes; cell types (see “Experimental Procedures”). For some experiments with myocytes, the nominally Ca-free DME/F-12 base medium (Sigma) was used. (6) also noted that in these cell lines, ouabain-induced expression of c-fos was neither Ca-dependent nor affected by depletion of PKC. body of older literature (45) showing inhibitory effects of ouabain on transformation and proliferative growth of several mammalian cell types and indicating Ouabain is a cardiac glycoside that acts by inhibiting the Naâº/Kâº-ATPase sodium-potassium ion pump (but it is not selective). The costs of publication of this article were defrayed in part by the payment of Control reaction mixtures for the detection of specific binding contained 100-fold excess of unlabeled The probes were applied to Nytran membrane through dot-blot At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na /K -ATPase with epidermal growth factor receptor Compound GDC, an Isocoumarin Glycoside, Protects against LPS-Induced Inflammation and Potential Mechanisms In Vitro. In similar experiments (not shown), it was established that repletion of medium Ca did not have a significant effect on phenylephrine's or PMA's effect on c-fos expression. not shown when smaller than symbol size. Objectives were to evaluate effects of ouabain-induced inhibition of Na +, K +-ATPase in equine blastocysts. Values are mean ± S.E. As shown in Fig. It performs several functions in cell physiology.. 6 showed that ouabain-induced increase in steady state concentrations of c-fos mRNA was due, at least in part, to increase in transcriptional rate of c-fos. Equal counts of P-labeled RNA from different groups were used for hybridization. The purified Na/K-ATPase (2 μg) and purified Src were incubated in the presence of either 10 μM ouabain or 1 and 10 μM MB7 for 15 min and assayed for Src activation as described in âExperimental Proceduresâ. 1 and Fig. serum. When time-dependent changes in c-fos mRNA in response to 0.1 mM ouabain were measured (Fig. stimulation of AP-1. It performs several functions in cell physiology.. NLM Then were washed, and ouabain binding was performed as described in the MATERIALS AND METHODS. 2) is consistent with the fact that of the two isoforms (α and α) of Na/K-ATPase present in neonatal rat cardiac myocytes, the predominant one is the relatively ouabain insensitive α isoform(24, 25). the important role of the sodium pump in these processes. apparatus, denatured, and immobilized by UV-cross-linking. No attempt was made to quantitate these effects Read "Brain (Na + , K + )âATPase and Noradrenergic Activity: Effects of Hyperinnervation and Denervation on HighâAffinity Ouabain Binding, Journal of Neurochemistry" on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at ⦠Ouabain Concentration-Response Curve Effect of Ouabain on Basal Tone and for Na+-K+-ATPase Agonist-Induced Contractions The expression of both a1 and a2 isoforms necessitated Effect of ouabain on basal tone was determined by to examine whether the pulmonary artery plasmalemmal exposure of the arterial rings to different concentrations of the Na+-K+-ATPase isoforms were differently sensitive ⦠The nuclei were collected, lysed, and digested with RNase-free DNase 20-HETE (1 μM) had a similar effect as ouabain and increased [Na +] i from 19 ± 1 to 30 ± 1 mM. shown to regulate several cardiac genes, including those of contractile proteins(51, 52, 53). 23â25). medium supplemented with 10% fetal bovine serum. of one. Autoradiograms obtained at −70°C were scanned with a Bio-Rad densitometer. to explore the effects of these drugs on the expressions of cardiac early-response genes. Refs. Their studies on HeLa and some This enzyme (the sodium pump) catalyzes the coupled active transport of Na and K across the plasma membranes of most animal cells (1). Previous studies have demonstrated that both inotropic and toxic concentrations of ouabain This cell (7) reported that in vascular smooth muscle cells, ouabain did not affect the expression of c-fos in the absence of serum, but that the mitogenic effects of serum, including the inductions of early-response genes, were are accompanied by significant dissipation of the normal Na and K gradients and are most likely due to the well-established inhibitory effects of low intracellular K on protein synthesis(45). Growth hormone and Integrative Comp. While these effects may be the consequence of activation of phospholipase C by ouabain-induced increases in intracellular eCollection 2018. Treatment with ouabain increased the number of myocytes beating regularly. Methods CONSTRUCTION OF LOOPS Experiments were carried out in 18 healthy, de-wormedmongrel dogs weighing between 15 and 30 kg. We then evaluated whether fish oil treatment prevented the diabetesâinduced changes. Cells were incubated in the presence of 0.1 mM ouabain and 3B) is similar to the differential effects of other stimuli (e.g. Ca(2, 3, 4, 21, 22, 23), our data showed significant increases in intracellular Ca concentrations of these myocytes in parallel with ouabain-induced inhibition of Rb uptake (Fig. ouabain clearly affected myocyte contractility as expected (see “Experimental Procedures”), although we did not quantitate day â1). increase in intracellular Ca is obtained without large changes in intracellular Na and K concentrations(2, 3, 4, 21, 22, 23). proto-oncogenes. Would you like email updates of new search results? Lanes 1 and 3, P-labeled probe; lanes 2 and 4, labeled probe plus 100-fold excess of unlabeled probe. 2019 Jun 6;19(1):119. doi: 10.1186/s12906-019-2532-6. Section 1734 solely to indicate this fact. of other cardiac genes by the resulting transcription factors, and stimulation of myocyte growth. For Northern blot analysis, total RNA was isolated using TRI reagent (Molecular Research Center, Inc.) as recommended by the 2) The observation that 0.1 mM ouabain causes significant inhibition of the pump (Fig. And sustained increase of c-fos is independent of serum ( Fig rather the. Data clearly indicate that ouabain increased a nuclear protein that could bind to a probe containing the AP-1 binding.... Prior report relating the early-response genes, increases protein content and stimulates growth... Of cardiac early-response genes to the sodium pump is that of GAPDH mRNA to for... The PI3K/Akt pathway evidence, there seem to be cardiac-specific regulatory effects of ouabain, phenylephrine, and the was... Restored the effect of ouabain, i.e had little effect on Na-K-ATPase in. Cells in any of the effects of MB7 and ouabain on expressions proto-oncogenes... Any of the hypertrophied failing heart with a hypertrophic stimulus exposures were analyzed to ensure the. W, Chu K. Inflammation COVID-19 is an emerging, rapidly evolving situation the increased mRNAs were proportional the... Of these issues are in progress to determine whether or not ouabain exerts effects... Digoxin and ouabain binding was performed as described before ( 11 ) the exposure of a Na+/K+-ATPase-Src-caveolin in. Ouabain interaction with cardiac Na/K-ATPase reveals that the same signal responsible for the positive inotropic action of ouabain was but! 30 kg 7 independent experiments run in triplicate described that ouabain increased the number myocytes... Concentrations for 45 min zhang Y, Yan G, Sun C Bernklau... And 3, P-labeled probe ; lanes 2 and 4, decapsulated, the! Quantitate these effects on myocyte c-fos expression in serum-free and serum-containing media rats the effect of ouabain intracellular! Cholesterol Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na+/K+-ATPase signaling.... Data are means t SE of 7 independent experiments run in triplicate dna-protein complexes were separated on 4.5... Isolated caveolae preparations as it did in live cells before surgery sufficient data to compare with! On intracellular Ca ( Fig done in the treatment of the inhibitors of that... Increases in c-fos and c-jun mRNAs are shown in the MATERIALS and.... College of Ohio, P. O of any other stimulus the Src-Ras-p42/44 MAPK cascade a time-dependent.... Affect viability up to fourfold prefer-ence for α2/ 3 over 1 ( ). Other stimuli ( e.g run in triplicate were prepared and cultured as described the!, phenylephrine, and protects from apoptosis Degeneration in Guinea Pigs nondenaturing 4.5 % polyacrylamide gels in mM... Similar to this enzyme, the precise chemical structure and its pathway of response to ouabain enzyme ceases to,... Steroid/Ouabain-Binding site of the film isolated as described before ( 11 ) ( see “ results )... Signaling complex phenylephrine and PMA features are temporarily unavailable experiments showing ouabain effects determined. The ouabain effect on na-k atpase group these issues are in progress to determine the effects different. The transcriptional regulation of c-fos is independent of serum and ouabain are almost.! The nominally Ca-free DME/F-12 base medium ( Sigma ) was used with cardiac Na/K-ATPase reveals the., with up to 24 h of incubation and normalized to that of Cayanis et.! Antibody ( 11 ) ( 15 ) and labeled by T4 DNA using. Salmon gill ” ) rats the effect of Fatty Acids on [ 3 h ] binding! And ouabain-induced changes in c-fos mRNA was blocked when myocytes were cultured on glass coverslips loaded... Potential Mechanisms in Vitro cholesterol depletion abolished ouabain-induced recruitment of Src to the Na+/K+-ATPase signaling complex beating regularly )... Were repeated in several human cell lines ( e.g ± S.E use in experiments whether hitherto... A myosin heavy chain antibody ( 11, 12 ) BMC Complement Altern Med we then whether... Plössl K, schmid C, Nan L, Messaggio E, Lanzani C, a incubation! Steady state intracellular Ca in the graph hypertrophied failing heart with a myosin heavy chain antibody 11! Ca to the sodium pump is that of Cayanis et al therefore, precise... Or higher concentrations of ouabain, phenylephrine, and ouabain are apparent ( see “ results ” ) responsible the... Genes to the sodium pump is that of Cayanis et al within linear! + âATPase ( NKA ) is similar to the Src-Ras-p42/44 MAPK cascade Spiral ganglion cell Degeneration in Pigs! Be modulated by physiological ouabain concentrations on myocyte growth ( Molecular Research Center, Inc. COVID-19 is an emerging rapidly... To correct for differences in sample loading and transfer scanned with a hypertrophic stimulus a long-term potentiation response, K.! With 100 μM ouabain and furosemide inhibited 86 Rb + uptake by myocytes and intracellular., clearly establishing the necessity of ouabain-induced inhibition of pump-mediated Rb uptake was assayed described. Rna was counted ouabain or related cardiac glycosides are highly specific inhibitors of +... Gradient of sodium into ⦠ouabain in nanomolar concentration elicits a long-term potentiation....:2668-74. doi: 10.3390/ijms19071948 mRNAs are shown in Fig experiments as described under “ Procedures.... The regulation of Na+, K+-ATPase by ouabain were used, exposure times did not exceed min. This suggests a role of intracellular Ca ( Fig Na ( + ) -ATPase α subunit is highly in... Complex in the course of induction of c-fos was more pronounced than that on c-jun ( Fig,!, i.e but it is not selective ) kinase using ouabain effect on na-k atpase -P ATP! Steroid/Ouabain-Binding site of the Na, K-ATPase inhibition by ouabain was associated with think isoform. L, Messaggio E, Lanzani C, Manunta P. Int J Mol.... 47 ):34249-58. doi: 10.1167/iovs.61.5.1 types ( see “ ouabain effect on na-k atpase Procedures )...