Ouabain Assembles Signaling Cascades through the Caveolar Na+/K+-ATPase

Wang, Haojie; Haas, Michael; Liang, Man; Cai, Ting; Tian, Jiang; Li, Shengwen; Xie, Zijian

doi:10.1074/jbc.m313239200

Cited by 250 publications

(307 citation statements)

References 49 publications

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“…Cholesterol depletion was carried out by incubating the cells in DMEM containing 10 mM methyl-␤-cyclodextrin (M␤-CD) for 30 min at 37°C as we previously described (Wang et al, 2004). After the cells were washed twice with serum-free medium, they were used for the experiments.…”

Section: Cholesterol Depletionmentioning

confidence: 99%

“…Pig LLC-PK1 cells, mouse SYF and SYFϩSrc cells were obtained from American Type Culture Collection and were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and penicillin (100 U/ml)/streptomycin (100 g/ml) as we previously described (Wang et al, 2004). When cell cultures reached ϳ80% confluence, LLC-PK1 cells were serum-starved for 24 h and used for the experiments.…”

Section: Cell Preparation and Culturementioning

confidence: 99%

“…Lysates from LLC-PK1 cells, or other identified cell types, were cleared by centrifugation at 16,000 ϫ g for 15 min at 4°C, and the supernatants (1 mg) were either immunoprecipitated using various antibodies or incubated with different GST fusion proteins (Wang et al, 2004). The immunoprecipitates or GST pulldown products were dissolved in sample buffer, separated on 7% SDS-PAGE, transferred to an Optitran membrane and probed with a monoclonal anti-Na/K-ATPase ␣1, or anti-IP3R2, or anti-PLC-␥1 antibodies.…”

Section: Immunoprecipitation and Gst Pulldown Assaymentioning

confidence: 99%

“…Third, Na/K-ATPase is found to interact directly with neighboring membrane proteins and organized cytosolic cascades of signaling complexes to transmit the ouabain signal to different intracellular compartments (Xie and Cai, 2003). Specifically, we have shown recently that the Na/K-ATPase is concentrated in caveolae together with its signaling partners and that binding of ouabain to the caveolar Na/K-ATPase activated the Na/K-ATPase signaling complex, resulting in tyrosine phosphorylation of multiple proteins including epidermal growth factor receptor (EGFR; Wang et al, 2004). Finally, like other receptors, activation of the signaling function of the Na/K-ATPase by ouabain induces the endocytosis of the enzyme (Liu et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…In view of these recent advances, we tested the hypotheses that the Na/K-ATPase has scaffolding function, capable of assembling a calcium-regulatory complex and that ouabain regulates the function of this complex by activation of protein tyrosine kinases. Pig LLC-PK1 cells were chosen for this work because we have previously used these cells as a model to dissect how the Na/K-ATPase is involved in signal transduction (Liu et al, 2004;Wang et al, 2004). These cells were derived from renal proximal tubules and only express ouabain-sensitive ␣1 isoform of Na/KATPase.…”

Section: Introductionmentioning

confidence: 99%

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Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

Yuan

Cai

Tian

et al. 2005

MBoC

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202

209

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We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-␥1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase ␣1 subunit interacts with PLC-␥1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-␥1 and IP3 receptors together to form a Ca 2؉ -regulatory complex. This notion is supported by the following findings.

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Section: Cholesterol Depletionmentioning

confidence: 99%

Section: Cell Preparation and Culturementioning

confidence: 99%

Section: Immunoprecipitation and Gst Pulldown Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

Yuan

Cai

Tian

et al. 2005

MBoC

Self Cite

202

209

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Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain‐sensitive Na/K‐ATPase

Smolyaninova

Dergalev

Kulebyakin

et al. 2012

Cell Biochemistry & Function

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It is known that ouabain, a selective inhibitor of Na/K-ATPase, can cause not only activation of signal cascades, which regulate the cell viability, but also can cause free radical accumulation, which can evoke the oxidative stress. We have shown that nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.

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Ouabain‐induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells

Лопачев

Lopacheva

Osipova

et al. 2016

Cell Biochemistry & Function

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Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

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Ouabain Assembles Signaling Cascades through the Caveolar Na+/K+-ATPase

Cited by 250 publications

References 49 publications

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain‐sensitive Na/K‐ATPase

Ouabain‐induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells

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