Determination of total (Na++K+)-ATPase activity of isolated or cultured cells

Xie, Zijian; Wang, Yahong; Ganjeizadeh, Mehdi; McGee, Richard; Askari, Amir

doi:10.1016/0003-2697(89)90470-3

Cited by 60 publications

(51 citation statements)

References 26 publications

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“…Na/K-ATPase Activity Assay-Na/K-ATPase enzymatic activity was determined as we described previously (14). Briefly cells were collected from the cultures in Tris-EGTA buffer (pH 7.2) and briefly sonicated.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Liang

Cai

Tian

et al. 2006

Journal of Biological Chemistry

149

230

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We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several ␣1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing ␣1-specific small interference RNA. Although the ␣1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabaininduced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat ␣1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat ␣1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat ␣1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases. Na/K-ATPase was discovered by Skou (1) as the molecular machinery of the cellular sodium pump. It belongs to a family of evolutionarily ancient ATPases that couple the hydrolysis of ATP to membrane ion translocation (2, 3). A major difference between the Na/K-ATPase and other ATPases is its ability to bind cardiotonic steroids such as ouabain. Studies from many laboratories have now established that the binding of ouabain to this enzyme not only inhibits the ATPase activity but also stimulates protein tyrosine kinases such as Src (4, 5). The activated Src in turn transactivates epidermal growth factor receptor, resulting in the assembly and activation of multiple signaling cascades such as the ERK1/2 2 and phospholipase C-␥/ protein kinase C pathways (5, 6).Because several laboratories have demonstrated that the activation of Src is essential for ouabain-induced changes in many cellular activities including the regulation of intracellular calcium, gene expression, and cell growth (6 -9), we have recently examined whether the Na/K-ATPase interacts directly with Src to form a functional signaling receptor (10). Using in vitro glutathione S-transferase pulldown assays we have identified that the second and the third intracellular domains of the Na/K-ATPase ␣1 subunit interact with the Src SH2 and the kinase domains, respectively. Functionally these interactions keep Src in an inactive state, and binding of ouabain to this inactive Na/K-ATPase⅐Src complex frees and then activates the associated Src (10). These new findings suggest that the cellular Src-interacting Na/K-ATPase may play an...

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Liang

Cai

Tian

et al. 2006

Journal of Biological Chemistry

149

230

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“…1; Ref. 20), and significant stimulation of MAPKs occurs when 10 -20% of the enzyme is inhibited by ouabain. Although the effects of ouabain on MAPKs correlate well with its binding to Na ϩ /K ϩ -ATPase in these cells, there is always a possibility that 10 -100 M ouabain may affect other cellular proteins in addition to Na ϩ /K ϩ -ATPase.…”

Section: And Egfrmentioning

confidence: 99%

“…Assay for Na ϩ /K ϩ -ATPase Activity and Intracellular Na ϩ Concentration-Whole cell total Na ϩ /K ϩ -ATPase activity was assayed in the presence of alamethicin as described previously (20). Briefly, cells were collected from the cultures and treated with alamethicin at a concentration of 0.1 mg/mg protein.…”

mentioning

confidence: 99%

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

Haas

Wang

Tian

et al. 2002

Journal of Biological Chemistry

Self Cite

263

260

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“…Each assay was done in the presence and absence of 1 mM ouabain, and the ouabainsensitive component was considered as Na ϩ -K ϩ -ATPase activity. When indicated, alamethacin was included in the assay as described before (46). Pi was measured with Malachite Green (17), using the commercially available Biomol Green reagent (BIOMOL Research Laboratories, Plymouth Meeting, PA).…”

Section: Methodsmentioning

confidence: 99%

β-Subunit of cardiac Na⁺-K⁺-ATPase dictates the concentration of the functional enzyme in caveolae

Liu¹,

Askari²

2006

American Journal of Physiology-Cell Physiology

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Previous studies showed the presence of a significant fraction of Na+-K+-ATPase α-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na+-K+-ATPase with its signaling partners. Because both α- and β-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na+-K+-ATPase, we have examined the relative distribution of two major subunit isoforms (α1 and β1) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na2CO3 or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of α1-subunits and 80–90% of β1-subunits. Use of Na2CO3 was shown to inactivate Na+-K+-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained ∼75% of total myocyte Na+-K+-ATPase activity. Sealed isolated caveolae exhibited active Na+ transport. Confocal microscopy supported the presence of α,β-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na+-K+ fluxes, and the sites where the pumping and signaling functions of Na+-K+-ATPase are integrated. Preferential concentration of β1-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of α1 and β1 in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na+-K+-ATPase in cardiac caveolae.

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Determination of total (Na++K+)-ATPase activity of isolated or cultured cells

Cited by 60 publications

References 26 publications

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

β-Subunit of cardiac Na⁺-K⁺-ATPase dictates the concentration of the functional enzyme in caveolae

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Determination of total (Na++K+)-ATPase activity of isolated or cultured cells

Cited by 60 publications

References 26 publications

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

β-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

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β-Subunit of cardiac Na⁺-K⁺-ATPase dictates the concentration of the functional enzyme in caveolae