Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding

Lücking, K.; Nielsen, Jesper M.; Pedersen, Per Amstrup; Jørgensen, P. E. V.

doi:10.1152/ajprenal.1996.271.2.f253

Cited by 40 publications

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“…1 also show the presence of mRNA for the ␣1 and ␣3 isoforms, but not that for ␣2, again paralleling the results found with reticulocyte RNA (1). It should be noted that the presence of ␣3 in the kidney control has been found before (26).…”

Section: Resultsmentioning

confidence: 90%

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Hoffman

Wickrema

Potapova

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the ␣1, ␣3, ␤2, and ␤3 subunit isoforms and the ␥ modulator were present. Northern analysis of the erythroid progenitor cells again showed that ␤2 but not ␤1 or ␣2 isoforms were present. The erythroid cells display a unique ␤ subunit expression profile (called ␤-profiling) in that they contain the message for the ␤2 isoform but not ␤1, whereas leukocytes and platelets are known to have the message for the ␤1 but not for the ␤2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of ␣1, ␣3, (no ␣2), ␤2, ␤3, and ␥ together now with clear evidence that ␤1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that ␣1, ␣3, ␤1, ␤2, ␤3, and ␥ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the ␣1 and ␣3 isoforms of the ␣ subunit, the ␤1, ␤2, and ␤3 isoforms of the ␤ subunit, and the ␥ modulator.

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Section: Resultsmentioning

confidence: 90%

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Hoffman

Wickrema

Potapova

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The different results most likely reflect ␣-subunit isoform-and cell-specific regulation. In proximal tubule cells, insulin increases the Na ϩ affinity (Féraille et al, 1994) of the predominantly expressed ␣1-isoform of Na ϩ ,K ϩ -ATPase (McDonough et al, 1994;Lü cking et al, 1996), whereas in skeletal muscle cells, insulin increases the cell surface expression (Hundal et al, 1992;Marette et al, 1993) of the predominantly expressed ␣2-isoform (Hsu and Guidotti, 1991).…”

Section: Discussionmentioning

confidence: 99%

Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Féraille

Carranza

Gonin

et al. 1999

MBoC

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Phosphorylation of the ␣-subunit of Na ϩ ,K ϩ -ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na ϩ ,K ϩ -ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the ␣-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat ␣-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86 Rb uptake in opossum kidney cells expressing mutant rat ␣1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86 Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

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“…This latter aspect is crucial when assessing the physiological relevance of nanomolar concentrations of ouabain in rat kidney where only the ouabain-resistant ␣1 isoform has been detected (27).…”

Section: Expression Of Na-k Atpase and Signaling Molecules In Total Rmentioning

confidence: 99%

Organ Hypertrophic Signaling within Caveolae Membrane Subdomains Triggered by Ouabain and Antagonized by PST 2238

Ferrandi

Molinari

Barassi

et al. 2004

Journal of Biological Chemistry

133

166

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In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 g/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nM and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (؉11%, p < 0.05), and kidney weight (؉9%, p < 0.01). These effects in vivo are associated with a significant enrichment of ␣1, ␤1, ␥a Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat ␣1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 g/kg/day in vivo or 10 ؊10 -10 ؊8 M in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.Until recently, the main, if not unique, function ascribed to the integral membrane protein Na-K ATPase is the maintenance and regulation of the electrochemical gradient across the cell membrane in all tissues (1). Ouabain and other steroidal cardenolides (2) or bufadienolides (3) are considered to be the specific inhibitors of the Na-K ATPase activity. However, in recent years, several studies have indicated that Na-K ATPase can also act as a signal transducer in response to the interaction with its natural ligand ouabain (4). This finding originates mainly from studies carried out on cultured rat cardiomyocytes or renal tubular cells based on effects on cell growth and hypertrophy of ouabain in the micromolar range. At these rather high concentrations, which, however, do not seem to affect the bulk intracellular Na ϩ and Ca 2ϩ concentrations (5), ouabain activates: (a) tyrosine phosphorylation of the epidermal growth factor receptor (EGFr), 1 Src, and p42/44 mitogenactivated protein kinase (MAPKs) in both neonatal rat cardiac myocytes and A7r5 cells (4, 6); (b) the same signaling pathway within the cellular membrane microdomain of caveolae in isolated perfused rat heart (7); and (c) slow intracellular Ca 2ϩ oscillations in rat tubular cells that favor the association of Na-K ATPase with the inositol 1,4,5-trisphosphate receptor (InsP 3 ...

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Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding

Cited by 40 publications

References 0 publications

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Organ Hypertrophic Signaling within Caveolae Membrane Subdomains Triggered by Ouabain and Antagonized by PST 2238

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Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding

Cited by 40 publications

References 0 publications

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Organ Hypertrophic Signaling within Caveolae Membrane Subdomains Triggered by Ouabain and Antagonized by PST 2238

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Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10