Analysis of Na<sup>+</sup>,K<sup>+</sup>-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

Bertorello, Alejandro M.; Komarova, Yulia; Smith, K. John; Leibiger, Ingo B.; Efendiev, Riad; Pedemonte, Carlos H.; Borisy, Gary G.; Sznajder, Jacob I.

doi:10.1091/mbc.e02-06-0367

Cited by 54 publications

(49 citation statements)

References 35 publications

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“…They observed that dopamine enhanced Na pump mobility towards the PM, except for 'perinuclear' Na pump (see figure 3 in Bertorello et al, 2003). Although the observed perinuclear Na + /K + -ATPase may be adjacent, immobile ER Na pump, it may also simply be enzyme targeted to the NE.…”

Section: Namentioning

confidence: 98%

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Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Galva

Artigas

Gatto

2012

Journal of Cell Science

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Summary Na + /K + -ATPase, an integral membrane protein, has been studied for over a half century with respect to its transporter function in the plasma membrane, where it expels three Na + ions from the cell in exchange for two K + ions. In this study, we demonstrate a functioning Na + /K + -ATPase within HEK293 cell nuclei. This subcellular localization was confirmed by western blotting, ouabain-sensitive ATPase activity of the nuclear membrane fraction, immunocytochemistry and delivery of fluorescently tagged Na + /K + -ATPase a-and bsubunits. In addition, we observed an overlap between nuclear Na + /K + -ATPase and Na/Ca-exchanger (NCX) when nuclei were immunostained with commercially available Na + /K + -ATPase and NCX antibodies, suggesting a concerted physiological coupling between these transporters. In keeping with this, we observed an ATP-dependent, strophanthidin-sensitive Na + flux into the nuclear envelope (NE) lumen loaded with the Na-sensitive dye, CoroNa-Green. Analogous experiments using Fluo-5N, a low affinity Ca 2+ indicator, demonstrated a similar ATP-dependent and strophanthidin-sensitive Ca 2+ flux into the NE lumen. Our results reveal an intracellular physiological role for the coordinated efforts of the Na + /K + -ATPase and NCX to actively remove Ca 2+ from the nucleoplasm into the NE lumen (i.e. the nucleoplasmic reticulum).

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Section: Namentioning

confidence: 98%

“…In order to study the effects of dopamine on the trafficking of Na pump, Bertorello and co-workers (Bertorello et al, 2003) expressed GFP-tagged a-subunit in A549 cells. They observed that dopamine enhanced Na pump mobility towards the PM, except for 'perinuclear' Na pump (see figure 3 in Bertorello et al, 2003).…”

Section: Namentioning

confidence: 99%

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Galva

Artigas

Gatto

2012

Journal of Cell Science

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“…It is noteworthy that dynein kinetics after low Vt and high Vt are similar to the kinetics of Na ϩ , K ϩ -ATPase. Cytoplasmatic dynein is an essential component of microtubules, and disruption of the microtubule network has recently been demonstrated to inhibit vesicle motion toward the plasma membrane and prevent a dopamine-induced increase in the Na ϩ , K ϩ -ATPase activity in alveolar epithelial cells (32). This decrease in cytoplasmic dynein could contribute to an impaired ability of the injured ATII cells to augment Na ϩ , K ϩ -ATPase activity and the associated fluid clearance in high-Vt ventilation.…”

Section: Cell Membrane Proteinsmentioning

confidence: 99%

Impact of Low and High Tidal Volumes on the Rat Alveolar Epithelial Type II Cell Proteome

Hirsch¹,

Hansen²,

Sapru³

et al. 2007

Am J Respir Crit Care Med

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Rationale: Mechanical ventilation with high tidal volumes leads to increased permeability, generation of inflammatory mediators, and damage to alveolar epithelial cells (ATII). Objectives: To identify changes in the ATII proteome after two different ventilation strategies in rats. Methods: Rats (n ϭ 6) were ventilated for 5 hours with high-and low tidal volumes (VTs) (high VT: 20 ml/kg; low VT: 6 ml/kg). Pooled nonventilated rats served as control animals. ATII cells were isolated and lysed, and proteins were tryptically cleaved into peptides. Cellular protein content was evaluated by peptide labeling of the ventilated groups with 18 O. Samples were fractionated by cation exchange chromatography and identified using electrospray tandem mass spectrometry. Proteins identified by 15 or more peptides were statistically compared using t tests corrected for the false discovery rate. Measurements and Main Results: High VT resulted in a significant increase in airspace neutrophils without an increase in extravascular lung water. Compared with low-VT samples, high-VT samples showed a 32% decrease in the inositol 1,4,5-trisphosphate 3 receptor (p Ͻ 0.01), a 34% decrease in Na ϩ , K ϩ -ATPase (p Ͻ 0.01), and a significantly decreased content in ATP synthase chains. Even low-VT samples displayed significant changes, including a 66% decrease in heat shock protein 90-␤ (p Ͻ 0.01) and a 67% increase in mitochondrial pyruvate carboxylase (p Ͻ 0.01). Significant differences were found in membrane, acute phase, structural, and mitochondrial proteins. Conclusions: After short-term exposure to high-VT ventilation, significant reductions in membrane receptors, ion channel proteins, enzymes of the mitochondrial energy system, and structural proteins in ATII cells were present. The data supports the two-hit concept that an unfavorable ventilatory strategy may make the lung more vulnerable to an additional insult.Keywords: acute lung injury; alveolar epithelium; corticosterone Acute lung injury is a major cause of morbidity (1) and mortality, and mechanical ventilation is critical to survival of most patients with acute lung injury. However, mechanical ventilation with high tidal volumes (Vts) is known to have deleterious side effects. The importance of ventilator-induced lung injury caused by mechanical ventilation has been established by several experimental and clinical studies (2, 3). Avoidance of excessive Vts Mechanical ventilation with high tidal volumes leads to increased permeability, generation of inflammatory mediators, and damage to alveolar epithelial cells that may contribute to ventilator-induced lung injury. What This Study Adds to the FieldUsing a proteomic approach, significant reductions of key proteins in alveolar type II cells from rats were identified after short-term positive-pressure ventilation.during mechanical ventilation reduces the risk of ventilatorinduced injury, and application of a lower Vt strategy to ventilate patients with acute respiratory distress syndrome has been shown to lead to a greater than 2...

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“…We generated an alveolar epithelial cell line that expresses the Na + /K + -ATPase a1-subunit tagged with GFP and, using these cells, we reported that GPCR agonist stimulation increases the directional movement of ATPase-containing vesicles (Bertorello et al, 2003). In the present study, we investigated the role of myosin-V in the recruitment of the Na + /K + -ATPase to the plasma membrane.…”

Section: Introductionmentioning

confidence: 97%

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

Lecuona

Минин

Trejo

et al. 2009

Journal of Cell Science

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Stimulation of Na+/K+-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na+/K+-ATPase recruitment to the plasma membrane by increasing the average speed of Na+/K+-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na+/K+-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na+/K+-ATPase-containing vesicles, as well as the Na+/K+-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na+/K+-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na+/K+-ATPase to the plasma membrane and thus increased activity.

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Analysis of Na⁺,K⁺-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

Cited by 54 publications

References 35 publications

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Impact of Low and High Tidal Volumes on the Rat Alveolar Epithelial Type II Cell Proteome

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

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Analysis of Na+,K+-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

Cited by 54 publications

References 35 publications

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Impact of Low and High Tidal Volumes on the Rat Alveolar Epithelial Type II Cell Proteome

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

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Product

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Analysis of Na⁺,K⁺-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells