Endocytosis inhibition protects the isolated guinea pig heart against ouabain toxicity

Núñez‐Durán, Harol; Atonal, Fausto; Contreras, Paola; Melendez, Eloy

doi:10.1016/0024-3205(96)00025-2

Cited by 9 publications

(6 citation statements)

References 15 publications

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“…When cells were chased in the presence of 1 μM ouabain, the α1-subunit was swiftly internalized within 1 hour and remained inside the cell for the next few hours without degradation (data not shown). These data match well those of previous studies in which ouabain treatment induces the internalization of the enzyme (Cook et al, 1982;Griffiths et al, 1983;Lamb and Ogden, 1982;Nunez-Duran et al, 1996;NunezDuran et al, 1988).…”

Section: Different Lifetimes Of the α-And β-Subunits In The Plasma Mesupporting

confidence: 82%

Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

Yoshimura

Iwasaka

Schwarz

2008

Journal of Cell Science

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The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (α1) and auxiliary (β1 and β3) subunits of Na+/K+-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the β-subunit is required for the cell-surface expression of the α-subunit, the plasma membrane contains more α-subunits than β-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the β-subunits was much faster than that of the α1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the β1-subunit. Double knockdown of the β1- and β3-subunits by RNAi resulted in the disappearance of these β-subunits but not the α1-subunit in the plasma membrane. All these results indicate that the α- and β-subunits of Na+/K+-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.

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Section: Different Lifetimes Of the α-And β-Subunits In The Plasma Mesupporting

confidence: 82%

Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

Yoshimura

Iwasaka

Schwarz

2008

Journal of Cell Science

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“…PAO is a protein-tyrosine phosphatase inhibitor known to inhibit actin polymerization (32). In addition to these agents, incubation at 4 o C was also used since endocytosis is temperature dependent (33). Figure 4 shows the effect of Cytochalasin-D, Latrunculin-A and PAO on fDT-2 internalization.…”

Section: Discussionmentioning

confidence: 99%

Culture conditions influence uptake and intracellular localization of the membrane permeable cGMP-dependent protein kinase inhibitor DT-2

Garcı́a²,

Ke³

et al. 2005

Front Biosci

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The membrane-permeable peptide DT-2 which utilizes the HIV-Tat membrane translocation sequence is known to inhibit cGMP-dependent protein kinase (PKG) effectively in vitro and in various cell lines and tissue preparations. However, the uptake characteristics of DT-2 have not been studied in detail. We investigated the intracellular uptake and localization of fluorescein-labeled DT-2 (fDT-2) in cultured C6-glial cells and vascular smooth muscle cells (VSMCs) as well as VSMCs in intact arteries. To avoid fixation-induced fluorescence, live unfixed cells and arteries were incubated with fDT-2 and examined using conventional and confocal fluorescence microscopy. In non-differentiated cultured VSMCs, uptake appeared vesicular with nuclear exclusion, consistent with an endocytotic internalization mechanism. Inhibition of endocytosis by phenylarsine oxide (PAO), low temperature or disruption of actin polymerization by cytochalasin-D or lantrunculin-A showed a residual non-endocytotic fDT-2 translocation with diffuse cytosolic and nuclear uptake. Similarly, differentiated contractile VSMCs within the medial layer of intact cerebral arteries also showed a distinctively different, more diffuse cytosolic uptake and time dependent nuclear localization. To verify the morphology dependency of fDT-2 uptake, VSMCs were reconstituted in fibrillar collagen matrices. The cells adopted a differentiated morphology and fDT-2 translocation was similar to cells in intact arteries. These results demonstrate that VSMCs cells utilize distinct cellular uptake mechanisms depending on their phenotype.

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“…First, cooling decreases the activity of the Na + pump and diminishes contributions of pump inhibition changes in the contractile state induced by the CS. Second, we had demonstrated previously that ouabain could gain entrance to heart cells via an energyrequiring mechanism (Núñez-Durán et al, 1988, 1996. Cooling greatly slows or prevents this route of Fig.…”

Section: Inotropic Effects Of Hydrophobic and Hydrophilic Cs At Low Tmentioning

confidence: 96%

Evidence for an intracellular site of action in the heart for two hydrophobic cardiac steroids

Núñez‐Durán¹,

Fernández²

2004

Life Sciences

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Endocytosis inhibition protects the isolated guinea pig heart against ouabain toxicity

Cited by 9 publications

References 15 publications

Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

Culture conditions influence uptake and intracellular localization of the membrane permeable cGMP-dependent protein kinase inhibitor DT-2

Evidence for an intracellular site of action in the heart for two hydrophobic cardiac steroids

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