A simple and rapid method for the determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle

Nørgaard, Aage; Kjeldsen, Keld; Hansen, Otto; Clausen, Torben

doi:10.1016/s0006-291x(83)80154-5

Cited by 107 publications

(63 citation statements)

References 17 publications

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“…Monolayers were treated with glycolaldehyde (0 or 20 mM) for 15 min; basolateral fluid was removed and replaced with 3H-ouabain binding assay buffer which was composed of 250 mM sucrose, 3 mM MgSO4, 3 mM Na2HPO4, 1 mM Na3VO4, 10 mM Tris-HCl, and 0.5% BSA at pH 7.2 and containing 3H-ouabain at concentrations ranging from 0.125 to 2 pM (19,(25)(26)(27). Monolayers, in 6.5-mm Transwells, were allowed to bind 3H-ouabain for 2 hr at 37°C.…”

Section: Measurement Of Specific 3h-ouabain Bindingmentioning

confidence: 99%

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Robison

Zhou

Kim

1996

Environ Health Perspect

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Pulmonary injury from nitrogen dioxide (NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. With the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon alpha-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 +/- 2.7 and 234.0 +/- 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 +/- 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86Rb uptake (an index of Na+,K(+)-ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations > or = 5 mM led to an inhibition of ouabain-sensitive 86Rb uptake, while lower concentrations had no effect. the effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+,K(+)-ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified. Images Figure 1. Figure 2. Figure 3. Figure 4.

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Section: Measurement Of Specific 3h-ouabain Bindingmentioning

confidence: 99%

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Robison

Zhou

Kim

1996

Environ Health Perspect

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“…Alternatively, hypothyroidism might be involved as Tahiliani & McNeil (1985) have reported depressed plasma levels of both T3 and T4 in 6-week streptozotocin-diabetic rats and hypothyroidism has been found to increase cardiac sympathetic activity whether measured by [3H]-NA turnover or, as here, by tissue NA decline following AMPT (Landsberg & Axelrod, 1968). Na', K+-ATPase activity has been reported to be depressed by hypothyroidism and increased by T3 administration (Norgaard et al, 1983). The untreated diabetic rats in the present study may, therefore, have had reduced neuronal Na', K+-ATPase activity due to hypothyroidism which resulted in the observed increase in NA turnover.…”

Section: Discussionmentioning

confidence: 66%

Tissue noradrenaline and the polyol pathway in experimentally diabetic rats

Lucas

Qirbi

1989

British J Pharmacology

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1 The effects of a six week period of streptozotocin-induced diabetes on tissue catecholamines and on in vivo noradrenaline turnover were assessed in rats. 2 Noradrenaline concentrations measured in heart ventricle, terminal ileum, vas deferens, spleen and adrenal tissue from the diabetic rats were all found to be elevated compared to those found in control rat tissues. The adrenaline contents of the adrenal glands were also raised in these animals. 3 Noradrenaline turnover in heart ventricle, terminal ileum and vas deferens was estimated from the decline in tissue content of the amine following inhibition of its synthesis with a-methyl-ptyrosine. Turnover was found to be increased in all three tissues. 4 The involvement of the polyol pathway in the above changes was investigated by examining the effects of continuous treatment with an aldose reductase inhibitor, Statil (ICI 128436) or dietary myo-inositol supplementation. Either treatment was found to prevent or reduce the increases in tissue noradrenaline and in its turnover. Myo-inositol treatment also partially prevented the rise in adrenal adrenaline. 5 It is concluded that the elevation of tissue catecholamines and of noradrenaline turnover by diabetes was related to myo-inositol depletion secondary to excessive sorbitol synthesis. Possible mechanisms for the observed increase in noradrenaline turnover could involve Na', K+-ATPase depression.

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“…Ca 2+ /K + -ATPase and 5'-nucleotidase activity [22] and [ 3 H]ouabain binding were performed as described previously [23] . Protein was assayed according to Bradford [24] using ovalbumin as a standard.…”

Section: Cell Surface Biotinylationmentioning

confidence: 99%

Insulin induced translocation of Na+/K+-ATPase is decreased in the heart of streptozotocin diabetic rats

et al. 2009

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Aim: to investigate the effect of acute insulin administration on the subcellular localization of na + /K + -AtPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats. Methods: Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. na + /K + -AtPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-Omethylfluorescein-phosphatase activity. Results: In control rat heart muscle α1 isoform of Na + /K + ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P<0.05) in plasma membrane and α2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2-but not α1-subunits was detected. α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and β1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. the insulin-induced translocation was wortmannin sensitive.Conclusion: This study demonstrate that insulin influences the plasma membrane localization of Na + /K + -AtPase isoforms in the heart. α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates na + /K + -ATPase α1-and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

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A simple and rapid method for the determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle

Cited by 107 publications

References 17 publications

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Tissue noradrenaline and the polyol pathway in experimentally diabetic rats

Insulin induced translocation of Na+/K+-ATPase is decreased in the heart of streptozotocin diabetic rats

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