BACKGROUNDThe catalytic α and smaller β subunits of the plasma membrane Na,K-ATPase occur in various molecular forms (α1, α2, α3, β1 and β2). The α isoforms of the enzyme have varying affinities for ...ouabain and exist in different tissues with particular distribution patterns.
OBJECTIVETo document the existence of isoforms of the Na,K-ATPase in cultured human umbilical vein endothelial cells.
METHODSMicrosomal fractions were prepared by differential ultracentrifugation from primary cultures of human umbilical vein endothelial cells and from such cells obtained after three passages. Na,K-ATPase activity was assayed using the coupled assay method and sensitivity to ouabain was determined in the presence of varying concentrations of ouabain. Specific antibodies for the various Na,K-ATPase isoforms were used to label these different proteins by immunocytochemistry in endothelial cells and by Western blotting in isolated membranes.
RESULTSIn plotting the dose–response curves for Na,KATPase activity in response to ouabain we assumed the existence of two independent sites exhibiting different affinities for ouabain (in the μmol/l and the nmol/l ranges). The contribution of low-affinity sites was threefold that of high-affinity sites. After three passages in culture, a specific increase in Na,K-ATPase activity of the high-affinity sites was observed compared with that of cells from primary cultures. Confocal microscopy revealed the existence of β1, β2, and α1 subunit proteins in human umbilical endothelial cells. Staining for α3 isoform was less pronounced and no obvious α2 was detected.
CONCLUSIONThese findings suggest that human umbilical vein endothelial cells contain β1, β2, a large amount of α1 isoform with an apparently low affinity for ouabain, and a lesser amount of high-affinity sites, which may correspond to the α3 protein.
We investigated the effect of focal cerebral ischaemia on the activity and the affinity of the ouabain sites of Na
+,K
+-ATPase in the mouse. The Na
+,K
+-ATPase activity was decreased by 38% as ...early as 30 min following ischaemia. In the sham group, the dose–response curves for ouabain disclosed three inhibitory states which contribute, respectively, 24.9±6.7%, 39.1±7.5% and 36.0% of the total activity (low affinity, LA; high affinity, HA and very high affinity, VHA, respectively). Their computed IC
50 values are, respectively: 1.3×10
−3 M, 4.5×10
−6 M and 2.9×10
−9 M. Surprisingly, in ischaemic cortices, only two sites for ouabain were detected. The first site exhibits a LA (IC
50=2.0×10
−4 M) but its relative contribution to the total activity (46.1±5.2%) is twice that noted for the LA site in non-ischaemic tissues. The second site presents an affinity intermediate between those of HA and VHA sites of the sham group (IC
50=1.7×10
−7 M) and contributes 53.9% to the total activity. Loss in the specific activity of the second site explains that of the total activity. The most likely explanation in the presence of only two ouabain sites of Na
+,K
+-ATPase following ischaemia may be a change in ouabain affinity of
α
2 and/or
α
3 isoforms, as the presence of all three
α isoforms has been observed by Western blotting. These results suggest that ischaemia induces intrinsic modifications in Na
+,K
+-ATPase which result from perturbations in membrane integrity and/or association of the
α isoforms of this enzyme.
1 Laboratoire de Recherche
Cardiologique, 2 Centre de
Résonance Magnétique Biologique et Médicale,
Unité Mixte de Recherche 6612, Centre National de la Recherche
Scientifique (CNRS), and 3 Equipe
...Associée 2205, Faculté de Médecine, Université
de la Méditerranée, 13005 Marseille; and
4 Laboratoire de Neurosciences
Unité de Recherche Associée 1829, CNRS, Caen, France
We tested the hypothesis that a fish oil (FO)
diet promotes positive inotropy of ouabain without increased toxicity.
For 2 mo, two groups of adult male rats were fed
1 ) a regular food diet supplemented
with dietary long-chain polyunsaturated fatty acid from FO or
2 ) a regular food diet (control).
The responsiveness to ouabain was evaluated for the two groups in
Langendorff-perfused hearts, by
31 P nuclear magnetic resonance
spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum
positive inotropy achieved with ouabain was nearly two times higher in
the FO than in the control group and was not associated with
significant changes in energetics. Alteration of function and energetic
metabolism and inhibition of Na-K-ATPase in response to 3 × 10 4 M ouabain were delayed
in the FO group. This study demonstrates that dietary FO, by a cardiac
membrane incorporation of n -3
polyunsaturated fatty acid, promotes positive inotropy of ouabain
without toxicity and changes in cardiac metabolism.
fatty acid; nutrition; sodium-potassium-adenosine
5'-triphosphatase
We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet ...supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control).
Diabetic neuropathy has been associated with a decrease in nerve conduction velocity, Na,K-ATPase activity and characteristic histological damage of the sciatic nerve. The aim of this study was to ...evaluate the potential effect of a dietary supplementation with fish oil (n-3) fatty acids on the sciatic nerve of diabetic rats. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (n = 20) were fed a nonpurified diet supplemented with either olive oil (DO) or fish oil (DM), and control animals (n = 10) were fed a nonpurified diet supplemented with olive oil at a daily dose of 0.5 g/kg by gavage for 8 wk. Nerves were characterized by their conduction velocity, morphometric analysis and membrane Na,K-ATPase activity. Nerve conduction velocity, as well as Na,K-ATPase activity, was improved by fish oil treatment. A correlation was found between these two variables (R = 0.999, P < 0.05). Moreover, a preventive effect of fish oil was observed on nerve histological damage endoneurial edema, axonal degeneration (by 10–15%) with demyelination. Moreover, the normal bimodal distribution of the internal diameter of myelinated fibers was absent in the DO group and was restored in the DM group. These data suggest that fish oil therapy may be effective in the prevention of diabetic neuropathy.
Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+, K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced ...diabetes on hepatic Na+, K+-ATPase catalytic α1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic α1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the α1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+, K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.
Na+/K+‐ATPase during diabetes may be regulated by synthesis of its α and β subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in ...rats the effect of streptozotocin‐induced diabetes on liver Na+/K+‐ATPase. We then evaluated whether fish oil treatment prevented the diabetes‐induced changes. Diabetes mellitus induced an increased Na+/K+‐ATPase activity and an enhanced expression of the β1 subunit; there was no change in the amount of the α1 and β3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No α2 andα3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes‐induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+‐ATPase activity can be associated with the enhanced expression of the β1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high‐affinity site for ouabain (IC50 = 10−7 m) could be explained by the presence of (αβ)2 diprotomeric structure of Na+/K+‐ATPase or an as yet unknown α subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.
The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and ...alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha 1, exhibited high Na+ sensitivity Ka of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13; the ouabain high-affinity isoform, alpha 2, had two Na+ sensitivities, a high (Ka of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (Ka of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha 3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.
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