Regeneration influences expression of the Na+, K+-atpase subunit isoforms in the rat peripheral nervous system

Neural injury triggers changes in the expression of a large number of gene families. Particularly interesting are those encoding proteins involved in the generation, propagation or restoration of electric potentials. The expression of the Na+, K+-ATPase subunit isoforms (alpha, beta and gamma) was studied in dorsal root ganglion (DRG) and sciatic nerve of the rat in normal conditions, after axotomy and during regeneration. In normal DRG, alpha1 and alpha2 are expressed in the plasma membrane of all cell types, while there is no detectable signal for alpha3 in most DRG cells. After axotomy, alpha1 and alpha2 expression decreases evenly in all cells, while there is a remarkable onset in alpha3 expression, with a peak about day 3, which gradually disappears throughout regeneration (day 7). beta1 Is restricted to the nuclear envelope and plasma membrane of neurons and satellite cells. Immediately after injury, beta1 shows a homogeneous distribution in the soma of neurons. No beta2 expression was found. Beta3 Specific immunofluorescence appears in all neurons, although it is brightest in the smallest, diminishing progressively after injury until day 3 and, thereafter, increasing in intensity, until it reaches normal levels. FXYD7 is expressed weakly in a few DRG neurons (less than 2%) and Schwann cells. It increases intensely in satellite cells immediately after axotomy, and in all cell types at day 3. Transient switching of members of the Na+, K+-ATPase isoform family elicited by axotomy suggests variations in the sodium pump isozymes with different affinities for Na+, K+ and ATP from those in intact nerve. This adaptation may be important for regeneration.     

The alpha1 isoform of Na+,K+-ATPase in rat soleus and extensor digitorum longus.

A novel immunochemical method was used for determination of the concentration of Na,K-adenosine triphosphatase (ATPase) containing the ouabain-insensitive alpha1 peptide in rat m. soleus and extensor digitorum longus (EDL). Homogenates of soleus and EDL from 4-week or 10-11-week rats were run on sodium dodecyl sulphate (SDS) gels and in parallel lanes was run a well-characterized preparation of Na,K-ATPase isolated from rat kidney that is known to contain only the alpha1 isoform. After electroblotting to PVDF membranes blots were incubated with the alpha1 specific monoclonal antibody 3B, then with an 125I-coupled secondary antibody, and finally the specific labelling of adjacent alpha spots was analysed by means of an electronic autoradiography system (Packard InstantImager). As the alpha1 content of reference Na,K-ATPase was known from the specific Na+-dependent 32P-phosphorylation capacity, the alpha1 content of adjacent alpha spots in homogenates from soleus and EDL could be calculated. In soleus and EDL from 4-week rats an alpha1 concentration of 135-220 pmol (g tissue)(-1) was found, dependent on the conditions of the experiments but without significant differences between the two types of muscle. In 10-11-week rats a significantly lower concentration of 70-80 and 40-60 pmol (g tissue)(-1) in soleus and EDL, respectively, was found. Ouabain-insensitive Na,K-ATPase containing the alpha1 peptide may thus represent 15-25% of the total number of pumps in skeletal muscle if another 20-30% has to be added to the pool known from (3H)ouabain binding.     

Na^＋，K^＋—ATPase研究进展

Na+ ,K+ ATPase广泛分布于多种细胞的细胞膜上 ,是维持细胞内外Na+ ,K+ 浓度梯度的关键酶。Na+ ,K+ ATPase由α,β和γ亚基组成 ,在不同组织 ,不同发育阶段表达不同的亚型。激素等通过蛋白激酶A、蛋白激酶C、酪氨酸激酶等调节Na+ ,K+ ATPase的活性。小的跨膜蛋白结合Na+ ,K+ ATPase的特异亚基 ,调节其活性。Na+ ,K+ ATPase不仅参与离子转运、蛋白转运、维持离子自稳平衡 ,而且在脊椎动物胚胎发育、神经元导向、中枢神经系统发育、细胞形态维持、细胞粘附等方面发挥作用 ,甚至可作为喹巴因的受体 ,参与信号传递。     

Role of oxidative stress in ischemia-reperfusion-induced changes in Na+,K(+)-ATPase isoform expression in rat heart

The aim of this study was to assess whether depression of cardiac Na+,K(+)-ATPase activity during ischemia/reperfusion (I/R) is associated with alterations in Na+,K(+)-ATPase isoforms, and if oxidative stress participates in these I/R-induced changes. Na+,K(+)-ATPase alpha1, alpha2, alpha3, beta1, beta2, and beta3 isoform contents were measured in isolated rat hearts subjected to I/R (30 min of global ischemia followed by 60 min of reperfusion) in the presence or absence of superoxide dismutase plus catalase (SOD+CAT). Effects of oxidative stress on Na+,K(+)-ATPase isoforms were also examined by perfusing the hearts for 20 min with 300 microM hydrogen peroxide or 2 mM xanthine plus 0.03 U/ml xanthine oxidase (XXO). I/R significantly reduced the protein levels of all alpha and beta isoforms. Treatment of I/R hearts with SOD+CAT preserved the levels of alpha2, alpha3, beta1, beta2, and beta3 isoforms, but not that of the alpha1 isoform. Perfusion of hearts with hydrogen peroxide and XXO depressed all Na+,K(+)-ATPase alpha and beta isoforms, except for alpha1. These results indicate that the I/R-induced decrease in Na+,K(+)-ATPase may be due to changes in Na+,K(+)-ATPase isoform expression and that oxidative stress plays a role in this alteration. Antioxidant treatment attenuated the I/R-induced changes in expression of all isoforms except alpha1, which appears to be more resistant to oxidative stress.     

Gentamicin inhibition of Na+, K+-ATPase in rat kidney cells

Na,K+-ATPase activity is decreased in homogenized renal tissue from GM-treated rats. This study examines whether the site of the active effect of GM on Na,K-ATPase activity in the kidney can be localized to the proximal convoluted tubules (PCT) where the drug is taken up and where it will produce necrosis. In rats treated with gentamicin (50 μg. kg^-1.day^-1 i.m.) for 7 days, PCT Na,K-ATPase activity was reduced as compared to vehicle-treated rats but returned to control levels 7 days after treatment withdrawal. In another nephron segment, the medullary thick ascending limb of Henle (mTAL), where GM induced lesions are uncommon, Na,K-ATPase activity was the same in GM- and vehicle-treated rats treatment. To study the in vitro effect of GM, dissected PCT and mTAL segments from untreated rats were preincubated for 30 min with GM 10^-3m , a dose similar to the tissue concentration in chronically treated rats. In tubule segments that were permeabilized to allow the drug to enter the cells, GM 10^-3m significantly inhibited Na,K-ATPase activity both in PCT and mTAL. In non-permeabilized mTAL segments GM did not inhibit Na,K-ATPase activity. GM inhibition of Na,K-ATPase activity in permeabilized PCT segments persisted after the tubules were rinsed in GM free medium. GM does not inhibit Na,K-ATPase partly purified from the renal cortex. Conclusion. Gentamicin inhibits Na,K-ATPase activity in renal tubule cells when it has access to the cytoplasm. Treatment with GM will therefore cause a selective inhibition of Na,K-ATPase in the proximal tubule cells.     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

Heterogenous Na+, K+-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissues

Na⁺, K⁺-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissue was measured using indirect immunofluorescence and confocal laser scanning microscopy. All four major sites of aggregated lymphoid tissue, i. e. Peyer's patch, sacculus rotundus, caecal patch and appendix, were studied. Na⁺, K⁺-ATPase expression was localized to the basolateral surface of cells of the follicle-associated epithelium (FAE) and adjacent villous or surface epithelia (non-FAE), where increased expression during enterocyte migration was evident. In the FAE, expression of Na⁺, K⁺-ATPase appeared to be lower in the specialized M cells than in enterocytic-type cells, although expression in both cell types was lower than in adjacent non-FAE. Quantification of immunofluorescent staining of Na⁺, K⁺-ATPase by confocal laser scanning imaging showed a reduction of expression in the FAE to approximately 20–60% relative to that in the adjacent non-FAE. These results are consistent with a primary role of the FAE in mucosal immunity with minimal involvement in active solute absorption.     

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.     

Age differences in hormonal regulation of Na+, K+-ATPase activity in the rat renal cortex

Laboratory of Physiological Genetics, Institute of Cytology and Genetics, Siberian Brach, Russian Academy of Sciences, Novosibirsk. (Presented by Academician of the Russian Academy of Medical SciencesV. P. Lozov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 8, pp. 150–153, August, 1992.     

Digitalis-like compounds and Na+, K+-ATPase activity in bovine lens

 Digitalis-like compounds in bovine lens capsule, cortex and nucleus were determined quantitatively, following extraction, by their ability to inhibit [³H]ouabain binding to red blood cells. These compounds were found to be highly concentrated in the epithelium capsule and were significantly diminished in the cortex and nucleus. Na⁺, K⁺-ATPase density in the different regions was determined by [³H]ouabain binding to membranes and by autoradiography of lens slices. The highest concentration of [³H]ouabain-binding sites was observed to occur in membranes prepared from the epithelial cells of the capsule, and was almost 100- and 200-fold higher than the concentrations observed in membranes prepared from fiber cells of the cortex and nucleus, respectively. In the autoradiography studies, strong labeling of [³H]ouabain appeared in the epithelial cell zone, and only weak specific labeling appeared in the lens cortex and nucleus. Almost all (99%) of the Na⁺,K⁺-ATPase specific activity was found to be in the capsule epithelium and only 0.5% was measured in the cortex and no activity was detected in the nucleus. These results indicate that the digitalis-like compounds and Na⁺, K⁺-ATPase are concentrated in the lens capsule epithelium and are present only at low levels in the cortex and nucleus, thus implying that the lens capsular epithelial layer is the major region of the lens responsible for the homeostasis of ions and water in this tissue. Received: 20 September 1996 / Received after revision and accepted: 17 October 1996     

Training in inhibitory avoidance causes a reduction of Na+,K+-ATPase activity in rat hippocampus

Compelling evidence has indicated the involvement of Na(+),K(+)-ATPase in the mechanisms of synaptic plasticity. In the present study, we investigated the effect of inhibitory avoidance training on Na(+),K(+)-ATPase activity, at different times after training, in the rat hippocampus. Male adult Wistar rats were trained in a step-down inhibitory avoidance task and compared to those submitted to isolated footshock (0.4 mA) or placed directly onto the platform. Na(+),K(+)-ATPase activity decreased, by 60%, in hippocampus of rats sacrificed immediately after the isolated footshock, as well as immediately (0 min) and 6 h after training; this effect was not present 24 h after training. We also verified that enzyme activity was not altered in rats killed after just being on the platform. These findings suggest that Na(+),K(+)-ATPase activity may be involved in the memory consolidation of step-down inhibitory avoidance in the hippocampus.     

大鼠糖尿病早期肾脏皮质Na+,K+-ATP酶α1-亚单位的表达

目的：探讨大鼠糖尿病早期肾脏肥大和肾脏皮质Na^ ，K^ -ATP酶α1-亚单位mRNA表达的变化。方法：采用单剂量腹腔注射链脲佐菌素(streptozocozin,STZ)诱发大鼠糖尿病，分别在糖尿病第1、3、7天测定肾脏肥大指数，并以RT-PCR技术检测肾脏皮质Na^ ，K^ -ATP酶α1-亚单位mRNA的表达。结果：大鼠糖尿病早期血糖显著升高，肾脏肥大，肾脏皮质Na^ ，K^ -ATP酶α1-亚单位mRNA的表达明显降低。结论：肾脏皮质Na^ ，K^ -ATP酶α1-亚单位mRNA表达的改变可能是糖尿病早期肾脏肥大的一个因素。     

Gentamicin inhibition of Na+,K(+)-ATPase in rat kidney cells.

Na,K(+)-ATPase activity is decreased in homogenized renal tissue from GM-treated rats. This study examines whether the site of the active effect of GM on Na,K(+)-ATPase activity in the kidney can be localized to the proximal convoluted tubules (PCT) where the drug is taken up and where it will produce necrosis. In rats treated with gentamicin (50 micrograms.kg-1.day-1 i.m.) for 7 days, PCT Na,K(+)-ATPase activity was reduced as compared to vehicle-treated rats but returned to control levels 7 days after treatment withdrawal. In another nephron segment, the medullary thick ascending limb of Henle (mTAL), where GM induced lesions are uncommon, Na,K(+)-ATPase activity was the same in GM- and vehicle-treated rats treatment. To study the in vitro effect of GM, dissected PCT and mTAL segments from untreated rats were preincubated for 30 min with GM 10(-3) M, a dose similar to the tissue concentration in chronically treated rats. In tubule segments that were permeabilized to allow the drug to enter the cells, GM 10(-3) M significantly inhibited Na,K(+)-ATPase activity both in PCT and mTAL. In non-permeabilized mTAL segments GM did not inhibit Na,K(+)-ATPase activity. GM inhibition of Na,K(+)-ATPase activity in permeabilized PCT segments persisted after the tubules were rinsed in GM free medium. GM does not inhibit Na,K(+)-ATPase partly purified from the renal cortex. Conclusion. Gentamicin inhibits Na,K(+)-ATPase activity in renal tubule cells when it has access to the cytoplasm. Treatment with GM will therefore cause a selective inhibition of Na,K(+)-ATPase in the proximal tubule cells.     

Androgens stimulate preoptic area Na+,K+-ATPase activity in male rats

This paper describes the effects of castration and testosterone replacement on the Na+,K+-ATPase activity levels of the cerebral cortex (CC), preoptic-suprachiasmatic region (POSC) and mediobasal hypothalamus (MBH) in male rats. Na+,K+-ATPase activity was estimated as the ouabain-sensitive fraction of ADP and AMP generation rate, measured by high-pressure liquid chromatography (HPLC) with UV detection, from a standard incubation mixture containing 3 mM ATP. Orchidectomy, performed 4 weeks before sacrifice, decreased ATPase activity of MBH. Testosterone propionate treatment (50 micrograms/day X 2 days) to castrated animals resulted in a 4-fold increase in enzyme activity in the POSC, an effect that might be related to the behavioral effects of androgens. None of the treatments seemed to influence the enzyme activity of the cerebral cortex.     

Axonal transport of Na+,K+-ATPase identified as a ouabain binding site in rat sciatic nerve

Na+,K+-ATPase levels were measured in different segments of rat sciatic nerves by in vitro binding of [3H]ouabain. Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve, indicating an anterograde and retrograde axoplasmic transport of Na+,K+-ATPase. Accumulation of Na+,K+-ATPase at the ligature was time dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of the ligature was also visualized by autoradiographic studies in longitudinal section of sciatic nerves using [3H]ouabain.     

Modulation of Na+,K+-ATPase activity is of importance for RVD

AIM: This study was performed to examine the role of Na+,K+-ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). METHODS: The studies were performed on COS-7 cells transfected with rat Na+,K+-ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. RESULTS: Ouabain-mediated inhibition of Na+,K+-ATPase resulted in a dose dependent decrease in the rate of RVD. Total 86Rb+ uptake as well as ouabain dependent 86Rb+ uptake, used as an index of Na+,K+-ATPase dependent K+ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS-7 cells expressing rat Na+,K+-ATPase, where Ser23 in the catalytic alpha1 subunit of rat Na+,K+-ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na+,K+-ATPase exhibited a significantly lower rate of RVD and showed no increase in 86Rb+ uptake during RVD. CONCLUSION: Taken together, these results suggest that a PKC-mediated transient increase in Na+,K+-ATPase activity plays an important role in RVD.     

Effects of warm acclimation on Na+,K+-ATPase alpha-subunit expression in chloride cells of Antarctic fish

The teleosts Trematomus bernacchii thrive in southern oceanic waters with temperatures below 0 degrees C. These fish have serum osmolalities almost double those found in fish of temperate waters, thereby lowering their serum's freezing point and the energy needed for ionic homeostasis. Upon warm acclimation to 4 degrees C, T. bernacchii decrease their serum osmolality and increase the Na+,K+-ATPase activity in their gills. Na+,K+-ATPase alpha1-, alpha2-, and alpha3-subunit isoforms are expressed in the gills of T. bernacchii and it is thought that Na+,K+-ATPase subunit composition in chloride cells changes with warm acclimation. Using immunohistochemistry, we compared the number of chloride cells expressing various alpha-isoforms of the Na+,K+-ATPase in the gills of cold- and warm-acclimated T. bernacchii. We found no change in the number of alpha2- or alpha3-immunopositive cells in warm-acclimated fish gills or in the number of cells immunopositive for the Na+,K+,2Cl- cotransporter. However, the number of pan-alpha-immunopositive (recognizing all three alpha-isoforms) and alpha1-immunopositive cells both increased in warm-acclimated fish. This suggests that changes in the number of alpha1-isoform-expressing chloride cells could contribute to the increased Na+,K+-ATPase activity that occurs with warm-acclimation.     

A brain Na+, K+-ATPase inhibitor (endobain E) enhances norepinephrine release in rat hypothalamus

We have shown that synaptosomal membrane Na+, K+-ATPase activity is stimulated or inhibited by norepinephrine according to the presence or absence of a brain soluble fraction. Gel filtration of such soluble fraction has allowed the separation of two fractions, peaks I and II, able to stimulate and inhibit Na+, K+-ATPase activity, respectively. Peak II behaves much like ouabain, which has suggested the term endobain. From peak II, a subfraction termed II-E (endobain E), which highly inhibits Na+, K+-ATPase, has been separated by anionic exchange chromatography in a Synchropack AX-300 column. We determined the in vitro effect of endobain E obtained from rat cerebral cortex on neuronal norepinephrine release by incubating rat hypothalamic tissue in the presence of [3H]norepinephrine. Neuronal norepinephrine release was quantified as the factor above basal [3H]norepinephrine released to the medium at experimental and three post-experimental periods. Endobain E was found to increase norepinephrine release in a concentration-dependent fashion, reaching 200%, equivalent to the effect achieved with 400 microM ouabain. Ouabain effect persisted along three post-experimental periods whereas that of endobain E remained only during the first post-experimental period. These results led us to conclude that endobain increases norepinephrine release in hypothalamic neurons at the presynaptic nerve ending level, an effect resembling that of ouabain. It is postulated that endobain E may enhance catecholamine availability in the synaptic gap, leading to an increase in noradrenergic activity.