Adrenocorticoid regulation of Na+, K+-ATPase in adult rat kidney: effects on post-translational processing and mRNA abundance

The mechanisms by which adreno-corticoid hormones regulate Na⁺, K⁺-ATPase in adult kidney were studied in adrenalectomized (Adx) rats. Five days after adrenalectomy, Na⁺, K⁺-ATPase activity was significantly reduced in the renal cortex homogenate (C = 13.0±0.8 vs. Adx = 7.1±0.7 μmol Pi mg^-1 protein h^-1) and in renal microsomes (C = 30.3 ± 1.9 vs Adx = 14.6 ± 1.3 μmol Pi mg^-1 protein h^-1). Glucocorticoid replacement treatment of adrenalectomized rats with betamethasone (20 μg kg^-1 body wt twice daily for 5 days) effectively counteracted the observed reduction in Na⁺, K⁺-ATPase activity. In cortical homogenate the protein level of α1 and β1 subunits measured in immunoblots was not significantly different in Adx and control rats, indicating that 5 days after adrenalectomy the α1 and β1 subunits were present in renal cortical cells to almost normal extent but could not be assembled into a transmembrane functional unit. In support of this conclusion we found that the protein level of both the α1 and β1 subunits was significantly lower (P < 0.001 for both subunits) in microsomes from Adx than in control rats. The mRNA abundance for α1 and β1 subunits were not lower in Adx as compared to control rats 1 and 5 days after surgery. However, if Adx rats were given a single dose of betamethasone (600 μg kg^-1 body wt), a significant 2-fold increase in both α1 and β1 mRNAs was observed (P < 0.05 for both subunits). These data suggest that glucocorticoids can upregulate the mRNA of both Na⁺, K⁺-ATPase subunits but that the low renal Na⁺, K⁺-ATPase activity in adult Adx rats is mainly due to loss of glucocorticoid regulation of the post-translational processing of the enzyme.     

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.     

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.     

Localization and activity of Na+,K(+)-ATPase in the ductuli efferentes of the rat.

The Na+,K(+)-ATPase enzyme through its p-nitrophenyl phosphatase activity was localized in the ductuli efferentes of rats. Enzymatic activity was demonstrated along the cytoplasmic side of the plasmalemma of the ductular epithelial cells. The most intense deposition of reaction products was found on the plasmalemma delimiting the lower lateral and basal regions of the cells. The plasma membranes forming the microvilli, apical junctional complexes were devoid of reaction product while the midlateral membranes showed a weak reaction. The enzyme reaction was potassium-dependent and was abolished by addition of 10 mM ouabain to the incubation media. Enzyme activity decreased significantly from proximal to distal regions of the ductules (8,101.47 +/- 274.53, 6,658.95 +/- 269.53 and 4,668.10 +/- 575.41 pmoles p-nitrophenol/mm/h, respectively in initial, conus vasculosus and terminal zones). A unified model for water absorption is proposed in the efferent ductules based upon this data and that of others.     

Coordination of ATP production and consumption in brain: parallel regulation of cytochrome oxidase and Na+, K(+)-ATPase.

Previous studies have shown that most neuronal ATP is produced by oxidative metabolism, and consumed by Na+,K(+)-ATPase. We hypothesized that the distribution of Na+,K(+)-ATPase in brain would correlate with that of the energy-producing enzyme, cytochrome oxidase (CO). We localized these enzymes in monkey hippocampus and striate cortex by histochemistry and immunohistochemistry. Their distributions were generally similar, although some differences were observed. We also studied regulation of enzyme levels, using monocular impulse blockage with tetrodotoxin (TTX) to alter neuronal activity in the visual system. Parallel changes in CO and Na+,K(+)-ATPase activity were induced in striate cortex. These results provide further evidence that neuronal energy demands regulate CO levels and distribution.     

Ouabain-insensitive, vanadate-sensitive K(+)-ATPase of rat distal colon is partly similar to gastric H+,K(+)-ATPase.

A membrane fraction from rat distal colon contained both ouabain-sensitive and -insensitive K(+)-ATPase activities, which were measured under Na(+)-free conditions. About 38% of the ouabain-insensitive K(+)-ATPase activity was inhibited by vanadate. It was determined whether the ouabain-insensitive, vanadate-sensitive K(+)-ATPase in the colon is similar or identical to gastric H+,K(+)-ATPase. This colonic K(+)-ATPase activity was inhibited completely by monoclonal antibody HK4001, which inhibits the hog gastric H+,K(+)-ATPase activity but not Na+,K(+)-ATPase or Ca(2+)-ATPase. The colonic ATPase activity was inhibited partly by SCH 28080, which is a specific reversible inhibitor of gastric H+,K(+)-ATPase. The colonic ATPase activity was stimulated by low concentrations of K+ (its half-maximal stimulating concentration was 1 mM) and inhibited by high concentrations of K+ (its half-maximal inhibiting concentration was 10 mM), indicating that high and low K+ affinity sites are present in the colonic enzyme as in gastric H+,K(+)-ATPase and that this enzyme is not fully operative under normal physiological conditions. Two other monoclonal antibodies, which inhibit the gastric H+,K(+)-ATPase activity, did not inhibit the colonic K(+)-ATPase activity. The present results suggest that the colonic ouabain-insensitive K(+)-ATPase is partly similar but not identical to the gastric H+,K(+)-ATPase.     

Exercise-induced cholesterol depletion and Na+,K(+)-ATPase activities in human red cell membrane.

Red cell membranes were isolated from blood samples obtained from athletes during exhaustive exercise on a bicycle ergometer and during the subsequent recovery period of 60 min. Plasma lactate levels were also determined. During exercise, cell membranes were progressively depleted of cholesterol and, at exhaustion, membrane cholesterol was less than 80% of the initial level. A parallel decline in Na+,K(+)-ATPase was also noted, while phospholipid reduction was around 5%. During recovery, the erythrocyte membrane cholesterol and Na+,K(+)-ATPase increased, but at a slow rate and were inversely proportional to plasma lactate content.     

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.     

Effects of L-phenylalanine on acetylcholinesterase and Na(+), K(+)-ATPase activities in adult and aged rat brain

The effect of different L-phenylalanine (Phe) concentrations (0.12-1.8 mM) on acetylcholinesterase (AChE), (Na(+), K(+))-ATPase and Mg(2+)-ATPase activities was investigated in homogenates of adult and aged rat whole brain at 37 degrees C. Adult and aged rat experiments were necessary in relation to phenylketonuria (PKU) since phenylketonuric patients usually discontinue their therapeutic special diet when they reach adulthood. Diet discontinuation results in the pathological increase of Phe concentration in plasma and consequently in brain. AChE activity in adult brain homogenates showed a decrease up to 18% (P<0.01) with 0.48--1.8 mM Phe preincubated for 1 h. Adult brain Na(+), K(+)-ATPase was stimulated by 30--35% (P<0.01) in the presence of 0.48--1.8 mM Phe. However, high Phe concentrations were not able to affect the activities of AChE and Na(+), K(+)-ATPase, when preincubated with aged brain homogenate for 3 h. Moreover, high Phe concentrations appeared unable to affect the activity of eel E. electricus pure AChE inhibited about 30% (P<0.001) by the free radical system H(2)O(2)/Fe(2+). Also, the antagonists of alpha- and beta-adrenergic receptors (phenoxybenzamine and propranolol, respectively) inhibited adult rat brain Na(+), K(+)-ATPase activity about 30--40% (P<0.01) and Phe was unable to change this action. It is suggested that: (a) The inhibitory effect of Phe on brain AChE and its stimulatory effect on brain Na(+), K(+)-ATPase are decreased with age; (b) These effects may be influenced by aging factors, such as free radical action and/or reduced density of alpha- and beta-adrenergic receptors in the tissue.     

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.     

Choroid plexus, brain and kidney Na+,K+-ATPase: comparative activities in fetal, newborn and young adult rabbits

We have studied maturation of brain barrier systems in fetal, newborn, juvenile and adult rabbits. We have compared choroid plexus and brain Na+,K+-ATPase levels in each age group, as well as serum and CSF Na+ concentration as a measure of the ability of the choroid plexus to generate a gradient from blood to CSF. The choroid plexus appears functionally mature at all ages studied, both in ability to produce a Na+ gradient and in ATPase levels. In contrast, brain ATPase levels rose markedly with age. Kidney ATPase measured for comparison showed a pattern similar to brain.     

Altered in vitro adaptive responses of lymphocyte Na+,K(+)-ATPase in patients with manic depressive psychosis.

When lymphocytes from healthy subjects are incubated in lithium (8 mM) or ethacrynate (1 microM) they show a time-dependent adaptive response, which consists of a significant increase in the number of Na+,K(+)-ATPase molecules in the lymphocyte membrane. We have studied the lymphocytes from nine euthymic drug-free patients with a history of manic depressive psychosis, and have found that this normal adaptive response was absent. It was also absent from the lymphocytes of euthymic patients taking lithium. We conclude that this altered in vitro adaptive response of lymphocyte Na+,K(+)-ATPase represents an enduring trait marker in manic depressive psychosis.     

Effects of electroconvulsive seizures on Na(+),K(+)-ATPase activity in the rat hippocampus

Although several advances have occurred concerning the use of electroconvulsive therapy, little progress has been made in understanding the mechanisms underlying its therapeutic or side effects. Na(+),K(+)-ATPase is an important enzyme of central nervous system, responsible for ionic gradient maintenance and consumption of approximately 40-50% of brain ATP. This work was performed in order to determine Na(+),K(+)-ATPase activity after acute and chronic electroconvulsive shock. Results showed an inhibition of Na(+),K(+)-ATPase activity in the hippocampus 48 h, 7, 30, 60 and 90 days after a single electroconvulsive shock. Chronic treatment diminished the enzyme activity in the hippocampus 7 and 30 days after electroconvulsive (ECS) sessions. Our findings demonstrated that Na(+),K(+)-ATPase activity is altered by ECS.     

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.     

Effect of polyphenolic compounds on the renal Na+,K(+)-ATPase during development and persistence of hypertension in rats

It has been suggested that polyphenolic substances provide protection against the risk factors of cardiovascular diseases. The present study was designed to investigate whether application of red wine polyphenols influences the kinetic properties of the renal Na+,K(+)-ATPase in rats with hypertension (164 +/- 8 mmHg) that was experimentally induced by the NO synthase inhibitor N(G.) -nitro-L- arginine methyl ester (L-NAME). Polyphenols in a dose of 40 mg kg(-1) day(-1) in drinking fluid induced different effects on the properties of the renal Na+,K(+)-ATPase depending on the mode of their administration. Preventive application of polyphenols during the development of hypertension (144 +/- 5 mmHg) partially protected the Na+,K(+)-ATPase molecule against hypertension-induced deterioration via increased capability of the enzyme to bind ATP and/or Na+ as suggested by decrease of Km and KNa, respectively, even to values lower than in controls. However, polyphenols did not prevent the hypertension-induced reduction of the number of active Na+,K(+)-ATPase molecules as shown by similar V(max) values as compared to the hypertensive L-NAME group. The above protection is probably secured by a NO-dependent mechanism as suggested by 150% increase of the NO synthesis. Additional treatment of already hypertensive animals with polyphenols (153 +/- 8 mmHg) resulted in partial restoration of the Na+,K(+)-ATPase affinities especially for sodium as indicated by significant diminution of KNa. However, polyphenols in this mode of application did not slow down the L-NAME-induced decrease in the number of Na+,K(+)-ATPase molecules in the kidney as suggested by additional significant decrease in V(max) values when comparing this group with the control group and also the hypertensive L-NAME group. In this case the polyphenols affected the Na,K-ATPase molecule in a NO-independent way as indicated by the fact that polyphenols failed to restore normal NO synthesis.     

Differential role of KIR channel and Na(+)/K(+)-pump in the regulation of extracellular K(+) in rat hippocampus.

Little information is available on the specific roles of different cellular mechanisms involved in extracellular K(+) homeostasis during neuronal activity in situ. These studies have been hampered by the lack of an adequate experimental paradigm able to separate K(+)-buffering activity from the superimposed extrusion of K(+) from variably active neurons. We have devised a new protocol that allows for such an analysis. We used paired field- and K(+)-selective microelectrode recordings from CA3 stratum pyramidale during maximal Schaffer collateral stimulation in the presence of excitatory synapse blockade to evoke purely antidromic spikes in CA3. Under these conditions of controlled neuronal firing, we studied the [K(+)]o baseline during 0.05 Hz stimulation, and the accumulation and rate of recovery of extracellular K(+) at higher frequency stimulation (1-3 Hz). In the first set of experiments, we showed that neuronal hyperpolarization by extracellular application of ZD7288 (11 microM), a selective blocker of neuronal I(h) currents, does not affect the dynamics of extracellular K(+). This indicates that the K(+) dynamics evoked by controlled pyramidal cell firing do not depend on neuronal membrane potential, but only on the balance between K(+) extruded by firing neurons and K(+) buffered by neuronal and glial mechanisms. In the second set of experiments, we showed that di-hydro-ouabain (5 microM), a selective blocker of the Na(+)/K(+)-pump, yields an elevation of baseline [K(+)]o and abolishes the K(+) recovery during higher frequency stimulation and its undershoot during the ensuing period. In the third set of experiments, we showed that Ba(2+) (200 microM), a selective blocker of inwardly rectifying K(+) channels (KIR), does not affect the posttetanus rate of recovery of [K(+)]o, nor does it affect the rate of K(+) recovery during high-frequency stimulation. It does, however, cause an elevation of baseline [K(+)]o and an increase in the amplitude of the ensuing undershoot. We show for the first time that it is possible to differentiate the specific roles of Na(+)/K(+)-pump and KIR channels in buffering extracellular K(+). Neuronal and glial Na(+)/K(+)-pumps are involved in setting baseline [K(+)]o levels, determining the rate of its recovery during sustained high-frequency firing, and determining its postactivity undershoot. Conversely, glial KIR channels are involved in the regulation of baseline levels of K(+), and in decreasing the amplitude of the postactivity [K(+)]o undershoot, but do not affect the rate of K(+) clearance during neuronal firing. The results presented provide new insights into the specific physiological role of glial KIR channels in extracellular K(+) homeostasis.     

Electrogenic pump (Na+/K(+)-ATPase) activity in rat optic nerve

Rat optic nerves were studied in a sucrose gap chamber in order to study the origin of a late afterhyperpolarization that follows repetitive activity. The results provide evidence for electrogenic pump (Na+/K(+)-ATPase) activity in central nervous system myelinated axons and demonstrate an effect on axonal excitability. Repetitive stimulation (25-200 Hz; 200-5000 ms) led to a prolonged, temperature-dependent post-train afterhyperpolarization with duration up to about 40 s. The post-train afterhyperpolarization was blocked by the Na+/K(+)-ATPase blockers strophanthidin and ouabain, and the substitution of Li+ for Na+ in the test solution, which also blocks Na+/K(+)-ATPase. The peak amplitude of the post-train afterhyperpolarization was minimally changed by the potassium-channel blocker tetraethylammonium (10 mM), and the Ca2(+)-channel blocker CoCl2 (4 mM). Hyperpolarizing constant current did not reverse the afterhyperpolarization. The amplitude of the hyperpolarization was increased in the presence of the potassium-channel blocker 4-aminopyridine (1 mM). In the presence of 4-amino-pyridine, the post-train hyperpolarization was much reduced by strophanthidin, except for a residual early component lasting several hundred milliseconds which was blocked by the potassium-channel blocker tetraethylammonium. This finding indicates that after exposure to 4-aminopyridine, repetitive stimulation leads to activation of a tetraethylammonium-sensitive K(+)-channel that contributes during the first several hundred milliseconds to the post-train afterhyperpolarization. The amplitude of the compound action potential elicited by a single submaximal stimulus during the post-train hyperpolarization was smaller than that of the control response.The decrement in amplitude was not present under identical stimulation conditions when the post-train hyperpolarization was blocked by strophanthidin, indicating that the hyperpolarization associated with repetitive stimulation reduced excitability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Na+,K(+)-ATPase, Ca2(+)-ATPase and some soluble enzymes related to energy metabolism in brains of patients with Alzheimer's disease

Hexokinase, lactate dehydrogenase, acylphosphatase, (Na+,K+)-ATPase and Ca2(+)-ATPase of selected areas from postmortem Alzheimer's disease brains were studied. Hexokinase and lactate dehydrogenase were significantly changed in all the examined subcortical nuclei. (Na+,K+)-ATPase activity was altered in several areas of Alzheimer's disease brains. No changes in Ca2(+)-ATPase and acylphosphatase were observed. The main alterations of the assayed enzymes were observed in subcortical areas but not in cortical areas of Alzheimer's disease brains.