Effect of carbacholine and serotonin on the ATPase activity in synaptosomes of the projection and association areas of the feline cerebral cortex]

Na, K-ATPase and Mg-ATPase activities were measured in the synaptosomes of the temporal auditory projection area and the frontal association area. Moreover, the effects of carbacholine and serotonin on those activities were investigated. Na, K-ATPase activity in the synaptosomes of the association area was shown to be reliably higher that in the synaptosomes of the projection area (11.02 +/- 0.45 vs 8.40 +/- 0.55 microM Pi/mg of protein hr; P less than 0.05). Mg-ATPase activity was higher in the second case as compared to the first one (11.40 +/- 0.38 vs 9.04 +/- 0.35; p less than 0.05). Carbacholine and serotonin (10(-8)-10(-3) M) were found to induce equal inhibition of Na, K-ATPase activity in the synaptosomes of both cortices (1 max = 25-30%, 1C50 = 0.2-0.3 microM) which is blocked respectively with atropine (10(-6) M) and methysergide (10(-6) M) and enhanced in presence of GTP (5.10(-5) M). The enzyme activity is also inhibited by the non-hydrolysable guanine nucleotide, GTP gamma S (10(-8)-10(-4) M), in the absence of the antagonists (1 max = 35-40%, 1 C50 = 0.02 microM). In the methysergide-containing medium serotonin exerts a dose-dependent stimulatory effect on Na, K-ATPase which is more pronounced in the synaptosomes of the association area (A max = 25%, A C50 = 0.05 microM). Mg-ATPase activity of membrane preparations is liable to be stimulated by both serotonin and carbacholine, stimulation being more pronounced in the synaptosomes of the association cortex as well (A max = 35%, A C50 = 0.2-0.3 microM). This effect is insensitive either to the antagonists of the corresponding receptors or to GTP. GTP gamma S does not cause alterations in the enzymatic activity. Na, K-ATPase is suggested to be coupled to muscarine and serotonin receptors in the synaptic membranes of both projection and association cortical areas via a GTP-binding protein. At the same time, the agonists of receptors mentioned above are presumably also capable to effect Mg-ATPase activity by the receptor-independent way.     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T₃ increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T₃-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.     

Oxidative Stress (Glutathionylation) and Na,K-ATPase Activity in Rat Skeletal Muscle

Background

Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation) on the Na,K-ATPase in rat skeletal muscle membranes.

Results

Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM) increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05) in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0–10 mM) increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.

Conclusion

This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.

Perspective

Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.     

The beta-adrenergic regulation of the Na, K-ATPase activity in the sarcolemma of the heart muscle

Using a sensitive potentiometric method the effect of isoproterenol upon the activity of Na, K-ATPase in cardiomyocytes has been studied. The activity of the enzyme in rat sarcolemma at isoproterenol-induced myocarditis decreases by 42%. A direct action of isoproterenol on the Na, K-ATPase activity in sarcolemma in vitro has been investigated. In the concentration range 10(-9)-10(-3) M (from receptor-binding up to cardiotoxic) a gradual decrease of the activity reaching the complete inhibition at 10(-3) M is revealed. Antagonist of beta-adrenoreceptors propranolol in concentrations required for displacing the agonist (10(-9) M) provides for the recovery of the Na, K-ATPase activity up to 76% of its normal activity. This action transforms into nonspecific inhibition at rising concentration of the antagonist. Possible mechanisms of the beta-adrenergic regulation effect in cardiomyocytes on Na, K-ATPase of the sarcolemma are discussed.     

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

Changes in the activity and regulatory properties of Na, K-ATPase from the myocardial sarcolemma in total graded ischemia

Na, K-ATPase activity of the rat and guinea-pig myocardial sarcolemma and its sensitivity to digoxin (DG) and carbamylcholine (CCh) were investigated during experimental ischemia. Ischemia was induced by the incubation of hearts in the air at 37 degrees C. This 15-, 30- and 45-min treatment led to a decrease in enzymatic activity which was similar in both animal species. Dose-related dependence of DG effect (10(-8)-10(-2) M) on sarcolemmal Na, K-ATPase activity of guinea-pig ischemic hearts did not differ from the control, whereas the rat enzyme sensitivity to glycosides rose with the progress of ischemia. CCh (10(-7)-10(-3) M) produced an inhibition of Na, K-ATPase activity which had reached 40% both in the rat and guinea-pig myocardial preparations. This effect was blocked by atropine (10(-6) M). The magnitude of enzyme responses to CCh declined depending on the duration of ischemia, with it being greater in guinea-pig sarcolemma than in rat membrane. The increased sensitivity of the rat Na, K-ATPase to CCh was also observed.     

Effect of adrenaline on the ATPase activity of suslik brain synaptosomes]

Action of adrenaline on ATPase activity of ground squirrel synaptosomes in vitro at 37 degrees and 17 degrees C was studied. It has been shown in experiments in vitro at 37 degrees C that adrenaline in a concentration of 5.10(-4) M influenced Mg and Na, K-ATPase of the synaptosomes in ground squirrel brain. The inhibition (42-72%) of Na, K-ATPase in the synaptosomes of the brain was seen during hibernation and in summer. The inhibition of Mg-ATPase (50%) was observed only in summer. The effect of adrenaline on the activity of Na, K-ATPase of synaptosome was seen in vitro as well as at 17 degrees (a 50% inhibition). It was shown that adrenaline in vitro at a concentration of 5.10(-4) M inhibited ATPases more than noradrenaline.     

Changes in the structural characteristics and ATPase activity of the erythrocyte membranes in angiographically verified coronary artery lesion

Structural characteristics and Na, K-ATPase activity of erythrocyte membranes were studied by the spin probe method in patients with an angiographically verified damage to the coronary arteries. The rise of the cholesterol/phospholipid ratio in patients with coronary heart disease was associated with the increased orderliness of the fatty acid chains of erythrocyte membrane lipids, leading to inhibition of Na, K-ATPase activity. The data point to the same line of changes in erythrocyte membranes and smooth muscle cells during the development of coronary heart disease.     

Changes in the Na, K-ATPase of neuronal membranes in penicillin-induced epileptic foci in the cerebral cortex of the rat]

The relationship between electrophysiological changes and Na, K-ATPase activity of neuronal membranes in sodium penicillin-induced epileptic foci was studied. Na,K-ATPase activity is inhibited both in the primary focus and in homotopic contralateral area during latent period and in the stage of forming epileptic activity. In the stage of marked convulsive activity Na, K-ATPase is inhibited only in the primary focus. It is shown that penicillin at a concentration range of 2 x 10(-6)--2 x 10(-3) M does not influence Na,K-ATPase activity of crude synaptosomes of the rat brain cortex. It is suggested that Na,K-ATPase inactivation may serve as a pathogenetic factor in the development of convulsive process.     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.     

Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.

A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and core material. Sucrase specific activity in the purified brush border plasma membranes was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochrome c reductase, nonspecific esterase, beta-glucuronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent 35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

The Na, K-ATPase activity of the separate structural components of the guinea-pig visual system in hypoxia

Different degree of sensitivity to acute hypoxia in vivo has been shown in guinea-pig pigmented epithelium, retina and visual cortex Na, K-ATPase. The highest degree of the enzyme activity inhibition has been noted in the pigmented epithelium (more than 3 times), lower inhibition-in visual cortex (26%) and the lowest-in the retina (18%). In contrast to Na, K-ATPase, hypoxia effect on Mg-ATPase resulted in both inhibition and activation of the enzyme in all 3 structures. Reoxygenation following acute hypoxia increases Na, K-ATPase activity inhibition in the retina and visual cortex. But under reoxygenation the enzyme activity is recovered in the pigmented epithelium. Preliminary administrations of vitamin E completely prevented Na, K-ATPase activity inhibition in all studied structures.     

Effect of catecholamines and metal chelating agents on the brain and brown adipose tissue Na,K-ATPase

Catecholamines stimulate Na,K-ATPase activity in the microsomal membranes of the brain and brown adipose tissue. This stimulation is apparent in the absence of soluble, cytosolic inhibitors and exhibits the same characteristics in both tissues: it occurs at high concentrations (10(-6)-10(-4) M) only; there is no difference in potency between isoprenaline, norepinephrine and epinephrine (EC50 = 1-2 X 10(-5) M); the D-stereoisomer of isoprenaline is equally as effective as the L-form; stimulation of Na,K-ATPase may also be achieved by the metal chelators EDTA, EGTA and desferal; the hydrophobic beta-blockers, propranolol and alprenolol, inhibit both the norepinephrine-stimulated and basal levels of enzyme activity at concentrations of 10(-5)-10(-3) M; phenoxybenzamine, an irreversible alpha-adrenergic blocker, inhibits basal Na,K-ATPase as well as norepinephrine-stimulated enzyme activity (EC50 = 2.5 X 10(-5) M). Because none of these observations can be related to the properties of the stereospecific adrenergic receptor (alpha or beta), it may be concluded that the catecholamine-Na,K-ATPase interaction is not mediated by the receptor. More probably, catecholamines may antagonize the Na,K-ATPase inhibition caused by some tightly membrane-bound metals (but not vanadium) via the ortho-catechol moiety of the catecholamine molecule. The stimulation of brown fat Na,K-ATPase by catecholamines does not have much relevance to the norepinephrine-stimulated thermogenesis in this tissue.     

Some properties of transport ATPases in functionally different muscles]

The properties of Ca-transporting system in sarcoplasmic reticulum membranes in fast and slow frog muscles as well as some properties of sarcolemma Na, K-ATPase of the same object were investigated. The rate of Ca2+ uptake, Ca-ATPase activity and Ca/ATP ratio for the reticulum of fast muscle demonstrated higher values than those for the reticulum of slow muscle. The rate of Ca2+ accumulation by the fragments of the rectus reticulum and Ca/ATP ratio were found to decrease under the influence of acetylcholine (0.05-5 mM). The transport system of the sartorius reticulum was found to be less sensitive to acetylcholine. The peak activity of Na, K-ATPase in femoral muscles of the frog occurred at 80 mM NaCl and 60 mM KCl, whereas in the rectus abdominal muscle it equalled 100 mM NaCl and 40 mM KCl. Thus, Na, K-ATPase activity in the slow muscle was predominantly higher than that in the mixed (femoral) muscles. If the sarcolemma preparations of the muscles of both types the inhibitory effect of acetylcholine on Na; K-ATPase was registered. The enzyme of slow muscles exhibited higher sensibility to acetylcholine.     

Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells. Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr. The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+. In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.     

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.