Ouabain-sensitive ATPase Activity Research Articles

Na, K-ATPase in the salivary gland of the ixodid tick Amblyomma hebraeum (Koch) and its relation to the process of fluid secretion.

Total and ouabain-sensitive ATPase activities were determined in the salivary glands of ticks throughout the feeding cycle. Activities were very low in unfed specimens. In the glands of feeding females, the activities rose until a maximum was reached for both ATPase components at approximately 200 mg. The activities remain low in males throughout the feeding period. These findings are discussed in relation to the fluid secretory process of the salivary glands.

The possible involvement of Na+ ions in corticosterone-induced hypercontractility in the rat anococcygeus muscle.

The possibility that corticosterone-induced hypercontractility in the rat anococcygeus muscle might be due to an intramuscular redistribution of Na+ was investigated. 2 The contractility of the isolated muscle to noradrenaline (NA) was directly dependent on the Na+ concentration of the Krebs solution. 3 There was a linear relationship between the maximum contractile response of the muscle to NA and the Na+ concentration of the Krebs solution. 4 Muscle contractility was also increased by the presence of ouabain (10(-4)M) in the bathing medium. 5 Muscles from rats treated with corticosterone exhibited increases in both total and ouabain-sensitive ATPase activity. 6 The relationship between corticosterone, Na+ distribution, and muscle contractility is discussed.

Alteration in intracellular sodium concentration and ouabain-sensitive ATPase in erythrocytes from hyperthyroid patients.

The activity of the ouabain-sensitive sodium-potassium-activated component of the ATP-hydrolyzing enzyme system (ouabain sensitive ATPase) was studied in the erythrocyte membranes of 10 patients with hyperthyroidism, and found to be decreased in all 10 patients. The mean ouabain-sensitive ATPase activity was 43 +/- 4 nmol Pi/mg tissue/h in the patients, compared with 69 +/- 5 nmol Pi/mg tissue/h in the erythrocyte membranes of 10 paired control subjects. The mean concentration of sodium within the erythrocytes was 10.8 +/- 0.9 nmol/liter of red blood cells in the patients and 7.2 +/- 0.3 nmol/liter of red blood cells in the controls. The decrease in ouabain-sensitive ATPase activity did not appear to be associated with a change in the ligand sensitivity of ATPase, nor was there a difference in the activity of the ouabain-insensitive component of ATPase. Serial studies to follow the effects of treatment of hyperthyroidism on red cell membrane ATPase were performed repeatedly in one of these patients. There was a significant inverse correlation between L-thyroxine (T4) and ouabain-sensitive ATPase, as both variables returned to normal. Normal erythrocyte membranes were assayed for ATPase activity in the presence of varying concentrations of L-triiodothyronine (T3) and T4, and following pre-incubation with T4. No significant effect on erythrocyte membrane ATPase was demonstrated in either series of experiments. It can be concluded from these studies that the decreased sodium efflux in the erythrocytes of patients with hyperthyroidism is associated with a decrease in the activity of the ouabain-sensitive component of ATPase in the erythrocyte membrane. The failure to reproduce this effect in vitro suggests that it does not represent a direct effect of the thyroid hormones on the mature erythrocyte membrane.

Effect of ozone on erythrocyte membrane adenosine triphosphatase

Human erythrocyte membrane fragments were exposed to O 3 over varying lengths of time. Ozone was found to have a deleterious effect on the ouabainsensitive ATPase (EC 3.6.1.3) in the membrane fragments. After 1 min of exposure to O 3, which was generated at a rate of 4.0 μmol/min, ouabain-sensitive ATPase activity decreased to 26% of the control. Ouabain-insensitive ATPase was found to be unaffected by O 3 exposure under the test conditions. Additions of ascorbic acid or cysteine, prior to O 3 exposure, partially protected the enzyme from inactivation. However, the inactivating effect of O 3 could not be reversed by addition of either ascorbic acid or cysteine after exposure. Superoxide dismutase or catalase did not afford significant protection. The enzyme could not be protected by Ellman's reagent. The inactivating effect of O 3 on the ouabain-sensitive ATPase was also demonstrated in exposure of intact erythrocytes. No detectable change was observed in glycolytic activity in the hemolysate prepared from O 3-treated erythrocytes, however. It was postulated that inactivation of the membrane ATPase by O 3 may be responsible for the destructive effect of O 3 on the red cell.

Post-tetanic hyperpolarization, sodium-potassium-activated adenosine triphosphatase and high energy phosphate levels in garfish olfactory nerve.

1. While much is now known about the Na-K-ATPase and the posttetanic hyperpolarization of nervous tissue, they have yet to be studied together in the same preparation. 2. The post-tetanic hyperpolarization was studied in desheathed garfish olfactory nerve. The rate constant of decay of the post-tetanic hyperpolarization was determined by monitoring difference potentials after stimulation at 1/sec for 2-3 min. 3. In membrane fractions prepared from these nerves, the ouabain-sensitive ATPase activity (Na-K-ATPase) was determined by spectrophotometric measurements. 4. Both the post-tetanic hyperpolarization and the Na-K-ATPase showed a similar sigmoidal dependence on K+ concentration. The sequence of cation specificities measured at the K-site of the enzyme was the same as that determined by post-tetanic hyperpolarization measurements in whole nerve. 5. The rate constants of the enzyme showed a dependence on Na+ concentration that paralleled the way in which the post-tetanic hyperpolarization rate constants varied as a function of the number of impulses. When Na+ was completely replaced by Li+, neither enzyme activity nor post-tetanic hyperpolarization could be measured. 6. The pH optimum for enzyme activity was between pH 7-0 and 7-8, while the optimal pH for post-tetanic hyperpolarization was above pH 8-0. 7. Metabolite levels in preparations of this nerve studied in vitro correspond to levels found in vivo. 8. High energy phosphate levels were measured fluorometrically in extracts of nerve samples that had been stimulated in air at 1/sec for various intervals. 9. During the first 2 min of stimulation, there was a significant accumulation of inorganic phosphate, and the ATP/ADP.Pi ratio dropped appreciably. 10. The accumulation of ATPase products was commensurate with the approach of post-tetanic hyperpolarization rate constants to their maximum level. This provides direct evidence for an ATPase functioning in active Na+ transport in nerve. 11. The garfish Na-K-ATPase is sensitive to the ATP/ADP ratio of the incubating medium, but is relatively insensitive to orthophosphate, Pi. The fall in post-tetanic hyperpolarization rate constants observed with continued nerve stimulation may have been partially due to the falling ATP/ADP ratio measured in nerve under similar conditions.

Changes in Ouabain-Sensitive Adenosine Triphosphatase Activity in Gills of Coho Salmon (Oncorhynchus kisutch) During Parr–Smolt Transformation

The effects of incubation temperature, pH, sodium, potassium, and ATP concentration, and ouabain on the activity of Na+–K+-activated ATPase of the gills of seawater-adapted juvenile coho salmon (Oncorhynchus kisutch) were determined. The temperature and pH optima were 40 C and 7.4, respectively. The apparent Km for ATP at equimolar Mg++ concentration was 0.2 mM at Na+ and K+ concentrations of 100 and 20 mM, respectively. Maximal enzyme activity for Na+ concentration of 10.50 and 100 mM occurred at K+ concentrations of 12.5, 15.0, and 20.0 mM, respectively. The Ki for ouabain was 2 × 10−6 M and 7 × 10−6 for K+ concentrations of 10 and 20 mM, respectively.A large portion (up to 60%) of the ouabain-sensitive ATPase activity in freshwater fish was activated by sodium ions in the absence of potassium ions (Na+-activation). Exposure to sea water resulted in a large increase in total ouabain-sensitive activity and a sharp decrease in the proportion of sodium activation. These changes occurred within 14 days after transfer to full strength sea water.On a seasonal basis, total ouabain-sensitive enzyme activity in juvenile freshwater coho was low (less than 5 μmol Pi/mg N per h) to the end of November, increased to a peak (over 125 μmol Pi/mg N per h) in mid-January, and subsequently declined by late February. A slow, steady rise in activity occurred during the smoking period of March and April and the relative contribution of sodium ions to the total activity declined in this period.

Inhibition of Ouabain-sensitive ATPase by the Saliva of Patients with Cystic Fibrosis of the Pancreas

A study has been made of the effect of saliva from children with cystic fibrosis of the pancreas (CFP) on various components of the ATP hydrolyzing enzyme system. The ouabain-sensitive ATPase activity of erythrocyte membranes prepared from intact erythrocytes preincubated with CFP saliva was 35 +/- 4 nmol Pi/mg fry wt membrane suspension/hr, compared with 48 +/- 7 nmol Pi/mg dry wt membrane suspension/hr when the erythrocytes were preincubated with control saliva. A calcium-activated component of ATPase was decreased from 202 +/- 30 nmol Pi/mg dry wt membrane suspension/hr in erythrocytes preincubated with control saliva, to 151 +/- 17 nmol Pi/mg dry wt membrane suspension/hr when the incubation was carried out with CFP saliva. In a second series of experiments, ultrafiltered saliva was added directly to an ATPase assay. The saliva from children with CFP brought about a mean decrease in ouabain-sensitive ATPase of 16% compared with control saliva.

Membrane fractions of rabbit granulocytes. 1. The presence of ouabain-sensitive ATPase and the electrophoretic patterns

In one or two membrane fractions isolated from the homogenate of casein-induced rabbit granulocytes ouabain-sensitive ATPase activity was found definitely. Electrophoretic patterns of these membrane fractions showed no significant differences in staining for carbohydrate and protein.

Microsomal [formula omitted] ATPase from frog skin epithelium. Cation activations and some effects of inhibitors

A method is described for the extraction of microsomal ouabain-sensitive (Na + + K +)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na + + K +)-stimulated activity in the range of 30–40 nmol · mg −1 · min −1 at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg 2+, Na + and K +. These preparations also contain Mg 2+-dependent or Ca 2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na +, K + or Li +. The activations of the ouabain-sensitive ATPase activity by Mg 2+, Na +, and K + are similar to those described in other tissues. It is found that Li + does not substitute for Na + as an activator but in high concentrations does produce partial activation in the presence of Na + with no K +. These results are pertinent to the reported observations of ouabain-sensitive Li + flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li + on the sodium pump.

Changes in erythrocyte membrane ouabain-sensitive adenosine triphosphatase after renal transplantation.

1. Intracellular electrolytes, and erythrocyte membrane adenosine triphosphatase (ATPase) activity, was studied in twenty patients after renal transplantation. 2. The mean ouabain-sensitive ATPase activity in the erythrocyte membranes of the transplant patients was 122 nmol of inorganic phosphorus (Pi) h-1 mg of tissue-1 (SEM 14), compared with 62 nmol of Pi h-1 mg of tissue-1 (SEM 8) in a group of paired, healthy controls. 3. The increase in ouabain-sensitive ATPase was most marked in the 4 months after transplantation. However, a significant increase in ouabain-sensitive ATPase persisted for more than 8 months after transplantation. 4. This increase in ouabain-sensitive ATPase was associated with a decrease in intracellular sodium in the erythrocytes of the transplant patients.

Changes of sodium transport in erythrocytes of the maturing rabbit.

The red blood cells of New Zealand white rabbits have a low sodium and high potassium content. As the animals mature, the sodium concentration rises and the potassium content falls; studies of red cells from a group of five young and five mature animals revealed a highly significant increase of cell sodium with age that was associated with a significant fall in the rate of ouabain-inhibited active sodium efflux. This difference was still seen when the sodium concentration within the cells from old and young animals was equalized and elevated to saturating levels for active pump efflux. Total sodium efflux, however, increased significantly with age as did total sodium influx so that a steady state was reached. Ouabain-sensitive ATPase activity fell significantly in the cell membranes from older animals and ouabain-insensitive ATPase increased with age. The survival time of 51Cr-labeled red cells was significantly longer in old than in young animals and it is concluded that as the rabbit matures its red cells survive for a longer period and this is associated with the changes of sodium transport and ATPase activity that have been documented.

Effects of ouabain on the function and structure of a cell line (MDCK) derived from canine kidney. I. Light microscopic observations of monolayer growth.

MDCK is a transformed cell line derived from normal dog kidney. It exhibits qualities of renal tubular epithelium and of papillary adenocarcinoma. On solid substrates, MDCK forms confluent monolayers that are studded with multicellular blisters and cordlike structures. Ouabain, at a concentration of 4×10−8 m, abolishes blister formation within 24 hr; withdrawal of the drug brings about reappearance of the blisters. Ouabain-sensitive ATPase activity has been demonstrated in these cells by biochemical measurement. These findings suggest that blister formation in MDCK monolayer growth is associated with active transport of solutes accompanied by water and that 4×10−8 m of ouabain interferes with these transport systems. As an additional early effect, the drug decreases the frequency of mitosis. The exposure of MDCK monolayers to ouabain for several weeks results in profound cytological alterations. A marked eosinophilic stippling of the periphery of chronically exposed cells is seen with the light microscope. After prolonged exposure to ouabain, the cell population of MDCK monolayers becomes clearly heterogeneous, with frequent giant cells and foci of small, hyperchromatic proliferating cells. The small cells may indicate the appearance of ouabain-resistant strains.

(Na +K +)-activated ATPase in cattle erythrocytes

1. 1. In the presence of 150 mM Na +, increasing K + levels initially stimulated ( K + < 5 mM ) and then inhibited ouabain-sensitive ATPase activity in cattle erythrocyte ghosts. 2. 2. Ouabain-sensitive K + uptake measurements in high Na + medium showed that the external affinity for K + was about the same for high or low K + cells. 3. 3. K + inhibition of ATPase was greatest in ghosts derived from low K + cells, and least in ghosts from high K + cells, but a spectrum of sensitivities was found, correlating with the original red cell K + level. 4. 4. Sensitization with sheep anti-L reagent gave an increased overall ouabain-sensitive ATPase activity, with a small decrease in the apparent affinity for the K +-inhibition component.

Aldosterone, sodium depletion and hypothyroidism on the ATPase activity of rat colonic epithelium.

SUMMARY Transmucosal electrical potential difference and short-circuit current of an in-vitro preparation of mucosal epithelium of rat colon were almost abolished and active sodium transport ceased when ouabain (1 mmol/l) was present on the serosal side of the epithelium. A considerable fraction of the total ATPase activity in the mucosa was sodium dependent and concentrations of ouabain of 0·5 mmol/l or more completely eliminated the activity of this fraction. Adenosine triphosphatase activities in mucosa taken from normal, hypothyroid, aldosterone-treated and sodium-depleted rats were compared. The activity of the ouabain-sensitive, sodium-dependent fraction was similar in all groups except for the hypothyroid rats in which it was considerably reduced. There was no evidence from the present study that the increased active sodium transport by colonic mucosa, associated with aldosterone action or sodium depletion, depended upon a change in ATPase activity. In hypothyroidism, reduction of ouabain-sensitive ATPase activity may be responsible for the observed impairment of the sodium transport mechanism.

Decreased ouabain-sensitive adenosine triphosphatase activity in the erythrocyte membrame of patients with chronic renal disease.

1. Ouabain-sensitive ATPase (adenosine triphosphatase) activity was measured in the erythrocyte membranes of twenty patients with chronic renal disease. Decreased activity was found in nineteen of the twenty patients. The average decrease was 38%. 2. In three patients erythrocyte sodium concentration exceeded 9·5 mmol/l of RBC and these patients had the most marked decreases in ouabain-sensitive ATPase activity. 3. By contrast, in only two of 100 patients admitted to a general medical ward was erythrocyte sodium concentration greater than 9.5 mmol/l of RBC. 4. Ouabain-insensitive and calcium-activated components of erythrocyte membrane ATPase were unaffected by chronic renal disease.

Cation Transport in Erythrocytes of Normal and Porphyric Cows: Erythrocyte Membrane Adenosine Triphosphatase Activities

Congenital erythropoietic porphyria is characterized by the increased production of the type I porphyrins due to an enzymatic defect of the erythropoietic tissue. The disease in cattle is characterized by a haemolytic anaemia with a shortened red cell median survival time. The erythrocyte membrane Na-K-ATPase was investigated as a possible focus of an iutracorpuscular defect resulting in a shortened survival of the porphyric erythrocytes. Erythrocytes from porphyric cows had significantly more total, ouabain insensitive, and ouabain sensitive ATPase activity than normal erythrocytes. Erythrocyte ouabain sensitive and. total ATPase activities of the porphyric cows were inversely correlated with the volume percent (PCV) of erythrocytes (r = − 0.96, P

Detection and general properties of adenosinetriphosphatase activities from plasma membrane in rat epididymal adipose tissue

A highly active Mg 2+ or Ca 2+ adenosinetriphosphatase activity has been detected in rat epididymal fat cell ghosts. ATP is not a specific substrate for the enzyme activity; ITP, UTP, CTP and GTP were in fact equally effective. The bivalent cationdependent ATPase was significantly inhibited by oligomycin and by ethacrynic acid. 2,4-Dinitrophenol unaffected the reaction, whilst an ouabain inhibition was evident only in the presence of sodium azide. The ouabain sensitive ATPase activity corresponds to 38 per cent of the bivalent cation dependent ATPase. A sodium iodide purification procedure was then performed according to Nakao et al. as reported by Matsui and Schwartz. No (Na +−K +) ATPase activity was found in the purified enzyme, whereas a Mg 2+ or Ca 2+-dependent activity was increased about 5-fold. The purified enzyme is more affected by oligomycin, whereas DCCD ( N, N′-dicyclohexylcarbodiimide), an inhibitor of oxidative phosphorylation and of mitochondrial-stimulated ATPase activities is not active on the purified enzyme. Data concerning the properties of the extracted enzymes and the further sodium iodide purification procedure are given.

Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal mucosa

A careful washed preparation of intestinal brush border membranes from rat contained two-thirds of the sucrase activity of a starting homogenate and 2–4% of the ouabain-sensitive ATPase activity.

Characterization of Plasma Membrane Proteins in Mammalian Kidney: I. Preparation of a Membrane Fraction and Separation of the Protein

A simplified method has been developed for the separation of plasma membrane material from isotonic homogenates of rat kidney. When examined by electron microscopy, the preparation consists of membrane vesicles free of nuclei and essentially free of intact mitochondria. Membranes have been solubilized by sonic oscillation or with Lubrol W-X detergent. Gel filtrate chromatography of solubilized membranes on 6% agarose gel yields two major protein peaks. The first protein peak elutes in the void volume and contains macromolecules with a molecular weight exceeding 1,000,000. The second protein peak has an elution profile similar to proteins of approximately 45,000 molecular weight. Sodium-potassium-dependent, ouabain-sensitive ATPase activity is associated with fractions of the first peak. Protein material associated with the second peak is mostly insoluble when membranes are extracted with 1-butanol. The bulk of the protein material associated with the second peak is solubilized when membranes are extracted with NaI. When proteins eluted from agarose gel columns are subjected to polyacrylamide disc gel electrophoresis, a portion of the high molecular weight protein in the first column peak moves, separating into five protein bands. The sodium-potassium-dependent ATPase activity appears to be associated with the nonmobile portion. All proteins in the low molecular weight second peak move, separating into more than eight bands. Incorporation in vivo of radioactive leucine into membrane protein was measured. After column fractionation a considerable variation in radioactivity of membrane fractions was observed. The findings in this study indicate heterogeneity of membrane protein constituents.

The control of extrarenal excretion in the duck ( Anas platyrhynchos) with special reference to the pituitary-adrenal axis

Because of the relatively poor concentrating capacity of the nephron in marine birds, an extrarenal route for the excretion of excess electrolytes is necessary. The extrarenal excretory organs of these birds are the nasal glands, which are situated in the orbit and connect, via excretory ducts, with the anterior nasal cavity. Both neural and humoral motor pathways have been implicated in the control of nasal gland function. The former is cholinergic and may involve parasympathetic fibers of the VIIth cranial nerve. The latter involves the pituitary-adrenal axis and is mediated via the release of ACTH. Adrenocortical steroids and ACTH have been shown to augment the rate of extrarenal excretion when administered to the hypertonic saline-loaded duck. Unilateral adrenalectomy significantly attenuated the extrarenal response, and after total adrenalectomy it was completely abolished. The administration of cortisol to the saline-loaded adrenalectomized duck restored the extrarenal excretion to normal. Adenohypophysectomy of the duck 2–3 weeks prior to the administration of hypertonic saline almost completely prevented nasal gland secretion. Sham adenohypophysectomy, however, also reduced the response. Preliminary in vitro incubation studies have indicated that a corticotropin-releasing factor (CRF) may be present in the duck hypothalamus. Extracts of both cerebrum and spinal cord, however, also induced an increased ACTH release. Chronic experiments involving the maintenance of ducks on hypertonic saline have shown that the extrarenal excretory capacity of the nasal gland increased with the time of exposure. Furthermore, the changes in the Na + transporting properties of the nasal gland correlated both quantitatively and temporally with simultaneously observed changes in the ouabain-sensitive ATPase activity of the nasal gland. An acute rise and fall in the RNA concentration of the nasal gland tissue was observed during the first 24 hours of exposure to hypertonic saline, and the RNA:DNA and protein: DNA ratios of the tissue showed sustained increases throughout a 30-day period of maintenance on hypertonic saline. The possibility arises, therefore, that a DNA- and RNA-dependent induced protein synthesis is involved in the development of the increasing active Na + transporting capacity of the nasal gland during adaptation to hypertonic saline. Increased corticosterone secretory rates at this time, and the intracellular accumulation of corticosterone in the nasal gland subsequent to saline loading, lend credence to the possibility that adrenocortical hormones may be directly or indirectly involved in such an induction process. The role of acetylcholine, however, does not appear to be restricted to vasomotor responses, and it may be also involved in the control of the developing active transport mechanisms.