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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.
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PMID:Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin. 12 64

1. Theophylline (10 mM) and choleragen (1 x 10(-6) g ml.-1) abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mM) and ethacrynate (0.1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0.1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P less than 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0.1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl+ ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer + ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-1 Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choleragen-treated tissue gives rise to net fluid secretion.
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PMID:Fluid movements across rabbit ileum coupled to passive paracellular ion movements. 46 72

1. The intracellular Na activity of sheep heart Purkinje fibres has been measured using recessed-tip Na(+)-sensitive glass micro-electrodes.2. The internal Na activity was 7.2 +/- 2.0 mM (mean +/- S.D., n = 32) at the normal external Na concentration, [Na](o), in these experiments of 140 mM (equivalent to an external Na activity of 105 mM). The equilibrium potential for Na across the fibre membrane was therefore approximately + 70 mV.3. When the [K](o) was altered the internal Na activity changed, reaching a new level within about 20 min. Increasing the [K](o) from 4 to 25 mM decreased the internal Na by approximately 30%, while decreasing the [K](o) from 4 to 1 mM increased internal Na by 20%.4. The removal of external K produced an easily reversible increase in the internal Na with an initial rate equivalent to a concentration change of 0.24 +/- 0.07 m-mole/min (mean +/- S.D., n = 8).5. Ouabain produced increases in the internal Na activity that were only very slowly reversible. The threshold concentration for producing an increase was approximately 10(-7)M.6. When [Na](o) was reduced the internal Na activity fell rapidly with a single exponential time course (time constant 3.3 +/- 0.8 min, mean +/- S.D., n = 16) to a new, relatively stable level. The recovery of internal Na on return to the normal [Na](o) did not have a simple time course. It was normally complete within 10-30 min.7. The relationship of the stabilized level of the internal Na activity to the [Na](o) was approximately linear over the range 140-14 mM-[Na](o). When [Na](o) was reduced from 140 to 14 mM the internal Na activity fell by 72 +/- 5% (mean +/- S.D., n = 21).8. When the [Na](o) was reduced, the decrease in the internal Na activity was partially inhibited by Mn or by removal external Ca.9. When the [Ca](o) was altered over the range 0.2-16 mM the internal Na activity was reduced by approximately 50% for a tenfold increase in the [Ca](o).10. The relationship between internal Na and contractility is discussed.
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PMID:The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres. 59 21

1. Membrane potentials have been recorded from cells of seminiferous tubules of rats in vitro using micro-electrodes. The value in 808 impalements was -28-2 +/- 0-3 mV (mean +/- S.E.) at 33 degrees C. 2. Increasing the potassium concentration depolarized the cells, a tenfold increase in concentration causing a depolarization of 16 mV. Removal of sodium from the bathing solution caused a hyperpolarization of 3 mV at a potassium concentration of 5-9 m-equiv/l. Removal of chloride and replacement with impermeant anions had no effect on potential. Removal of calcium from the bathing solution caused a minor but significant depolarization. 3. Ouabain (10-3 M), dinitrophenol (2-5 times 10-4 M) or removal of glucose from the bathing fluid all caused depolarization. The membrane potentials of the cells were sensitive to temperature over the range 10-33 degrees C, the apparent activation energy for the reactions maintaining the potential being approximately 6 kcal/mole. 4. Membrane potentials in seminiferous tubules were independent of age of the animal, were insensitive to previous hypophysectomy and were insensitive to a number of hormones (FSH, LH, HCG, oxytocin). In high concentration prostaglandin E1 caused depolarization. 5. Acetazoleamide (4 times 10-5 M) caused a rapid, but reversible, depolarization of the tubular cells. This was also true in conditions when the HCO'3/CO2 buffer system was replaced with Tris-buffer. Another carbonic anhydrase inhibitor (p-sulphonamido-benzoic acid) had similar effects on cell potentials as acetazoleamide. These results are discussed in relation to the nature of the ionic secretion produced in the tubules. 6. Occasional cells showed phasic variations in membrane potential. A possible connexion between these variations and the contractile activity of the tubules is discussed.
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PMID:Intracellular potentials in cells of the seminiferous tubules of rats. 115 7

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.
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PMID:Model for the regulation of platelet volume and responsiveness by the trans-membrane Na+/K(+)-pump. 131 20

When perfused cortex-free ox adrenal medulla was stimulated to secrete catecholamine by infusion of 0.1 mM acetylcholine for 4 min, the oxygen consumption increased to a value which was 0.15 +/- 0.07 mumole O2/min/g wet weight (+/- S.D., N = 12) above the pre-stimulation value of 0.49 +/- 0.15 (P less than 0.001). 1.4 +/- 0.9 (+/- S.D., N = 12) mole of catecholamine was secreted per mole of enhanced O2 consumption in the 16 min following the start of the stimulation. The rate of ATP hydrolysis by the proton-translocating Mg-ATPase of the chromaffin granule may increase on fusing with the plasma membrane of the chromaffin cell during exocytosis. However, from the amount of catecholamine secreted, this was estimated to account for less than 17% of the oxygen consumption increase. The amount of catecholamine secreted by 4 min 0.1 mM acetylcholine stimulations correlated with the enhancement of oxygen consumption (r = 0.82, P less than 0.001) but, on stimulation with 60 microM veratridine for 4 min, O2 consumption enhancement was anomalously low. This dependence on mode of stimulation suggests that ATP consumption in exocytosis itself is an inadequate explanation. Ouabain-sensitive oxygen consumption rose from undetectable levels to 18 +/- 8% (+/- S.D., N = 4) of the basal respiration during prolonged 0.1 mM acetylcholine stimulation in the absence of Ca, indicating that Na,K-ATPase was not responsible for all of the oxygen consumption enhancement.
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PMID:Enhanced oxygen consumption in adrenal medulla on stimulation with acetylcholine. 298 35

The concentration-dependent effects of ouabain on the contractility of postnatally developing rat heart ventricles were studied. Ouabain caused a positive inotropic effect in right ventricular strips of neonatal rats up to the age of about 30 days but a negative inotropic effect in the adult cardiac tissue. When extracellular Ca concentration was lowered from 2.5 to 1.0 mmol/l and the rate of stimulation was simultaneously elevated from 0.2 to 1.0 Hz a clear positive inotropic effect was also generated in the adult rat heart. The positive inotropic effect of ouabain showed a biphasic developmental pattern: the contractile force first grew from birth to about 15 days of age but steeply declined near to the adult level during the 3rd postnatal week. The force response to ouabain occurred within two distinct dose-ranges. In the newborn only the high-dose (above 3 X 10(-6) mol/l) effect was seen but in rats older than 5 days a mixed low-dose (below 3 X 10(-6) mole/l)/high-dose effect was apparent. In both ranges the positive inotropic effect of ouabain seemed to be dependent on caffeine sensitive Ca store, perhaps the sarcoplasmic reticulum. It is suggested that during the 3rd postnatal week a transition from extracellular to intracellular Ca stores occurs in the rat heart, which is reflected as a changing inotropic effect of ouabain on the developing cardiac tissue.
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PMID:Postnatal changes in the inotropic effect of ouabain on the rat heart ventricle. 359 85

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5'-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 mumole per min per mg of protein under optimal conditions. The enzyme was Mg(++)-dependent and K(+)- or Na(+)-stimulated. Maximal activity was observed with a molar adenosine 5'-triphosphate (ATP) to Mg(++) ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5'-diphosphate. Inorganic pyrophosphate and the 5'-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.
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PMID:Adenosine triphosphatase in isolated membranes of Staphylococcus aureus. 423 Aug 57

1. A method to separate the epithelium from the underlying layers of the frog skin is described. The method is based on the combined use of collagenase and hydrostatic pressures.2. The potential difference and the short-circuit current values of isolated epithelia and whole skins are similar. Na net flux and short-circuit current are equivalent.3. The time course of changes in potential following rapid changes in composition of the bathing solutions shows that the barrier to K diffusion at the internal surface of the isolated epithelium is larger than the barrier to Na diffusion at the external surface.4. In the isolated epithelium there are 133 m-mole K(+) and 24.7 m-mole Na/l. cellular water. The amount of extracellular water was considered to be equal to the inulin space.5. Arginine vasopressin (0.1 u./ml.) markedly increased short-circuit current and potential difference in isolated epithelia. The amount of Na in the epithelium that equilibrated with Na in the external solution was not increased by the hormone.6. Ouabain (10(-4)M) reduced short circuit current and potential difference to values close to zero. The ouabain treated epithelia contained an increased amount of Na originating in the internal solution. On the other hand the amount of Na that originated from the external solution was not increased.7. The amount of epithelial Na that equilibrated with Na in the external solution was 0.009 mu-equiv/cm(2). This figure is about ten times smaller than the values found in whole skins.
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PMID:Sodium transport across the isolated epithelium of the frog skin. 432 24

1. Spleen slices pre-incubated for different periods at 4 degrees C in Krebs solution containing varying concentrations of calcium, up to 96 mM, lost their endogenous noradrenaline stores when reincubated in normal Krebs solution at 37 degrees C for 2 hr. Rate of loss of noradrenaline was roughly related to the calcium concentration of the pre-incubation medium and the pre-exposure time.2. Pre-treatment with isotonic barium or strontium (96 mM) Krebs solution also induced release of noradrenaline from spleen slices when re-exposed to normal Krebs solution. Barium was more effective than either calcium or strontium.3. The enhanced release induced by calcium pre-treatment occurred in the absence of calcium, with or without EGTA.4. Tissue calcium concentration of spleen slices was 0.68 m-mole/kg. Pre-treatment of slices with normal or 96 mM calcium-Krebs solution for 4 hr at 4 degrees C increased the calcium concentration to 2.57 and 9.9 m-mole/kg, respectively.5. Ouabain, which caused a dose-dependent release of noradrenaline, did not modify the release induced by calcium pre-treatment.6. Spleen slices prepared from cats anaesthetized with sodium pentobarbitone instead of ether were resistant to noradrenaline depletion by calcium pre-treatment.7. Evoked release of [(3)H]noradrenaline by high potassium from calcium-pre-treated slices did not occur in the absence of external calcium, even though the calcium pre-treatment enhanced the tissue concentration of this ion by nearly tenfold.8. Net uptake of noradrenaline in normal and in treated slices whose noradrenaline content was severely reduced by barium pre-treatment or sodium withdrawal was comparable.9. Specific activity of released and endogenous [(3)H]noradrenaline increased as the tissue stores of noradrenaline were reduced.10. It is suggested that the spontaneous loss of tissue noradrenaline after pre-treatment with high-calcium solution was due to inhibition of sodium-potassium-activated ATPase by intracellular accumulation of calcium ions. Evidence is presented to suggest that vesicles depleted of their endogenous transmitter by pre-treatment with calcium, strontium or barium, or by sodium withdrawal, are re-used for the storage and release of exogenous noradrenaline.
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PMID:Release of noradrenaline from slices of cat spleen by pre-treatment with calcium, strontium and barium. 477 3


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