Inhibitory Effect Of Ouabain Research Articles

EFFECTS OF OUABAIN ON HIGH-K INDUCED CONTRACTIONS OF VARIOUS SMOOTH MUSCLE TISSUES IN THE GUINEA-PIG

AbstractThe effects of ouabain on contraction, cellular Na concentration, and 45Ca uptake of various smooth muscle tissues depolarized by high-K (62.7 mM) solution were studied. In the taenia coli and ileum isolated from guinea-pig, ouabain (2×10−6 M – 5×10−B M) increased cellular Na concentration and strongly inhibited K-contraction. A good correlation between cellular Í a concentration and inhibition of tension was obtained both in the taenia coli and ileum. In the portal vein, aorta, and uterus, ouabain failed to induce relaxation and slightly increased the cellular. Na. A long-time exposure to ouabain (1×10−3 M) attenuated the response to high-K in the portal vein and uterus but not in the aorta. The inhibitory effect of ouabain in the taenia coli and ileum was antagonized by an increase in external Ca, and the inhibition was accompanied by a decrease in cellular 45Ca content. A possibility exists that excess cellular Na inhibits K-induced Ca influx, although there is a tissue-specific difference. The data also suggest that the inhibitory effect of Na-accumulation is dissimilar to that of verapamil with respect to the tissue-specific difference.

Effects of ouabain on high-K induced contractions of various smooth muscle tissues in the guinea-pig.

The effects of ouabain on contraction, cellular Na concentration, and 45Ca uptake of various smooth muscle tissues depolarized by high-K (62.7 mM) solution were studied. In the taenia coli and ileum isolated from guinea-pig, ouabain (2 x 10(-6) M - 5 x 10(-5) M) increased cellular Na concentration and strongly inhibited K-contraction. A good correlation between cellular Na concentration and inhibition of tension was obtained both in the taenia coli and ileum. In the portal vein, aorta, and uterus, ouabain failed to induce relaxation and slightly increased the cellular Na. A long-term exposure to ouabain (1 x 10(-3) M) attenuated the response to high-K in the portal vein and uterus but not in the aorta. The inhibitory effect of ouabain in the taenia coli and ileum was antagonized by an increase in external Ca, and the inhibition was accompanied by a decrease in cellular 45Ca content. A possibility exists that excess cellular Na inhibits K-induced Ca influx, although there is a tissue-specific difference. The data also suggest that the inhibitory effect of Na-accumulation is dissimilar to that of verapamil with respect to the tissue-specific difference.

Inhibition of lymphocyte activation by ouabain Interference with the early activation of membrane phospholipid metabolism

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of ( Na + + K +)-ATPase . Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. ( Na + + K +)-ATPase was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K + concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the ( Na + + K +)-ATPase .

Some Properties of the Polyamine Deprivation-inducible Uptake System for Methylglyoxal Bis(guanylhydrazone) in Tumor Cells.

Intracellular polyamine deprivation, produced by DL-alpha-difluoromethylornithine (DFMO), resulted in a striking enhancement of cellular transport of the natural polyamines and methylglyoxal bis(guanylhydrazone) (MGBG). In addition to the natural polyamines and MGBG, the uptake of other diamines and triamines was likewise enhanced in response to DFMO, although longer than three-carbon backbone was required for about 10-fold stimulation to occur. Intracellular deprivation of polyamines did not increase the affinity of the transport system for MGBG but greatly enhanced the maximum velocity of the drug transport. The uptake process of MGBG was temperature dependent and the activation energy (Ea = 67.5 kJ) for the uptake system was the same for both the polyamine-depleted tumour cells and for the untreated cells. The uptake of the drug appeared to be more dependent on the Na+-linked uptake, as indicated by the inhibitory effect of ouabain, than on energy production. Deprivation of putrescine and spermidine changed the intracellular distribution of MGBG since a major portion of the drug was concentrated in the microsomal fraction in polyamine-depleted cells.

Ouabain effects on hormonally-stimulated bone resorption and cyclic AMP content in cultured fetal rat bones.

Ouabain in concentrations from 20-100 micromoles produced a dose-related inhibition of in vitro stimulation of bone resorption by parathyroid hormone, 1,25-dihydroxyvitamin D3 and calcium ionophore A23187, as measured by 45Ca and [3H]-hydroxyproline release in 5-day cultures of fetal rat forelimb rudiments. The inhibitory effect on 45Ca release was completely reversed by subsequent incubation in ouabain-free medium. At a concentration of 100 micromoles ouabain virtually abolished active bone resorption; however, basal and stimulated bone cyclic AMP (cAMP) content were significantly increased above levels observed in the absence of ouabain. The increased cAMP content did not appear to be the result of phosphodiesterase inhibition. It is concluded that intact Na/K ATPase function is required for hormonally-stimulated bone resorptive processes and that the inhibitory effect of ouabain on bone resorption is produced at a point subsequent to cyclic AMP generation.

Calcium in the control of renin release.

The effect of Ca concentrations in the incubation medium and of estimated intracellular Ca concentrations on renin release was examined with use of pig renal cortical slices. In addition, the Ca requirement for the epinephrine stimulatory effect and for the ouabain inhibitory action on renin release was also tested. In mediums containing 5.9 mM K, variations in Ca concentration had no effect on renin release. In contrast, when the K concentration was 59 mM, a significant inhibition of renin release was attained with all concentrations of calcium. The inhibition of renin release in high K mediums by Ca was attributed to an increase in the intracellular Ca concentration. In addition, both the stimulatory effect of epinephrine and the inhibitory effect of ouabain on renin release required Ca in the medium. These results support the hypothesis that the control of renin secretion is mediated, in part, by changes in the intracellular concentration of Ca, most likely in the juxtaglomerular cells.

Na-K-ATPase and transepithelial sodium transport in the toad

1. 1. Studies have been conducted using the isolated toad skin and urinary bladder preparations in an attempt to evaluate the relationship between sodium, potassium activated adenosinetriphosphatase (Na-K-ATPase) system and transepithelial sodium transport. 2. 2. In both the skin and bladder, ouabain showed no significant inhibitory effect on the Na-K-ATPase activity at 23°C, while it exerted a potent inhibition of the active sodium transport at this temperature. On the other hand, at 37°C, both the Na-K-ATPase activity and the sodium transport were strongly inhibited by the glycoside. 3. 3. The inhibitory effect of ouabain on the sodium transport was reduced as potassium concentration in the serosal bathing medium was increased. 4. 4. There was no apparent correlation between the magnitude of sodium transport and the Na-K-ATPase activity of the skin regardless of whether the skin was treated with vasopressin (ADH), ouabain, or both ADH and ouabain. 5. 5. These results emphasize the difficulty associated with correlating sodium transport across epithelia with activity of Na-K-ATPase in a subcellular fraction.

Ionic dependence of luteinizing‐hormone‐induced steroidogenesis in the rabbit ovary.

1. Investigations were made into the influence of the ionic environment on the steroidogenic response of the rabbit ovary to luteinizing hormone (LH). 2. Removal of Ca2+ from the medium was without effect on the response to LH. A similar result was obtained in Ca2+-free medium containing EGTA. 3. A tenfold increase in [Ca2+]o to 25.6 mM, or the addition of La3+ or Eu3+ (0.25 mM) to medium containing the normal concentration of Ca2+, caused a marked inhibition of the response to LH. 4. Removal of Na+ from the medium, and replacement by choline, had no effect on the response to LH. Replacement of Na+ by Li+ inhibited the response to the hormone strongly, but the addition of 4 mM-Li+ to normal medium was without effect. 5. Removal of K+ from the medium inhibited LH-induced steroidogenesis, whereas a twentyfold increase in [K+]o to 100 mM had no effect. The response to LH was also unaffected by the absence of Cl-. 6. Ouabain (10(-4) M) inhbited the response to LH, but nupercaine (10(-4) M) was without effect. 7. The inhibitory effect of ouabain was reversed by the addition of 2 mM-NADP+ to the medium. In contrast, the inhibitory effect of Eu3+ persisted in the NADP+-rich medium. 8. It is suggested that the intracellular ratio of Na+ or Li+) to K+ is important for the expression of the steroidogenic response of the ovary to LH. Altered concentrations of these ions might affect the formation or availability of NADP+. The inhibitory effects of high [Ca2+]o and lanthanide ions, however, are probably due to inhibition of hormone-stimulated adenyl cyclase.

Inhibitory Effect of Ouabain on Epinephrine-Induced Stimulation of Adrenal Corticosterone Secretion in Rats

Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.

Histochemically demonstrable sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) activity in the rat lens

The rat lens epithelium was subjected to histochemical examination in order to demonstrate sodium-potassium-activated adenosine triphosphatase reaction. The histochemical technique was specifically adapted to demonstrate the sodium-potassium activated fraction of the enzyme activity. The precise localization of the enzyme activity in the lens epithelium was demonstrated. It was found that fixation in 2·5% glutaraldehyde for 30 min gave relatively good cytological preservation, but did not unduly inhibit the enzyme activity. The incubation solution contained 3 m m-ATP, lead and magnesium as well as 70 m m-sodium and potassium. When the inhibitory effect of ouabain was to be demonstrated, this treatment had to be carried out prior to fixation. Sodium-potassium-activated adenosine triphosphatase activity was localized on the epithelial cell membranes of the lens. The precipitate was observed on those parts of cell membranes where two adjoining cells were in contact with each other. The cytoplasm of the epithelial cells was negative, whereas the lens fibres seemed to contain some precipitation, which, however, did not fulfill the specifications for the enzyme and was thus considered not to demonstrate the real activity. The results obtained indicated that sodium-potassium activated ATPase, which has been supposed to be responsible at least partly for the ion and water transport of the lens, acts on the epithelial cell mambranes, but what direction this pumping mechanism takes is not yet clear.

Lymphocyte monovalent cation metabolism: Cell volume, cation content and cation transport

AbstractMechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium‐24 and potassium‐42 between thymus water and plasma water were very similar to the distribution ratios of non‐radioactive isotopes (sodium‐23 and potassium‐39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady‐state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady‐state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain‐sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm2/min × 10−8) than in CLL (3.24 ± 0.45 mmoles/cm2/min × 10−8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain‐sensitive sodium accumulation and ouabain‐insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half‐maximal ouabain inhibition of sodium entry was 7.5 × 10−3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain‐sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin‐sensitive (sodium‐potassium) ATPase: (a) steady‐state monovalent cation gradient (K ∼ 20:1, Na ∼ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady‐state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain‐insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady‐state cation content. Ouabain‐sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto‐ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain‐sensitive ATPase and monovalent cation transport in the function of lymphoid cells.

Quantitative aspects of the interaction between ouabain and (Na + + K +)-activated ATPase in vitro

The inhibitory effect of ouabain on (Na + + K +)-activated ATPase (Mg 2+-dependent, (Na + + K +)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na +, Mg 2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na + + K +)-activated ATPase reaction was subsequently started by the addition of a KCl solution. When the ouabain-enzyme reaction was brought to near equilibrium prior to the estimation of the ATPase activity, it was kinetically reversible, although overwhelmingly in favor of the ouabain-enzyme complex. This method resulted in a significant shift of the log concentration-response curve to the left. The concentration of ouabain to inhibit 50 % of the (Na + + K +)-activated ATPase activity was 0.12 μM, whereas it was 0.52 μM with the conventional method for the ATPase assay. Hence, the specific binding of ouabain to the (Na + + K +)-activated ATPase molecule was a slow process. This modified method was not suitable for the study of the effect of p-chloromercuribenzoate on (Na + + K +)-activated ATPase since the presence of ATP during the preincubation period protected the enzyme from the SH-blocking reagent. With the modified method, the effect of K + to antagonize ouabain inhibition of (Na + + K +)-activated ATPase was markedly reduced, indicating that the well-documented effect of K + was on the velocity of the ouabain-enzyme complex formation rather than on that of the ouabain-inhibited ATPase reaction. The ouabain-enzyme reaction was competitive with respect to K + at KCl concentrations below 5 mM, although the competition by K + was not remarkable. Above this concentration, the reaction was non-competitive with respect to K +. Ouabain released from the ouabain-enzyme complex was rebound to the enzyme during an incubation in the presence of Na +, Mg 2+ and ATP more easily than ouabain added to the incubation mixture.

Interspecies differences in the inhibitory effect of ouabain on lymphocyte transformation

The pronounced sensitivity of the phytohemagglutinin (PHA)-induced transformation of human lymphocytes suggested further investigation of this phenomenon in other animal species that are useful in studies of the immune response. Pronounced interspecies differences in the effect of ouabain were observed. Whereas PHA-stimulated pig leukocytes showed the same high sensitivity characteristic of human leukocytes to concentrations greater than 10 −8 M, stimulated rat leukocytes cultures were relatively insensitive to ouabain at concentrations higher by three orders of magnitude.

The ouabain inhibition of sugar transport in kidney cortex cells

The inhibitory effect of ouabain on the active, Na-dependent transport of α-methyl- d -glucoside by slices of rabbit kidney cortex was studied: A Ki of 0.13 m m was found. The inhibition at low ouabain concentrations (0.02 m m ) could be abolished by increasing [K+]0 to 25 m m , whereas at higher concentrations of ouabain (up to 0.5 m m ), increased [K+]0 was ineffective. Evidence for binding of ouabain by kidney cortex was obtained: (a) preincubation of slices in the presence of ouabain produced a subsequent inhibition of cation and α-methyl-glucoside transport in ouabain-free media; (b) more than 80% of ouabain-3H taken up by the tissue could not be washed out. Binding of ouabain by the tissue showed the following characteristics: (a) at low concentrations of the cardiac glycoside (1–100 μ m ) the saturable binding component was depressed by the absence of Na+, and was abolished by increased [K+]0, 0o, and denaturation of tissue proteins at 80°; (b) binding of ouabain by the nonsaturable tissue component was linearly related to the concentration of the substance in the medium. The maximum number of ouabain-binding sites in rabbit kidney, cortex, corrected for the nonspecific binding of the glycoside, was determined to be 6×1015/g wet wt, and the association constant was of the order of 3×105 m −1; in rats, the value for the maximum number of ouabain-binding sites was at least one order of magnitude lower than in rabbits. The above results correlate with the characteristics of the interaction of ouabain with the (Na++K+) ATPase. It is concluded that the membrane ATPase participates (directly or indirectly) in the Na-dependent transport system for α-methylglucoside.

The effects of an inhibitor of Na plus -K plus -active transport on the secretory activity of rat submaxillary gland cell cultures.

The role of electrolyte transport in the metabolism of mucosubstance has been investigated using cell cultures derived from rat submaxillary gland. When added to the culture medium at a concentration of 1 mM, ouabain, an inhibitor of active alkali metal ion transport through cell membranes, causes extensive cytoplasmic vesiculation suggestive of an accumulation of secretory materials within the cell; the rough ER and cytoplasmic polysomes are not affected. Isotopic experiments support these conclusions by demonstrating that ouabain inhibits the secretion of radioactive mucosubstances by the cells; comparative studies with ouabain and puromycin (2×10−4M) indicate that the inhibitory effect of ouabain is not due to an inhibition of protein biosynthesis. A relationship between secretion and alkali metal ion movement is suggested which may be of significance in connection with the genetic disease, cystic fibrosis.

Effects of Na+ and K+ on the uptake of metaraminol by rabbit ventricular slices.

1. The ionic dependence of [(3)H]-metaraminol transport by rabbit ventricular slices was studied.2. Transport was Na(+) dependent. Choline, Li(+), K(+), Rb(+) or Cs(+) could not be substituted for Na(+).3. Transport was K(+) dependent. Rb(+) and Cs(+), but not Li, could be substituted for K(+), their relative potencies being K(+) = or >Rb(+)>Cs(+)>>Li(+). Higher concentrations of K(+), Rb(+), Cs(+) and Li inhibited [(3)H]-metaraminol transport.4. The inhibitory effects on transport of either a marked reduction in the concentration of Na(+) or of omission of K(+) were rapidly reversible on exposure of slices to Krebs solution.5. The inhibitory effect of ouabain on [(3)H]-metaraminol transport was markedly time dependent, being significantly increased by preincubation with ouabain. The inhibitory action of ouabain was not affected, however, by the Na(+) concentration present during preincubation.6. An inwardly directed Na(+) gradient did not increase [(3)H]-metaraminol transport in slices in which Na(+) pumping had been prevented by 10(-5)M ouabain, 4 degrees C, omission of K(+) or by metabolic inhibition.7. These findings provide additional evidence that the Na(+) and K(+) activated adenosine triphosphatase may participate in the transport of metaraminol and related amines. In the absence of Na(+) pumping, induced Na(+) gradients were unable to produce transport of amine as would be predicted by the co-transport model proposed by Bogdanski & Brodie (1969).

Role of potassium in the lipolytic hormone effect in rat adipose tissues.

1. The influence of potassium ions upon the lipolytic hormone effect in vitro was examined on epididymal white as well as interscapular brown adipose tissue slices.2. In either adipose tissue, the omission of potassium from incubation medium markedly, though not completely, diminished the catecholamine effect on lipolysis.3. Similar inhibition was obtained by the addition of ouabain. This inhibitory effect of ouabain was well overcome in white adipose tissue by increasing the potassium concentration in the medium, whereas in brown adipose tissue no interaction between ouabain and potassium was observed.4. Lipolytic action of theophylline was not affected at all by ouabain in both adipose tissues and was significantly increased in the absence of potassium in the medium.5. Catecholamine-stimulated increase in the respiration of brown adipose tissue was almost completely abolished by ouabain or potassium omission.6. It is suggested that two mechanisms may be involved in the enhanced lipolysis. It is possible that potassium ions play some essential role in the operation of these mechanims.

Insulin-like action of ouabain II. Primary antilipolytic effect through inhibition of adenyl cyclase

1. 1. In the absence of added glucose, ouabain had a strong inhibitory action upon hormone-stimulated lipolysis. By contrast, the presence of ouabain resulted in little or no inhibition of lipolysis induced by dibutyryl-3',5'-AMP or caffein, indicating that the inhibitory action was exerted at the level of the formation of cyclic AMP. 2. 2. The inhibitory action of ouabain on hormone-stimulated lipolysis in the absence of glucose can be reproduced by removing K+ from the incubation medium. The inhibitory effect of ouabain and that of K+ lack could be overcome by increasing the concentrations of the lipolytic hormone. 3. 3. Using an assay system for adenyl cyclase described here, it could be shown that the adenyl cyclase activity of adipose tissue pretreated with ouabain was reduced by 60–70% when compared to that of the untreated tissue. Similarly, the removal of K+ from the incubation medium resulted in a marked decrease of the adenyl cyclase activity. By contrast, ouabain did not influence the activity of phosphodiesterase. 4. 4. These results are consistent with a decreased formation of cyclic AMP induced by both ouabain and/or lack of K+. Althrough the mechanism of this effect is unknown, it is possible that the affinity between lipolytic hormones and adenyl cyclase is regulated by the concentrations of cations and that the inhibitory effect of ouabain on hormone-stimulated lipolysis may be the result of decreasing intracellular K+ levels. Furthermore, it would seem that the effects of insulin and of ouabain have some features in common, since the presence of both agents may lead to decreased levels of cyclic AMP and decreased intracellular concentrations of free fatty acids.

A comparison of microsomal (Na + + K +)-ATPase with K +-acetylphosphatase

Similar distributions of radioactivity were observed upon paper electrophoresis of pepsin-digested beef brain microsomes previously exposed to either [ 32P]ATP or acetyl [ 32P]phosphate in the presence of Na + and Mg 2+. ATP and acetylphosphate were each competitive inhibitors of the other as substrate for a K +-requiring acetylphosphate (acylphosphate phosphohydrolase, EC 3.6.1.7) and for a (Na + + K +)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3). Despite these similarities, there were marked differences in the inhibitory effect of ouabain, oligomycin, and exposure to N-ethylmaleimide on the two enzymes. The data suggest that the K +-acetyl-phosphatase activity of the beef brain may represent a different entity than the ( Na + + K +)-ATPase.

Partition of sodium fluxes in isolated toad oocytes.

1. The rate constant for Na efflux from the oocyte calculated from (d/dt) (ln [Na(*)](i)]) is only approximately 52% of that calculated from (d/dt)[(ln(d[Na(*)](i))dt)]. The difference may be interpreted by supposing that 48% of the internal Na of the oocyte is either bound to proteins or sequestered in cell organelles.2. The mean rate constant for Na efflux was 6.4 x 10(-3) min(-1) corresponding to an apparent Na efflux rate of 13.3 p-mole/cm(2).sec. When this is corrected for the increase in surface area produced by microvilli the true efflux rate is 1.1-1.3 p-mole/cm(2).sec.3. The action of ouabain (1-5 muM) appears to involve two different effects: (a) there is 48-65% inhibition of the membrane Na pump, and (b) there is a release of some of the sequestered Na in the cell.4. Removal of external K causes a 40% reduction in Na efflux although this value may be an underestimation owing to the presence of K which has leaked from the cell and may be retained near the cell surface.5. Raising the external K concentration to 15 mM reduces the inhibitory effect of ouabain by approximately a half.6. It was concluded that the Na pump in the toad oocyte may have a slightly lower level of activity than that in frog muscle, but that its general properties are similar to those in frog muscle and some other animal cells.