Ouabain Sensitivity Research Articles

Effect of thyroid status on the development of the different molecular forms of Na +,K +-ATPase in rat brain

The effect of thyroid status on the postnatal development of the two molecular forms of Na +,K +-ATPase, distinguished kinetically on the basis of their ouabain sensitivity, was examined in rat brain. Hypothyroidism induced by PTU from day 1 postnatally significantly reduced the Na +,K +-ATPase activity in cerebellum (22–30 days) but not forebrain, whereas hyperthyroidism (T 4 treatment from day 1) had no effect. The hypothyroidism-induced reduction in cerebellum was reflected by a 20–45% reduction in the activity of the α(+) form of Na +,K +-ATPase (high ouabain affinity) against control brains compared to a 60–70% reduction in the activity of the α form (low ouabain affinity). These results show that neonatally induced hypothyroidism leads to a selectively greater impairment of the ontogenesis of the activity of cerebellar α form of Na +,K +-ATPase. This may possibly reflect a retarded development of a selective cerebellar cell population containing predominantly the α form of the enzyme.

Ouabain-resistant cells cultured in a synthetic medium.

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.

Specific involvement of calmodulin and non‐specific effect of tropomyosin in the sensitivity to ouabain of Na+, K+‐ATPase in murine plasmocytoma cells

The Kd for ouabain for inhibition of Na+,K+-ATPase isolated from murine plasmocytoma MOPC 173 cells is 120 microM, but when isolated in the presence of EDTA, it is 100-fold lower (1.2 microM). Simultaneous addition of muscle tropomyosin and calcium to sensitive membranes restored the original insensitivity (tropomyosin bound to the membranes in an irreversible and saturable manner). For comparison 86Rb influx into intact cells, mediated by the Na+,K+-pump, is half-maximally inhibited at 50 microM ouabain. Calcium converts the enzyme to an insensitive form. This appeared to involve calmodulin because after extraction of calmodulin with EDTA and EGTA from sensitive membranes, they could not be made insensitive by the addition of tropomyosin and Ca2+. Addition of exogenous calmodulin to these calmodulin-depleted membranes was required, in addition to tropomyosin and Ca2+, to decrease the ouabain sensitivity. The involvement of calmodulin was further assessed by measuring the range of Ca2+ concentrations required to convert to the insensitive form. At saturating concentrations of tropomyosin, increasing free [Ca2+] up to 3 microM led to an heterogeneous population of Na+,K+-ATPase forms. The calcium dependency was a saturable process. The shift to the insensitive form was half maximal at 0.65 + 0.11 microM free Ca2+ and was abolished by the addition of troponin I or trifluoroperazine (0.1 mM). These results suggest that, in murine plasmocytoma cells, the intrinsic sensitivity of Na+,K+-ATPase to ouabain might be regulated by a calmodulin-dependent process within a submembrane contractile-like environment.

Mechanisms of Age-Related Differences in the Cardiotoxic Action of Digitalis

Age-associated ouabain sensitivity was investigated in isolated perfused guinea pig heart and the findings were correlated with its receptor function. Ouabain (0.1 mumol L-1) produced average positive inotropic responses of 22% in 15-day-old, 25% in 4-6 month-old, and 34% in 18-24-month-old guinea pig hearts. The time required to produce the greatest positive inotropic response was 28 min in 15-day- to 6-month-old guinea pig hearts and 20 min in 18-24-month-old guinea pig hearts. To cause arrhythmias and cardiac arrest, 2.1 mumol L-1 ouabain was required in age groups of 15 days to 6 months, and 1.2 mumol L-1 in age groups of 12-24 months. The time of onset of arrhythmias and cardiac arrest was also significantly less in older than in younger animals. Cardiac membranes from 12-24-month-old guinea pig hearts had significantly lower Na+, K+ -ATPase activity per mg of sarcolemmal protein and numbers of [3H]ouabain binding sites than those from 28-day- to 6-month-old hearts. The enzyme inhibition by 0.1 mumol L-1 ouabain was, however, less in younger than in older groups. The difference in the inhibition of enzyme was reflected in lower ouabain binding affinity (Kd) in younger animals. The data confirm the existence of an age-related difference in ouabain sensitivity and suggest that these differences may be due to changes in receptor function.

Characterization of ouabain-resistant mutants of the preadipocyte Ob17 clonal line Adipose conversion in vitro and in vivo

After exposure to a mutagenic drug (ethylmethanesulfonate), mutant clones of Ob17 preadipose cells resistant to the cytotoxic action of ouabain have been isolated. Their ability to grow in serum-supplemented medium containing ⩾3 mM ouabain is consistent with the decreased ouabain sensitivity of K + transport observed in one of these clones (Ob17-OR11). Ouabain-resistant mutants retain their ability to convert into adipose-like cells in vitro with a frequency similar to that of original Ob17 cells. Long-term maintenance of Ob17-OR11 cells in ouabain-enriched medium leads to a higher frequency of adipose conversion, which is linked to a rather specific and limited growth-promoting effect of the drug. Undifferentiated Ob17-OR11 cells, when injected subcutaneously into athymic mouse, give rise in vivo to a fat pad containing ouabain-resistant mature adipocytes. Therefore, Ob17-OR11 clone should be a useful tool to distinguish in vivo between intrinsic and extrinsic factors involved in the differentiation of adipose precursor cells.

Species variation in the ouabain sensitivity of cardiac Na +/K +-ATPase : A possible role for membrane lipids

The role of membrane lipid composition on the modulation of ouabain sensitivity of cardiac Na +K +-ATPase has been studied in vitro using several animal species. The animals can be grouped as ouabain-sensitive and ouabain-insensitive species. Ouabain-sensitive species (I 50; 0.5–2.2 μM) include sheep, marmoset, pig and the guinea pig, whilst rat and mouse form the ouabain-insensitive group (I 50; 100–105 μM). Although no species variation in the distribution of major phospholipid classes was observed, significant differences were apparent in the proportions of certain saturated and unsaturated phospholipid fatty acids. Thus, there was a marked increase in the relative proportion of docosahexaenoic (22:6, ω-3) acid in the Na +/K +-ATPase preparations from the rat and mouse compared to ouabain-sensitive species. Despite these differences, all animals had similar proportions of total saturated (∑SAT) and total unsaturated (∑Unsat) fatty acids. On the other hand, a good correlation between the unsaturation index of membrane lipids and I 50 value for ouabain was observed. It is proposed that acyl chain characteristics (unsaturation and/or chain length) rather than the head group of the phospholipid molecule play a major role in the modulation of Na +/K +-ATPase to inhibition by ouabain.

Increased ouabain sensitivity of cultured human fibroblasts from muscular dystrophy.

Muscular dystrophy is a disease characterized by wasting of muscle tissue in vivo and net loss of muscle cell protein in vitro. No comparable changes have been reported in other tissues, although all cells of affected individuals must carry the X-linked recessive mutation. On the hypothesis that predisposition to accelerated protein degradation might be latent in nonmuscle cells I investigated protein metabolism in skin fibroblasts from normal individuals and patients with Duchenne and Becker dystrophy. Under normal culture conditions rates of protein synthesis and protein degradation in the two groups of cultures were indistinguishable. Both types of cells responded to treatments that stimulate protein degradation and the extent of response was similar. Treatment with ouabain to reduce cell K+ content, and hence protein synthesis, had no effect on protein degradation in either group. Synthesis of protein was reproducibly more sensitive to ouabain in dystrophic than in normal strains, however, and the rate of protein synthesis was correlated with the steady-state K+ content. Eight out of nine dystrophic strains showed a greater sensitivity of K+ content to ouabain inhibition of the membrane Na+-K+ pump than four normal strains. This increased sensitivity could be conclusively attributed to increased efflux or decreased influx of K+, or to alterations in ouabain binding to intact cells. Others have observed membrane abnormalities in dystrophic muscle as well as in other cell types. Our findings may represent a physiological consequence of that abnormality.

Enhanced ouabain sensitivity of the heart and myocardial sodium pump in aged rats

Tolerance to arrhythmogenic actions of digitalis decreases with advanced age. To determine if aged myocardium has reduced glycoside tolerance, ouabain sensitivity and associated biochemical changes were examined in young (3-month), adult (8-month) and aged (26-month old) Fisher 344 rats, because age-related changes in physiology and biochemistry are well characterized in this strain of rats. Although rat are uniquely tolerant to digitalis, basic mechanisms leading to inotropic and toxic actions of digitalis are not different from other species. In aged anesthetized rats, intravenous infusion of ouabain produced arrhythmias and cardiac arrest at lower doses. Aged myocardium had lower Na,K-ATPase activity and fewer high affinity [ 3H]ouabain binding sites. Affinity of the ouabain binding sites on Na,K-ATPase was not altered. Despite these findings, sodium pump activity, estimated from the ouabain-sensitive 86Rb + uptake by ventricular muscle slices, was higher in the aged myocardium when the uptake was observed in the absence of ouabain and was more sensitive to inhibitory action of the glycoside. Differences in Na,K-ATPase and sodium pump data may be explained if sodium leak influx is increased in aged myocardium. Fewer sodium pumping sites and enhanced ouabain sensitivity of the sodium pump seem to be responsible for the reduced tolerance of aged heart to digitalis-induced toxicity.

K-p-nitrophenylphosphatase activity, Na and K content, Na permeability and membrane lipid composition in rabbit myocardium after cholesterol rich diet.

The aim of the present study was to investigate the effects of a cholesterol-rich diet on membrane function and lipid composition in rabbit myocardium. The activity and the ouabain sensitivity of the K-p-nitrophenylphosphatase (K-pNPPase), a partial reaction of the Na, K-ATPase, were diminished after a cholesterol/oil or pure cholesterol diet. The content of cholesterol, cholesterol esters and of several classes of phospholipids was enhanced in microsomes. A causal relationship is assumed between cholesterol accumulation and a decrease in membrane fluidity as well as in Na, K-ATPase activity. The intracellular Na content and the Na-Li-exchange rate were higher after the cholesterol diet. The increase in the Na content is supposed to be induced by a lower Na transport and a higher Na permeability. An enhanced Ca flux via the sarcolemma could be the consequence.

Interaction of Ca 2+ with (Na + + K +)-ATPase: Properties of the Ca 2+-stimulated phosphatase activity

Ca 2+ inhibited the Mg 2+-dependent and K +-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na + + K +)-ATPase. In the absence of K +, however, a Mg 2+-dependent and Ca 2+-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K +-stimulated phosphatase. The Ca 2+-stimulated phosphatase, like the K +-stimulated activity, was inhibited by either ouabain or Na + or ATP. Ouabain sensitivity was decreased with increase in Ca 2+, but the K 0.5 values of the inhibitory effects of Na + and ATP were independent of Ca 2+ concentration. Optimal pH was 7.0 for Ca 2+-stimulated activity, and 7.8–8.2 for the K +-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca 2+-stimulated phosphatase is catalyzed by (Na + + K +)-ATPase; (b) there is a site of Ca 2+ action different from the site at which Ca 2+ inhibits in competition with Mg 2+; and (c) Ca 2+ stimulation can not be explained easily by the action of Ca 2+ at either the Na + site or the K + site.

Ethanol and temperature effects on five membrane bound enzymes

The effects of ethanol on the activities of five membrane bound enzymes were determined using a crude membrane preparation obtained from cortex of long-sleep (LS) and short-sleep (SS) mice. These two mouse lines were selectively bred for differences in duration of ethanol-induced sleep time. The enzymes studied were two forms of NaK-ATPase, Mg-ATPase, 5′nucleotidase, and acetylcholinesterase. Arrhenius plots of the ethanol-temperature-enzyme activity studies indicate specificity in ethanol's actions. NaK-ATPase activity consists of two enzymes which were distinguished by sensitivity to ouabain. The Arrhenius plot of the high ouabain sensitivity enzyme (low K i) exhibited a transition temperature which was reduced twice as much by ethanol in LS membranes as in SS membranes. Ethanol did not affect the transition temperature of the high K i NaK-ATPase but the control (no ethanol) transition temperature was 2.7° higher in SS membranes. Arrhenius plots of Mg-ATPase activity did not exhibit a transition temperature and ethanol did not alter enzyme activity. Ethanol did not alter the transition temperatures of 5′nucleotidase or acetylcholinesterase but the control transition temperature for acetylcholinesterase was 2.3° higher in SS membranes. These results indicate specificity in ethanol's actions on membranes and that inhibition of the lipid-enzyme interactions for the low K i NaK-ATPase is correlated with the difference in sensitivity to ethanol seen between the LS and SS mice.

<b>Purification and characterization of Na<sup>+</sup>,K<sup>+</sup>-ATPase from the kidney of spontaneously hypertensive and Wistar Kyoto </b><b>rats </b>

Na+,K+-ATPase (EC 3.6.1.3) was purified to homogeneity from the kidney of spontaneously hypertensive rats (SHR), and their normotensive control rats (Wistar Kyoto rats; WKY. Purity of the enzyme was confirmed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, in combination with immunoblotting using anti-dog Na+,K+-ATPase antibody. The enzyme purified from SHR and WKY did not differ in respect to the molecular weight, subunit composition, specific activity, ouabain sensitivity, requirements for Na+ and K+ (and also for Mg2+ and Ca2+), and to the extent of heat denaturation. The results indicate that the difference in Na+,K+-pump so far reported between SHR and WKY is not attributable to any difference in biochemical properties of the purified Na+,K+-ATPase.

Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart.

Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.

A carrier enzyme basis for ammonium excretion in teleost gill. NH +4-stimulated Na-dependent ATPase activity in Opsanus beta

1. 1. Branchial Na + K +-ATPase specific activity is some 20% greater in hyposahne adapted Opsanus beta than in SW specimens. 2. 2. Ouabain insensitive ATPase (Mg 2+-ATPase) specific activities were similar, while whole body-activity differences in low salinity and SW adapted fish could be accounted for by the 30% difference in extractable gill protein. 3. 3. NH 4 + ion was 15% more effective at dephosphorylation of the microsomal Na-dependent phosphoenzyme than either Rb + or K +. and revealed a maximal ATPase affinity ( K m = 0.2 mM) within the physiological range of blood [K +]. 4. 4. Similar properties as pH optima, ATP and Mg 2+ K m 's. ouabain sensitivity, percent recoveries and subcell distribution indicated that the NH + 4-stimulation acts through the Na + K + -ATPase carrier enzyme and may be resonsible for the Na +/NH + 4 exchange in Opsanu beta.

Origins of the different sensitivities of [formula omitted] adenosinetriphosphatase preparations to ouabain

1. 1. Properties of the Na +,K +-ATPase preparations from rat and dog kidney medullae were compared. 2. 2. The two enzymes, with a 1000-fold difference in ouabain sensitivities, had similar subunit compositions and similar K 0.5 values and Hill coefficients for substrates and activators of several catalytic activities; suggesting that the structural differences of the two are limited to the ouabain binding domains. 3. 3. Experiments on the interactions of ouabain, Pi, and Mg 2+ with the enzymes showed that the two enzymes differed (a) in their inherent affinities for ouabain; and (b) in that Mg 2+ binding increased affinity for ouabain to a greater extent in the dog enzyme than in the rat enzyme.

Potassium-induced vascular relaxation in two kidney-one clip, renal hypertensive rats.

This study was designed to characterize potassium-induced relaxation in vascular smooth muscle in two kidney-one clip (2K-1C), renal hypertensive rats. Potassium-induced relaxation was evaluated in the isolated tail artery and in the isolated pump perfused renal vasculature. Both preparations relaxed in response to potassium after contraction induced by norepinephrine in potassium-free solution. Arterial preparations from hypertensive rats showed greater relaxation than did those from normotensive rats. Potassium-induced relaxation in tail arteries from hypertensive rats was more sensitive to ouabain inhibition than those from normotensive rats; the renal vasculature of hypertensive rats did not differ from controls with respect to ouabain sensitivity. Relaxation in response to potassium in isolated tail artery segments varied with the: 1. length of incubation in potassium-free solution; 2. concentration of added potassium; and 3. concentration of norepinephrine added during the potassium-free interval. The amplitude of potassium relaxation is believed to be a functional measure of the electrogenic sodium pump. These experiments support the hypothesis that vascular smooth muscle from 2K-1C renal hypertensive rats has increased electrogenic sodium pump activity, in vitro.

Characterization of a β-actinin-like protein in purified non-muscle cell membranes. Its activity on [formula omitted

Treatment by EDTA of purified plasma membranes from MF 2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of ( Na + + K +)- ATPase to ouabain. When added back with Ca 2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of M r 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca 2+ it was able to restore the original sensitivity to ouabain of ( Na + + K +)- ATPase from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca 2+ the same quantitative biological activity on ouabain sensitivity of the ( Na + + K +)- ATPase . A possible interaction between β-actinin, calmodulin and membrane-bound ( Na + + K +)- ATPase is discussed.

Cyclic AMP stimulation of Na-K pump activity in quiescent swiss 3T3 cells.

Recently, we have found that an increase in the intracellular level of cAMP acts as a mitogenic signal for Swiss 3T3 cells (Rozengurt et al., Proc. Natl. Acad, Sci. USA, 78:4392, 1981). The results presented in this paper demonstrate that addition of cAMP-elevating agents to confluent and quiescent cultures of Swiss 3T# causes a marked increase in the rate of 86Rb+ uptake but has no effect on the rate of cation efflux. The stimulation of ion uptake is mediated by the Na-K pump as shown by the ouabain sensitivity of the 86Rb+ fluxes. The increase in Na-K pump activity occurs whether cAMP is generated endogenously by stimulation of adenylate cyclase activity by cholera toxin, adenosine agonists, or PGE1 or added exogenously as 8BrcAMP. The stimulatory effect of these compounds on 86Rb+ uptake is potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity. Cholera toxin stimulates the Na-K pump in a dose-dependent manner; half-maximal effect is achieved at 0.7 ng/ml. The stimulation of ouabain-sensitive 86Rb+ uptake by cAMP-elevating agents reaches a maximum after 2-3 h of incubation. This contrasts with the rapid (within minutes) stimulation of the Na-K pump caused by serum and other mitogenic agents. Further, cAMP-elevating agents fail to increase Na+ influx into 3T3 cells whereas serum causes a marked increase in Na+ influx, under identical experimental conditions. These findings suggest that the stimulation of Na-K pump activity caused by increased cAMP levels contrasts mechanistically with the rapid control of pump activity by serum which is primarily mediated by increased Na+ entry into the cells.

Effects of butyrate on ouabain-sensitive respiration of hamster brown adipocytes.

Brown adipose tissue is an important site of cold-induced nonshivering thermogenesis in many mammals. The plasma membrane-bound Na+-K+-ATPase has been shown to be significantly involved in this thermogenesis although its exact role is unknown at present. Evidence that coupling of oxidative phosphorylation to electron transport may become loosened during thermogenesis has prompted an investigation of potential roles of the Na+-K+ pump that would be compatible with altered respiratory coupling. One such role is that of modulating norepinephrine (NE)-induced lipolysis and hence provision of free fatty acids to the mitochondria. Under such conditions, inhibition of the pump would reduce NE-induced respiration by limiting substrate availability. If, in fact, the primary role of the pump in NE-induced thermogenesis is to facilitate substrate availability, provision of exogenous substrate should bypass this involvement and ameliorate the ouabain inhibition of respiration. In the present study, this possibility was examined by determining the effect of an exogenous substrate, butyrate, on the contribution of the Na+-K+ pump to NE-stimulated respiration of isolated hamster brown adipocytes. Although exogenous butyrate was able to serve as a substrate for brown adipocyte respiration, its presence had no significant effect on the ouabain sensitivity of NE-induced rates of oxygen consumption. That is, ouabain (1 mM) inhibited the NE-evoked thermogenesis of the adipocytes by 77.7 +/- 6.5% in the absence of butyrate (2 mM) and by 73.4 +/- 9.9% in its presence. It appears, therefore, that the contribution of the Na+-K+ membrane pump to brown fat thermogenesis does not simply reflect modulation of NE-evoked lipolysis.

Ouabain sensitivity of Rb+ uptake in canine Purkinje fibre and ventricular muscle.

Ouabain inhibitable active Rb+ uptake was compared in canine Purkinje fibre and the left ventricular muscle to define the possible difference in electrophysiological sensitivity to ouabain in the two tissues. Ouabain inhibitable, active uptake of Rb+ was calculated from the difference between total Rb+ uptake assayed in the absence of ouabain and nonspecific Rb+ accumulation assayed in the presence of 10(-4) mol/l ouabain. A similar degree of active Rb+ uptake was inhibited by ouabain in both Purkinje fibre and the left ventricular muscle from the same animal, even though the Purkinje fibre took up more 86Rb+ than the ventricle. The specific tissue binding of ouabain in vitro was also not significantly different in the functionally different two tissues. It is concluded that there is no differential ouabain sensitivity of Rb+ uptake in canine Purkinje fibre and ventricular muscle.