Affinity For Ouabain Research Articles

Substitution of transmembrane residues with hydrogen-bonding potential in the alpha subunit of Na,K-ATPase reveals alterations in ouabain sensitivity.

The role of H-bonding amino acids as determinants of ouabain affinity in the Na,K-ATPase was examined. Site-directed mutagenesis was used to substitute 21 conserved amino acid residues in the sheep alpha-subunit transmembrane regions. The amino acids were changed from residues which possess side chains capable of forming H-bonds with specific cardiac glycoside moieties such as the lactone ring or sugar(s) to residues unable to participate in H-bonding. The effect of each of these amino acid replacements on the affinity of the Na,K-ATPase for ouabain was initially assessed by screening the altered enzymes for the ability to confer ouabain resistance when expressed in otherwise sensitive HeLa cells. Three of the substitutions (Tyr-108 to Ala, Cys-104 to Ala, and Cys-104 to Phe) were able to confer resistance to the normally sensitive HeLa cells. Stable cell lines, each expressing one of the altered enzymes, were further characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. Cell lines expressing the alpha 1-isoform substitution Y108A, C104A, or C104F contained a Na,K-ATPase activity which gave an I50 value for enzyme inhibition 9-, 6-, and 150-fold greater, respectively, than the endogeneous HeLa or sheep enzyme. These data show that Tyr-108 and Cys-104 of the alpha subunit are determinants of ouabain affinity. Cys-104 has also been shown to be a determinant of ouabain sensitivity in Xenopus laevis [Canessa, C. M., Horisberger, J.-D., Louvard, D., & Rossier, B. C. (1992) EMBO J. 11, 1681-1687].(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in retinal Na+, K(+)-ATPase in diabetes: streptozotocin-induced and Zucker diabetic fatty rats.

The temporal pattern of changes in the specific activities of retinal Na+, K(+)-ATPase (Na, K-ATPase) and Mg(2+)-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1, 2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1, 2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 10(3) M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10(-9) to 10(-3) M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the alpha 3-high ouabain affinity or the alpha 1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the alpha 3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the alpha 3 isozyme. For example, after four months of diabetes, the mean +/- SEM IC50 values were 12 +/- 3 nM in the STZ rats and 48 +/- 6 nM in the age-matched controls and 19 +/- 3 nM in the ZDF rats and 30 +/- 4 nM in the age-matched controls. In contrast, the activity of the alpha 1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50 values were unchanged. Moreover, the Hill coefficient for the alpha 3 isozyme was decreased in both diabetic groups while it was unchanged for the alpha 1 isozyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of dietary α-linolenic acid on functional characteristic of Na +/K +-ATPase isoenzymes in whole brain membranes of weaned rats

The influence of dietary fatty acids on Na + sensitivity and ouabain affinity of Na +/K +-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of α-linolenic acid (sunflower oil diet) or rich in α-linolenic acid (soya oil diet). The ( n − 3) deficiency induced by the sunflower oil diet led to an increase in the ( n − 6)/( n − 3) molar ratio in whole brain membranes. Na +/K +-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain. In rats fed sunflower oil diet, the ouabain titration displayed three inhibitory processes with markedly different affinities: low affinity (α1); high affinity (α2); and very high affinity (α3). Membranes of rats fed soya oil diet exhibited only two inhibitory processes, i.e., low affinity (likely α1 + α2) and high affinity (likely α2 + α3) with the low affinity form intermediate between the sunflower α and α2 forms, and the high affinity form intermediate between the sunflower α2 and α3 forms. In fact, the Na + response shows that the three isoenzymes have different Na + sensitivities. Regardless of the diet, α1 has a similar Na + sensitivity (less than 1 mM), whilst α2 and α3 are more sensitive in soya oil membranes compared to sunflower oil membranes (5.1 vs. 7.2 mM and about 11 vs. 22.5 mM, respectively). Thus, sodium appears to be a better criterion of heterogeneity than ouabain.

Heterogeneous Na+ sensitivity of Na+,K(+)-ATPase isoenzymes in whole brain membranes.

The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha 1, exhibited high Na+ sensitivity [Ka of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13]; the ouabain high-affinity isoform, alpha 2, had two Na+ sensitivities, a high (Ka of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (Ka of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha 3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na K - ATPase activity was 65.9 ± 18.7 and 38.6 ± 22.8 ( P < 0.001) nmol/mg protein × min for control and high-expression transfected cells, respectively. Consistent with this observation, [ 3H] ouabain binding to whole cells was significantly decreased from 3.7 ± 0.3 to 2.0 ± 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na +, K +-ATPase protein; in contrast, the activities of 5′-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na K - ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na K - ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.

Inhibition of α2/α3 sodium pump isoforms potentiates glutamate neurotoxicity

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na +, K +-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (α1) and high (α2/α3) affinity for ouabain, we were able to inhibit selectively the α2 (principally glial) and α3 (neuronal) catalytic subunits without affecting the α1 isoform. Brief exposures (5–60 min) to high ouabain concentrations (1–10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the α2/α3 isoforms (by 1 μM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [ 3H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reduction in α2/α3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude glutamate excitotoxicity. Since the distribution of NaP is highly heterogenous in the nervous system, with similar cell types varying greatly in isoform expression, constitutive levels of this isoenzyme could constitute a major factor in the survival of stimulated neurons. Further, factors which directly affect pump activity, such as activation of protein kinase A, may modulate excitotoxicity.

Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding.

The Na,K-stimulated ATPase is inhibited by extracellular cardiac glycosides, which bind to the enzyme's alpha subunit. We used a monoclonal antibody, VG4, as a probe of the extracellular surface. The antibody was specific for Na,K-ATPase and bound to intact cells. The epitope was mapped to the first extracellular loop (H1-H2) of alpha, using a combination of techniques including trypsinolysis, N-terminal sequence of a fragment containing the determinant, and analysis of the effects of species-specific sequence differences. The antibody inhibited Na,K-ATPase activity under certain circumstances, indicating that the H1-H2 loop participates in conformational changes that are transmitted to the active site. Mutations in the H1-H2 loop have been shown by others to affect ouabain affinity. Ouabain and the antibody acted synergistically to inhibit the enzyme, which seemingly supported the hypothesis that the H1-H2 loop is an essential part of the cardiac glycoside binding site. Direct measurements of the binding of [3H]ouabain, however, indicated that VG4 enhanced rather than inhibited binding, presumably by promoting favorable conformation changes. The data suggest the possibility that the cardiac glycoside binding site may be intramembrane rather than extracellular.

The A2 isoform of rat Na +,K +-adenosine triphosphatase is active and exhibits high ouabain affinity when expressed in transfected fibroblasts

The α isoforms of the Na +,K +-ATPase (Na + pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly characterized the ouabain sensitivity of the rat A2 (α2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein. 86Rb + flux studies confirm that A2 is Functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.

3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.

The cardiac glycoside ouabain inhibits Na,K-ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild-type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K-pump in Xenopus oocytes injected with wild-type and mutated alpha 1 and wild-type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K-ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000-fold compared with wild-type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.

Sodium-independent effect of aldosterone on initial rate of ouabain binding in A6 cells.

To estimate the early effect of aldosterone on the number of active Na(+)-K+ pumps at the basolateral membrane of amphibian tight epithelia, we have measured the initial rate (at 4 min) of [3H]ouabain binding to the basolateral membrane of intact monolayers of A6 cells grown on permeable supports. Within 3 h, aldosterone induced a threefold increase of the Na transport and, simultaneously, a twofold increase of the binding rate of ouabain. Because the affinity of ouabain, estimated either by equilibrium binding studies or inhibition kinetics, was not modified by aldosterone, the effect on the initial rate of ouabain binding was due to an increase in the number of binding sites. This effect on ouabain binding was not prevented by 10 microM amiloride, which reduced the transepithelial sodium transport below control level. By contrast, the effect of aldosterone on ouabain binding was abolished by cycloheximide (5 micrograms/ml) or actinomycin D (2 micrograms/ml), doses which inhibited the aldosterone-dependent sodium transport response. These data suggest that aldosterone elicits an early, sodium-independent, protein synthesis-dependent increase in the expression of active Na(+)-K(+)-ATPase molecules.

High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function.

Na,K-ATPase of cultured bovine lens epithelial cells: H 2O 2 effects

Na,K-ATPase function was studied in cultured bovine lens epithelial cells under confluent and non-confluent conditions. The affinity of the Na,K-ATPase for the cardiac glycoside, ouabain, differs between the confluent and non-confluent cultures. The confluent cells have a higher affinity for ouabain than do the non-confluent cells. The ouabain affinity of the confluent cells is similar to that for the Na,K-ATPase isolated from the bovine axolemma and the bovine lens cortex. The ouabain affinity of the non-confluent cells is similar to that for the Na,K-ATPase of the renal medulla and bovine lens epithelium. Similar results are not found with confluent and non-confluent MDCK cells. H 2O 2 treatment of confluent and non-confluent lens epithelial cell cultures has differing effects on the Na,K-ATPase function. In the confluent cell preparations, H 2O 2 affects K +-dependent dephosphorylation of the intermediate phosphoenzyme. In the non-confluent preparations, H 2O 2 appears to inhibit K +-occlusion.

Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex

The effect of the pineal gland on the ouabain high-affinity binding sites ( K d=3.1±0.4 nM, B max=246.4±18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased B max (940.7±42.8 fmol/mg protein) and K d (7.6±1.5 nM) while melatonin injection (100 μg/kg b.wt.) counteracted these effects, restoring kinetic parameters ( K d=1.9±0.05 nM; B max=262.2±29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC 50 values.

Brain Na +,K +-ATPase regulation in vivo: reduction in activity and response to sodium by intracerebroventricular tetrodotoxin

We investigated the effects of intracerebroventricular infusion of tetrodotoxin on activity and function of brain Na +,K +-ATPase. Infusion of 1 or 3 μg/h for 2, 4 or 7 days by osmotic minipump reduced the number of Na +,K +-ATPase sites as measured by ouabain binding in cerebral cortex. Tetrodotoxin infusions substantially reduced the functional transport capacity of Na +,K +-ATPase, measured by the maximal increase in synaptoneurosomal 86Rb + uptake in the presence of monensin. The effects were maximal at 4 days, with a possible partial recovery of activity at 7 days. Results of ouabain inhibition curves suggested that the effect of tetrodotoxin was not specific for enzyme with high or low affinity for ouabain.

High affinity ouabain binding of the alpha 2 isoform of Na,K-ATPase in transfected mammalian cells

High affinity ouabain binding of the alpha 2 isoform of Na,K-ATPase in transfected mammalian cells

Effects of acute and chronic uremia on active cation transport in rat myocardium

As abnormalities of active cation transport could contribute to the genesis of uremic cardiomyopathy, we investigated myocardial sodium pump function in rats with acute renal failure (ARF) and with a model of experimental chronic renal failure (CRF) that has metabolic similarities to advanced chronic uremia in humans. CRF rats were hypertensive and had left ventricular hypertrophy (33% higher heart:body weight ratio; P less than 0.01) at four weeks compared to pair-fed sham-operated rats. Importantly, both ouabain- and furosemide-sensitive 86Rb uptake rates were unchanged in left ventricular myocardial slices from CRF, and the intracellular sodium concentration was not different from that of control rats even though skeletal muscle sodium was increased, as we found previously (J Clin Invest 81:1197, 1988). Insulin-stimulated, ouabain-sensitive 86Rb influx was also preserved. There also were no abnormalities in myocardium cation transport in rats with ARF. However, [3H]ouabain binding was decreased 45% in CRF rats (P less than 0.01); it was unchanged in acute uremia. Decreased ouabain binding in chronic uremia was due entirely to fewer low affinity [3H]ouabain binding sites (the binding affinity for ouabain was unaffected). We conclude that in chronic, (but not acute) renal failure, sodium pump number is reduced in myocardium but intracellular sodium is unchanged and active cation flux rates are maintained. These results emphasize that in rats with chronic uremia, intracellular sodium homeostasis is preserved in myocardium, despite the presence of marked abnormalities of active cation transport in skeletal muscle that are characteristic of chronic uremia.

A comparison of the affinities of rat (Na + + K +)-ATPase isozymes for cardioactive steroids, role of lactone ring, sugar moiety and KCl concentration

Binding experiments at equilibrium were performed to study pharmacological properties of isozymes of (Na + + K +)-ATPase from rat tissues. Experiments were performed on brain ( α 3 isozyme), kidney ( α 1 isozyme) and heart microsomes ( α 1 and α 2 isozymes). Affinity of series of ouabain and digoxin derivatives was studied in competition experiments. It was observed that: (i) ouabain and digoxin had higher affinity (P < 0.01) for α 3 isozyme ( K d of 0.071 ± 0.004 and 0.066 ± 0.001 μM, respectively) than for α 1 isozyme ( K d of 15.9 ± 0.8 and 1.78 ± 0.46 μM, respectively) and α 2 isozyme ( K d of 0.26 ± 0.0 and 0.15 ± 0.06 μM, respectively); (ii) saturation of the C 20C 22 bond on the C 17β lactone ring present in ouabain and digoxin markedly decreased the drug affinity for all isozymes (P < 0.01); and (iii) suppression of the C 3β osidic chain decreased the affinity of ouabain and digoxin to a higher extent for α 2 and α 3 than for α 1 (P < 0.01). The presence of 10 mM KCl in the incubation medium decreased ouabain affinity for the α 1 isozyme to a much higher extent ( K d increase of about 20-fold) than for the other isozymes ( K d increase of about 2-fold). The results show that the isozymes of (Na + + K +)-ATPase from rat tissue are differently sensitive to changes in the substituents of the cardioactive steroids and to the presence of 10 mM KCl.