All human Na+-K+-ATPase alpha -subunit isoforms have a similar affinity for cardiac glycosides

Jiangnan Wang1, Jeffrey B. Velotta1, Alicia A. McDonough1, and Robert A. Farley1,2

Departments of 1 Physiology and Biophysics and 2 Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Three alpha -subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (Kd) for all samples calculated using the single-site model was 18 ± 6 nM (mean ± SD). No difference in Kd was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only ~50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha -isoforms are similar to each other, although calculated Kd values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha -subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

sodium pump; ouabain binding


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC GLYCOSIDES such as digoxin are positive inotropic agents that specifically inhibit the activity of the sodium pump (Na+-K+-ATPase). In cardiac myocytes, a decrease in cellular sodium pump activity reduces Ca2+ efflux through the sodium gradient-coupled Na+/Ca2+ exchanger, leading to increased sarcoplasmic reticulum filling. Upon stimulation, more sarcoplasmic reticulum calcium can be released, leading to increased cytoplasmic Ca2+ concentrations and cardiac contractility, compared with untreated hearts. Because of this positive inotropic effect, cardiac glycosides are widely used in the treatment of congestive heart failure. The therapeutic plasma concentration range of cardiac glycosides is very narrow (1.3-2.6 nM), and concentrations above this range lead to toxic reactions, including cardiac arrhythmias. Sensitivity to cardiac glycosides increases as the heart fails and with aging, resulting in toxic reactions even within the nominal therapeutic concentration range (1, 10). Two mechanisms for the different responses to cardiac glycosides have been proposed. In one mechanism, the sodium pump isoforms that are present in human heart have different affinities for the drugs, and the therapeutic responses and toxic responses are due to inhibition of subsets of pumps with different affinities for the drugs (reviewed in Ref. 20). In the second mechanism, the total cellular pump abundance decreases both in heart failure and with age, making cells more sensitive to the effects of pump inhibition (1).

The affinity of the sodium pump for cardiac glycosides is determined by the catalytic alpha -subunit, and there are three different isoforms of the alpha -subunit of Na+-K+-ATPase, alpha 1, alpha 2, and alpha 3, in human heart (18, 19, 21, 23, 24). A second subunit, beta , is required for sodium pump function, but beta  appears to play a limited role, if any, in cardiac glycoside binding to the pump. Most studies of cardiac glycoside binding affinity in human heart have been done by measuring either [3H]ouabain equilibrium binding or [3H]ouabain association and dissociation kinetics in the presence or absence of K+. Some reports have described multiple ouabain receptor populations in membrane fractions from human hearts consistent with the existence of sodium pumps with different sensitivities to cardiac glycosides. Equilibrium dissociation constants (Kd) of 2.5-4.8 nM and 17 nM (5) or 20 nM and 170 nM (6) measured in the absence of K+ have been reported. Other studies have reported only a single population of high-affinity sites with Kd values of 11 nM (14), 3.7 nM (3), or 27 nM (16). Reasons for these different results are not obvious, but differences in methodology may be involved (20). Shamraj et al. (18) measured dissociation kinetics of [3H]ouabain from Na+-K+-ATPase complexes in human heart and detected two populations with different dissociation rate constants (k1 = 0.05/min, k2 = 0.01/min). The slower dissociation rate constant was the same as the dissociation rate constant for ouabain from human kidney membranes, suggesting that the alpha 1-isoform of human Na+-K+-ATPase has a higher affinity for cardiac glycosides than the alpha 2- or alpha 3-isoforms. This conclusion differs from the pattern in rodents, where the alpha 1-isoform has a lower affinity (Kd near 10-5 M) (21) than that of alpha 2- or alpha 3-isoforms, which have Kd values of ~10-7 and 10-9 M, respectively (15). Understanding whether the human Na+-K+-ATPase isoforms have significantly distinct affinities for cardiac glycosides has particular significance for the development of new inotropic drugs. If the Na+-K+- ATPase alpha -isoforms do not have significantly different affinities, then the likelihood of developing drugs that would retain beneficial inotropic effects while reducing toxic responses is small.

Recently, Crambert et al. (4) expressed human alpha - and beta -isoform combinations in Xenopus oocytes and measured Kd values in the absence of K+. The alpha 1beta 1 and alpha 3beta 1 complexes had Kd values of ~5 nM, and the alpha 2beta 1 complex had a Kd of ~22 nM. However, it is not known whether these Kd values are influenced by the membrane composition of the Xenopus oocyte expression system. To determine Kd values of the human sodium pump isoforms in situ, we have measured the equilibrium binding of ouabain to a panel of human tissues and cell lines that express one or multiple isoforms. Human kidney and epithelial cell lines contain only the Na+-K+-ATPase alpha 1-isoform, human skeletal muscle contains primarily alpha 2- and some alpha 1-isoforms, and human heart and brain express alpha 1-, alpha 2-, and alpha 3-isoforms. In addition, the association and dissociation rate constants for the interaction of ouabain with human cardiac tissue were measured. To determine whether the isoforms had significantly different affinities for ouabain, we fit the data with equations for single vs. multiple populations of binding sites, and the quality of the fits was assessed with an F statistic. The analysis indicated that ouabain binding to all of the human tissue and cell line samples can be described equally well by either model. Thus it appears that all three Na+-K+-ATPase alpha -subunit isoforms in their native membranes bind ouabain with approximately the same affinity. From this result it seems unlikely that the beneficial and harmful effects of cardiac glycosides in humans arise because of differences in affinity of the sodium pump isoforms for the drugs. These results support a model in which the decreased total cellular sodium pump abundance in heart failure is responsible for rendering cells more sensitive to cardiac glycosides.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue samples. Nonfailing human hearts (n = 10) were obtained from organ donors after brain death caused by traumatic injury; failed human hearts (n = 2) were obtained during cardiac transplantation. All human heart samples were obtained from R. H. G. Schwinger (University of Cologne, Germany). The cardioplegic solution was a modified Bretschneider solution containing (in mM) 15 NaCl, 10 KCl, 4 MgCl2, 180 histidine-HCl, 2 tryptophan, 30 mannitol, and 1 potassium dihydrogen oxoglutarate. Samples were dissected and frozen at -80° pending analysis. Samples of human brains (n = 3) and kidneys (n = 2) were obtained in an anonymous fashion at University of Southern California from ice-cold surgical pathology tissue that would otherwise have been discarded at surgery. Human muscle (n = 4) was obtained from the Cooperative Human Tissue Network, Western Division (Case Western Reserve University, OH). Human blood was obtained from hematologically normal adult volunteers. These procurement methods and investigations were approved by the local human subjects institutional review boards of both the University of Southern California and the University of Cologne.

Membrane preparation. Samples of left ventricular tissue and skeletal muscle (0.1-0.25 g) were rapidly thawed, dissected free of all visible fat and vessels, weighed, and then homogenized (1:20 wt/vol) with a Polytron (Brinkmann Instruments) at a setting of 5 for 2 min on ice in a solution of 5% sorbitol, 25 mM imidazole/histidine, pH 7.4, and 0.5 mM Na2EDTA containing the proteolytic enzyme inhibitors 1 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM 4-aminobenzamidine dihydrochloride. For preparation of membranes from human brain, kidney, and cultured cells, tissues were homogenized 1:10 (wt/vol) with a motor-driven glass-Teflon homogenizer for 1 min on ice in the same solution used to prepare cardiac membranes. Homogenates were spun at 6,000 g for 15 min at 4° to remove debris and unhomogenized tissue, and then the supernatants were spun at 100,000 g for 60 min. Pellets were suspended in 2-4 ml of homogenization buffer, and membranes were stored at -80° pending analysis. Human erythrocyte ghosts were prepared by hypotonic lysis in 5 mM sodium phosphate, pH 8. Protein was determined by Lowry method (11).

Immunoblot analysis. Immunoblot analysis was conducted as previously described (23). Membrane protein was resolved by SDS-PAGE, and gels were electrophoretically blotted onto Immobilon-P membrane. Blots were probed with one of the following antibodies: 464.6, a mouse monoclonal against alpha 1 (1:100) from M. Kashgarian (Yale University); McB2, a mouse monoclonal against alpha 2 (1:100) from K. Sweadner (Harvard University) (20); anti-TED, a rabbit polyclonal against alpha 3 (1:200) from T. Pressley (Texas Tech University). All blots were prepared and processed as previously described (23), and 125I-labeled protein A and autoradiography were employed for quantitation of antibody antigen complexes.

Equilibrium ouabain binding. Membrane protein (4 mg) was incubated at 37° in 50 mM Tris · HCl, 4 mM H3PO4, and 4 mM MgCl2, pH 7.4, with 0.2-1.0 nM [3H]ouabain (15-19.8 Ci/mmol) and increasing concentrations of nonradioactive ouabain (0-1 µM) for 60-120 min. Nonspecifically bound ouabain was estimated as [3H]ouabain binding in the presence of 100 µM nonradioactive ouabain. The reaction was stopped by incubation on ice for 15 min, and membranes were separated from buffer by vacuum filtration on 0.22-µm GSTF filters (Millipore). The filters were washed immediately two times with 3 ml of ice-cold water, and radioactivity was determined by scintillation counting. Data were analyzed by using a self-competition binding model (9) modified to include two populations of sites with different Kd values.

Association and dissociation kinetics. To obtain the [3H]ouabain dissociation rate constant, a suspension of membranes was preincubated for 60 min with 0.25 µM [3H]ouabain, and then at time 0, 100 µM nonradioactive ouabain was added to the reaction. At various times, 200-µl aliquots were removed for filtration to determine the amount of bound ouabain. The dissociation rate constant (kd) was obtained from the slope of the semilogarithmic plots of bound ouabain vs. time. To measure the rate of ouabain binding, 2.5 mg of membrane protein were added to 5 ml of 50 mM Tris · HCl, 4 mM H3PO4, 4 mM MgCl2, pH 7.4, and 0.25 µM [3H]ouabain at 37°. Aliquots of 200 µl were removed at times up to 20 min and rapidly filtered on Millipore GSTF 0.22-µm filters. Nonspecific binding was determined in the presence of 100 µM nonradioactive ouabain. The association rate constant (ka) was obtained as described by De Pover and Godfraind (5). Briefly, the observed rate constant (kobs) was obtained from the slope of a plot of log {[(B0 - Bt)/B0] × 100} vs. time, where B0 is the amount of ouabain bound at equilibrium and Bt is the amount of ouabain bound at each time t. The observed rate constant is related to the association rate constant by the equation 2.303 kobs = ka[O] + kd, where [O] is the concentration of [3H]ouabain.

Statistical analysis. Comparisons between single-site and two-site binding models were accomplished by using an F statistic (2). Data are reported in Tables 1 and 2 as means ± SD.

                              
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Table 1.   Equilibrium binding of [3H]ouabain to membrane fractions from human heart, brain, skeletal muscle, kidney, erythrocytes, HeLa cells, and Caco-2 cells


                              
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Table 2.   Association and dissociation rate constants for [3H]ouabain binding to human heart, brain, and kidney membranes

Materials. All chemicals were of reagent grade or higher. [3H]ouabain was obtained from NEN (Boston, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the tissue samples for analysis were obtained after surgery and pathology, the integrity of the sodium pumps in the membrane samples was assessed by immunoblot analysis before ouabain binding (23). This also allowed verification of the isoform distribution in the samples analyzed. Typical immunoblots are provided in Fig. 1, and the isoform distribution for each tissue and cell line is summarized in Table 1. The relative abundance of alpha 1 in the samples is brain approx  kidney > heart > skeletal muscle approx  Caco-2 approx  HeLa cells > red blood cells (RBC). The relative alpha 2 abundance is brain approx  skeletal muscle > heart, and the alpha 3 relative abundance is brain > heart. Comparisons of the abundance of different isoforms within each tissue are not possible from these immunoblots because of the differing affinities of the antibody probes for the proteins.


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Fig. 1.   Immunoblot analysis of human membrane fractions. Three sets of membrane samples were resolved separately by SDS-PAGE, blotted onto Immobilon-P, and then probed with antisera to either alpha 1, alpha 2, or alpha 3. The source and amount of membrane protein analyzed (in µg) are indicated (RBC, red blood cells; sk musc, skeletal muscle). Within each isoform assay, the film exposure time was the same.

To obtain a comprehensive survey of [3H]ouabain binding to the three isoforms of the Na+-K+-ATPase, we measured equilibrium binding in membrane fractions from human tissues and cell lines that express alpha 1 alone (kidney, erythrocytes, Caco-2 cells, and HeLa cells), predominantly alpha 2 with alpha 1 (skeletal muscle), or combinations of alpha 1 with alpha 2 and alpha 3 (brain and heart). The results of the binding measurements are summarized in Table 1, which shows Kd values calculated from a single-site model in which all alpha -isoforms are considered to have equal affinity for ouabain. The results for human heart membranes have been separated into those obtained from nonfailing and failing hearts. Previous assays of analogous samples have shown no difference in Kd values between failing and nonfailing samples (17), and the data in Table 1 confirm this result. The Kd values for equilibrium ouabain binding to human samples that express only alpha 1 are statistically indistinguishable and are in agreement with published values for ouabain binding to HeLa cells (7) and erythrocytes (8). Cultured Caco-2 cells also express only Na+-K+-ATPase alpha 1-subunits, but the Kd value measured for these membranes was about twofold less than the value found for the other samples. As discussed below, a two-site model in which two populations of sites with different affinities are present was also tested; however, this model did not improve the quality of the fit to the binding data. Preincubation of membranes from human heart or skeletal muscle with 0.6 mg/ml sodium deoxycholate did not increase the amount of ouabain bound, and this treatment increased binding to human brain samples only about 1.6-fold. The absence of large increases in ouabain binding after incubation with detergent indicates that the membrane samples consisted predominantly of leaky vesicles in which the reagents had access to both sides of the membrane.

Because of conflicting reports concerning the presence of one or multiple population(s) of ouabain binding sites in human heart, the equilibrium binding of ouabain to each sample was evaluated with both a single-site model and a two-site model (9). The results from each fit were compared using an F statistic (2), which compares models with different numbers of parameters and degrees of freedom. The F statistic tests determine whether the weighted sums of squared deviations have been sufficiently reduced to justify fitting the data with additional parameters. The equilibrium binding data were fit with both models, and the quality of the fits was compared. Figure 2 shows typical binding curves obtained from several different samples. In these experiments, increasing concentrations of nonradioactive ouabain were added to a fixed concentration of [3H]ouabain, and the amount of radioactivity bound to the membranes was determined by scintillation counting. The concentration of [3H]ouabain was usually <1 nM to avoid saturation of binding sites with low Kd values. Because of competition between the radiolabeled and the unlabeled ouabain, the number of disintegrations per minute (dpm bound) decreases at increasing concentrations of unlabeled ouabain, although the total amount of ouabain bound increases. The high background seen in the human RBC ghost sample is due to the large amount of protein in the sample (150 µg membrane protein) needed to measure binding to the small number of pumps found in the erythrocyte membrane. The lines drawn through the data points are the fits to a single-site binding model. In most experiments, the use of the more complex two-site model was not justified, as determined by the F statistic. The only cases where the two-site model was a better fit were two measurements of the same sample of nonfailing left ventricle and one of two measurements of the same brain sample. Because most of the samples for these tissues were best fit by a single-site model, this model was accepted as correct, and the Kd values from the fit to a single population of binding sites are summarized in Table 1.


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Fig. 2.   Equilibrium ouabain binding to membrane fractions from human tissue and cultured cells. Membranes from human tissues and cultured cells were equilibrated with [3H]ouabain, and binding to Na+-K+-ATPase was measured as described in MATERIALS AND METHODS. The lines through the data points are the nonlinear least-squares fit of the data by a self-competitive single-site model (9).

Bmax values for maximum ouabain binding are also summarized in Table 1. These values should be considered only approximate, however, because of possible variability in the collection and handling of the human tissue. Samples were collected at different times, may not have been handled identically, and were stored for different periods of time before analysis. These factors are not expected to have any effect on the Kd value for ouabain binding, but they could affect the abundance of functional Na+-K+-ATPase in each tissue sample. Despite this caveat, the relative abundance of ouabain binding sites found for membrane preparations from each tissue is consistent with the known or predicted abundance of Na+-K+-ATPase in these tissues. Brain membranes have the greatest abundance of the enzyme, roughly twice the amount found in kidney membranes. The cell lines derived from the colon (Caco-2) or cervical (HeLa) epithelial cancers have about half the number of pumps per milligram of membrane protein as kidney tissue, and cardiac tissue samples contain ~5-10% of the sodium pumps that brain does. Density of pump sites in failing hearts was 50% lower compared with nonfailing heart samples, similar to the 39% reduction previously reported by Schwinger et al. (17).

Estimates of equilibrium binding constants can also be obtained from measurements of association and dissociation rate constants. Thus the rates of ouabain binding and dissociation from human heart, brain, and kidney membranes were determined. Figure 3 shows the time course of [3H]ouabain binding to these membranes, and Fig. 4 shows the dissociation of [3H]ouabain. As shown in Fig. 3, ouabain binding to human heart, brain, and kidney membranes reached maximum levels by 10 min of incubation at 37°. Figure 3, inset, shows a semilogarithmic plot of the difference between amounts of ouabain bound at different times and the steady-state binding level. The straight lines obtained indicate that binding followed pseudo-first-order kinetics under these conditions. Half-maximum binding levels were reached in 60-90 s for all samples. The time course of dissociation of [3H]ouabain from human heart, brain, and kidney membranes (Fig. 4) could be fit equally well (r2 = 0.97) with a single-exponential decay or with two exponentials. The solid lines shown in Fig. 4 are fits to single-exponential equations, which are also shown next to the graph for each sample. The curvature in the fits to the dissociation data from heart and brain is due to the asymptotic approach of the lines to the fraction of ouabain that does not dissociate in these experiments. The association and dissociation rate constants were calculated as described in MATERIALS AND METHODS and are summarized in Table 2, along with the equilibrium dissociation constant Kd calculated from the rate constants and the percentage of total bound ouabain that does not dissociate. The results indicate that ouabain binding to this panel of human tissues is best characterized by a single population of binding sites, confirming the conclusions obtained from equilibrium binding measurements. The kinetic measurements suggest, however, that the affinity of the human tissues may be higher than the affinity calculated from the equilibrium measurements.


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Fig. 3.   Time course of ouabain binding to Na+-K+-ATPase in human heart, brain, and kidney membranes. Membranes from human heart (open circle ; n = 4), brain (; n = 2), and kidney (triangle ; n = 1) were incubated at 37° in 50 mM Tris · HCl, 4 mM H3PO4, 4 mM MgCl2, pH 7.4, and 0.25 µM [3H]ouabain. At the times shown, membranes were collected by rapid filtration. Inset: semilogarithmic plot of (B0 - B/B0) × 100 vs. time, where B is the amount of [3H]ouabain bound at each time point and B0 is the initial amount of bound ouabain.



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Fig. 4.   Time course of ouabain dissociation from Na+-K+-ATPase in human heart, brain, and kidney membranes. Membranes from human heart (n = 8), brain (n = 2 each in duplicate), and kidney (n = 1, assayed 9 times) were equilibrated at 37° with 0.25 µM [3H]ouabain, and aliquots were withdrawn for filtration at various times after addition of 100 µM nonradioactive ouabain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A systematic analysis of ouabain binding to human tissues and cultured cells was done to determine whether ouabain binding to different human Na+-K+-ATPase alpha -subunit isoforms is characterized by a single population of sites or by multiple populations with distinctly different affinities for the drug. The identities of the alpha -subunit isoforms present in each sample were confirmed with the use of subunit-specific antibodies, and a statistical test was used to compare a single-site binding model with a two-site binding model. Results from both equilibrium and kinetics data indicate that the different human alpha -subunit isoforms have very similar affinities for ouabain. In addition, it can be inferred that the different beta -subunit isoforms, as well as the presence or absence of the gamma -subunit, have little effect on ouabain binding because the different tissue and cell samples used for these measurements contained quite different isoform combinations. The mean dissociation constant for ouabain binding at 37° obtained from equilibrium measurements in the absence of K+ is 18 ± 6 nM (mean ± SD). The dissociation constant calculated from kinetic measurements, however, is ~2 nM. Although both of these values are consistent with published values of Kd between 4.6 and 22.0 nM for human isoforms expressed in Xenopus oocytes (4), the difference between them is statistically significant (P < 0.005).

The reasons why different Kd values are obtained by the two methods are not known, but the difference may be caused by a systematic overestimate of ouabain concentrations in the equilibrium binding assays. Ouabain concentrations were determined by using a published value of 18.8 mM-1 for the extinction coefficient at 220 nm (22). Nevertheless, if the ouabain solutions contained material that absorbed ultraviolet light at 220 nm but did not bind to the Na+-K+-ATPase, then the actual concentration of ouabain in the binding assays would have been lower than the calculated value, and the Kd values would have been overestimated. This would be a problem only in the equilibrium binding assays because the concentration of ouabain in the kinetic assays was at least 100 times higher than the Kd value. Alternatively, the kinetics measurements may have overestimated the dissociation rate constant or underestimated the association rate constant, resulting in artificially low Kd values. Consequently, although the results of these measurements clearly indicate that the different Na+-K+- ATPase alpha -subunit isoforms have similar Kd values for ouabain, the precise Kd values have been determined only within certain limits, which are discussed further below.

To answer the question as to whether the human Na+-K+-ATPase alpha -isoforms are characterized by a single Kd value or by multiple Kd values, we must be able to distinguish between receptor populations with different Kd values. Factors that would limit our ability to distinguish between populations with different Kd values are scatter in the data and the low abundance of isoforms characterized by Kd values significantly different from those of the higher abundance isoforms. In all tissues expressing multiple Na+-K+-ATPase alpha -subunit isoforms, each isoform was clearly visible on the Western blots, making it unlikely that contributions from any isoform to the binding data were missed. Scatter in the data is the most likely constraint on the precision of the measurements made during this investigation and may arise from variability in the tissue samples. A global analysis of the uncertainty in the mean Kd value reported in Table 1 indicates that the Kd values for the human alpha -isoforms have a 95% probability of being between 6 and 30 nM. For individual tissues and cell lines, the 95% confidence intervals all range between 5 and 37 nM. These results indicate that differences less than five- to eightfold in Kd values for different alpha -subunit isoforms would not have been detected in the experiments reported here.

Ouabain binding to human heart or brain has been characterized by multiple Kd values (5, 6) or by a single Kd value similar to the value obtained in this study (8, 16). In the experiments reported here, care was taken to keep the concentration of radiolabeled ouabain sufficiently low (generally <1 nM) that high-affinity sites could be detected by equilibrium binding. Membrane fractions were examined to avoid possible artifacts caused by nonequilibrium partitioning of ouabain into tissue samples. In addition, a panel of four different samples that contained only the alpha 1beta 1 Na+-K+-ATPase complexes were used as controls. In a very small number of samples, it was found that a binding model with two populations of binding sites with different affinities for ouabain fit the data better than a model with a single population of sites. Nevertheless, this was not consistently observed for any tissue. Binding models with either a single population of sites or with two populations of sites were fit to the data, and the resultant fits were compared with the use of the F statistic. This comparison was made to determine whether any improvement in the quality of the fit obtained using the more complicated model was justified, taking into consideration differences in the number of degrees of freedom in the two models. From this analysis it was clear that the simple model with a single population of binding sites was sufficient to characterize ouabain binding, even to tissues that contain multiple isoforms of the Na+-K+-ATPase alpha -subunit. This conclusion was reinforced by the results obtained from kinetics measurements of ouabain association and dissociation. For all tissues, the association rate constant was similar, near 5 × 106 M-1 · min-1, and the dissociation rate constant was also similar, around 10 × 10-3 · min-1. Although no evidence was obtained for a rapidly dissociating pool of ouabain from human heart (k = 0.05 min-1) (18), dissociation of ouabain from both brain and heart membranes was not complete (Table 2). This observation does not seem to be associated with the presence of alpha 2- and alpha 3-isoforms because the fraction of nondissociating ouabain is considerably smaller than the estimated 75% fraction of total Na+-K+-ATPase that is represented by these isoforms in rat brain (12). The relative abundance of alpha -isoforms in human brain is not known.

In this study, ouabain binding to isolated membranes was determined under conditions designed to optimize ouabain affinity, namely, in the absence of K+. Future efforts will be aimed at determining whether raising K+ to levels found in the plasma has a significant isoform-specific effect on ouabain binding affinity, either association or dissociation rates. The study of Crambert et al. (4) in Xenopus oocytes indicated that isoform-specific differences exist in the K+/ouabain antagonism: adding 5 mM K+ increased the Kd for alpha 1beta 1 three- to fourfold while increasing the Kd for alpha 2beta 1 and alpha 3beta 1 two- to threefold. If a significant isoform-specific difference in K+ antagonism in the human tissues is present, it may become evident as not only a change in Kd but also a better fit with the two-site model than with the one-site model.

As a result of these measurements, we conclude that the affinity of different Na+-K+-ATPase alpha -subunit isoforms for cardiac glycosides in human tissues and cell lines is very nearly the same. This conclusion is in agreement with results obtained from the heterologous expression of the pump in Xenopus oocytes and with several other studies of ouabain binding to different human tissues. One implication of this result is that both beneficial and harmful effects of cardiac glycosides in patients with congestive heart failure are due to drug binding to the same population of receptor sites. Because the affinity of each isoform for the drugs is the same, different responses in patients are likely to be due to different numbers of pump molecules in sensitive cells. Another implication of these results is that cardiac glycosides will bind with equal affinity to pumps expressed throughout the body, including the large pool of isoforms found in skeletal muscle (alpha 2, alpha 1). In patients with hypokalemia, large reductions in skeletal muscle alpha 2 sodium pump expression occur. This reduction could affect the amount of cardiac glycoside available to the heart during failure and may contribute to the seemingly random occurrence of toxicity associated with the use of cardiac glycosides (13).


    ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medical Sciences Grant GM-28673 (to R. A. Farley) and a Grant-in-Aid from the American Heart Association-Western States Affiliate (to A. A. McDonough).


    FOOTNOTES

Address for reprint requests and other correspondence: R. A. Farley, Dept. of Physiology & Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo St., MMR250, Los Angeles, CA 90033 (E-mail: rfarley{at}hsc.usc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 December 2000; accepted in final form 11 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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