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
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ABSTRACT |
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Three
-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
-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
-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
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INTRODUCTION |
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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 -subunit, and there are three different isoforms of
the
-subunit of Na+-K+-ATPase,
1,
2, and
3, in human
heart (18, 19, 21, 23, 24). A second subunit,
, is
required for sodium pump function, but
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
1-isoform of human Na+-K+-ATPase
has a higher affinity for cardiac glycosides than the
2-
or
3-isoforms. This conclusion differs from the pattern
in rodents, where the
1-isoform has a lower affinity
(Kd near 10
5 M) (21)
than that of
2- or
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
-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 - and
-isoform combinations in Xenopus oocytes and measured
Kd values in the absence of K+. The
1
1 and
3
1
complexes had Kd values of ~5 nM, and the
2
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
1-isoform, human
skeletal muscle contains primarily
2- and some
1-isoforms, and human heart and brain express
1-,
2-, and
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
-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.
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MATERIALS AND METHODS |
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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 1 (1:100) from M. Kashgarian (Yale University);
McB2, a mouse monoclonal against
2 (1:100) from K. Sweadner (Harvard University) (20); anti-TED, a rabbit
polyclonal against
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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Materials. All chemicals were of reagent grade or higher. [3H]ouabain was obtained from NEN (Boston, MA).
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RESULTS |
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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
1 in the samples is brain
kidney > heart > skeletal muscle
Caco-2
HeLa cells > red blood cells (RBC). The relative
2 abundance is
brain
skeletal muscle > heart, and the
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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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 1 alone (kidney, erythrocytes, Caco-2 cells, and HeLa cells), predominantly
2 with
1 (skeletal muscle), or
combinations of
1 with
2 and
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
-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
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
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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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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DISCUSSION |
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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 -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
-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
-subunit isoforms have very
similar affinities for ouabain. In addition, it can be inferred that
the different
-subunit isoforms, as well as the presence or absence
of the
-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 mM1 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
-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 -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
-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
-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
-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 1
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
-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
2- and
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
-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 1
1 three-
to fourfold while increasing the Kd for
2
1 and
3
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 -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 (
2,
1).
In patients with hypokalemia, large reductions in skeletal muscle
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).
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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