© 1995 American Heart Association, Inc.
Articles |
High- and Low-Affinity Sites for [3H]Dofetilide Binding to Guinea Pig Myocytes
From the Cardiovascular Research Group, The University of Calgary (Canada).
Correspondence to Dr Henry J. Duff, Department of Medicine, The University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada.
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Abstract |
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Abstract Dofetilide specifically blocks the rapid component of the delayed rectifier current (IKr) at nanomolar concentrations in a saturable manner, suggesting the presence of a receptor. We characterized two [3H]dofetilide binding sites to ventricular myocytes from adult guinea pigs by using a conventional filter assay. Scatchard analysis revealed two binding sites with different affinities: a high-affinity site (Kd, 2.8±0.3x10-8 mol/L; Bmax, 76±15 fmol/106 myocytes) and a low-affinity site (Kd, 1.64±0.4x10-6 mol/L; Bmax, 1620±260 fmol/106 myocytes) (n=11). Kinetic studies showed that there were two dissociation rate constants for [3H]dofetilide (0.02±0.005 min-1 [high-affinity site] and 0.22±0.064 min-1 [low-affinity site], n=4), although the observed association rate constant is equally well fit to a single- or two-site model. The ability of known IKr blockers to compete with [3H]dofetilide binding to both sites was assessed. E4031, clofilium, quinidine, and sotalol competed for binding at both sites. Disopyramide and NAPA only competed for a single binding site. The mean IC50 values for inhibition of binding to both the high- and low-affinity binding sites correlated with their concentrations required to inhibit IKr in electrophysiological studies. However, inhibition of [3H]dofetilide binding to the high-affinity site by class III antiarrhythmic drugs occurred at pharmacological concentrations, whereas suprapharmacological concentrations were required to inhibit binding to the low-affinity site. To demonstrate that the low-affinity site was not associated with IKr, [3H]dofetilide binding was assessed in rat ventricular myocytes, which do not express IKr electrophysiologically. In the rat, we observed specific binding of [3H]dofetilide only to the low-affinity site (Kd, 2.9±0.8x10-7 mol/L; Bmax, 248±75 fmol/106 myocytes; n=9). In addition, low concentrations of solatol (50 µmol/L) inhibit [3H]dofetilide only at its high-affinity site without affecting its low-affinity site. Thus, it can be concluded that the high-affinity [3H]dofetilide binding site is associated with IKr in guinea pig ventricular myocytes.
Key Words: [3H]dofetilide • delayed rectifier K+ current • ventricular myocytes
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antiarrhythmic effects of many class III drugs are due to the prolongation of repolarization.1 Voltage-clamp electrophysiological studies have demonstrated that class III antiarrhythmic drugs, such as dofetilide, block the K+ channel underlying IKr.2 3 4 5 6 Dofetilide specifically inhibits IKr at nanomolar concentrations in a saturable and reversible manner, suggesting the presence of a receptor for class III antiarrhythmic drugs associated with this channel. Development of a radioligand, which binds specifically to the delayed rectifying K+ channel, will facilitate the study of class III drug interaction with this channel.7 Chadwick et al7 have previously reported the presence of a single binding site for [3H]dofetilide in guinea pig myocytes. In preliminary studies, we attempted to replicate this work and found the presence of not one but two identifiable binding sites. The identification and characterization of both binding sites are relevant for a number of reasons: first, if binding actually occurs at two binding sites but is modeled to one site, the quantitative measurements of Bmax and Kd are likely to be inaccurate; second, if the ligand technique is used to examine regulation of binding site density and drug-induced changes in [3H]dofetilide binding affinity, then neglect of the second site and incorrect modeling can lead to incorrect interpretation of data. For example, if any given treatment produces a decrease in Kd of the low-affinity site and if the data are fit only to a single site, the binding isotherm will show a composite curve, which will be interpreted as a change occurring at the high-affinity site (associated with IKr). The purpose of the present study was to characterize the binding of [3H]dofetilide to highly viable guinea pig (a species expressing IKr) and rat (a species not expressing IKr) cardiac myocytes. Specifically, we assessed the number of sites to which [3H]dofetilide bound, whether these sites were associated with IKr, and whether there was only a single binding site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
[3H]Dofetilide and unlabeled dofetilide were provided by Pfizer Co. Clofilium was a gift from Eli Lilly; E4031, from Astra Hässle; sotalol, from Bristol-Myers Squibb Co; almokalant (H234) from Hassle Pharmaceutical; and quinidine, NAPA, disopyramide, lidocaine, isoproterenol, propranolol, carbachol, atropine, norepinephrine, phenoxybenzamine, adenosine, verapamil, and 4-aminopyridine, from Sigma Chemical Co. Stock solutions (10-2 mol/L) of the drugs were prepared in distilled water, with the exception of dofetilide, which dissolved in acidified solution (pH, >4); quinidine and lidocaine dissolved in acidified ethanol (50%). Collagenase was purchased from Worthington Biochemical Co.
Myocyte Preparation
Cardiac myocytes were isolated from adult male guinea pigs (250
to 350 g) and adult Sprague-Dawley rats (250 to 350
g).8 9 10 Guinea pigs and rats were treated with heparin (10
mg/kg) and then were anesthetized by an
intraperitoneal injection of phenobarbital (15
mg/kg). Animals were killed by cervical dislocation, and the heart was
rapidly removed. The aorta was quickly cannulated, and the heart was
perfused retrogradely in a Langendorff perfusion
apparatus. The heart was perfused and later incubated with
rinse and digestion solutions that were equilibrated with 95%
O2/5% CO2 at 37°C. The rinse solution
was based on Joklik's MEM supplemented with (mmol/L)
NaHCO3 24, MgSO4 1.2, DL-carnitine
1, and taurine 20. The digestion solution was prepared by adding 0.1%
fatty acid–free BSA and 0.065% collagenase to the
rinse solution. The heart was perfused first at 20°C for 5 minutes
with the rinse solution and then perfused at 37°C for 14 minutes with
the digestion solution. The ventricles were then removed, cut three
times with fine scissors, and incubated at 37°C for 15 minutes with
the digestion solution in a shaking water bath. Dispersed cells were
decanted into a plastic centrifuge tube, and the residual
tissue was shaken again with digestion solution. This resulted in near
total dispersion of the heart. The pooled myocytes were then filtered
through a 200-µm silk-screen mesh into a polypropylene tube. The
myocyte suspension was immediately diluted with 10 mL of
enzyme-free and Ca2+-free incubation solution
containing (mmol/L) KCl 45, KH2PO4 20,
MgCl2 5, KHCO3 0.5, potassium glutamate 50,
potassium aspartate 20, glucose 10, EGTA 1, and HEPES 10, along with
1% BSA (pH 7.4). Myocytes were allowed to sediment for 10 minutes
without centrifugation, and the pellet was harvested.
Myocytes were rinsed with the incubation solution three times.
Kinetic Binding Assays
The rate of dissociation of [3H]dofetilide from
its binding sites was measured by incubating myocytes for 45 minutes
with 10 nmol/L [3H]dofetilide in the absence or presence
of 3x10-5 mol/L unlabeled dofetilide at 37°C.
Dissociation of [3H]dofetilide was initiated by
volumetric dilution (1:20) with incubation solution containing
10-4 mol/L unlabeled dofetilide to block the reassociation
of [3H]dofetilide to its binding site. Incubation was
continued at 37°C, and reactions were terminated by rapid filtration
at successive times. The dissociation rate constant,
k-1, of [3H]dofetilide was
calculated from the following equation:
 | (1) |
The rate constant of association, k+1, of
[3H]dofetilide was measured by incubating myocytes with
10 nmol/L [3H]dofetilide in the absence or presence of
10-5 mol/L unlabeled dofetilide. Incubation was terminated
at successive time points by rapid filtration after volumetric dilution
(1:20). The unidirectional rate constant of association,
k+1, of [3H]dofetilide was
calculated from the following equation:
 | (2) |
 | (3) |
[3H]Dofetilide Equilibrium Binding
Myocytes (2x105 per assay) were incubated for 45
minutes at 37°C in a solution containing (mmol/L) KCl 45,
KH2PO4 20, MgCl2 5,
KHCO3 0.5, potassium glutamate 50, potassium aspartate 20,
glucose 10, EGTA 1, and HEPES 10, along with 1% BSA (pH 7.4) and with
10 nmol/L [3H]dofetilide in a final volume of 150 µL.
Reactions were terminated by adding 3 mL Tris buffer (mmol/L): Tris-HCl
25, NaCl 130, KCl 5.5, MgSO4 0.8, and glucose 10, along
with 50 µmol/L CaCl2 and 0.05% BSA [pH 7.4]) into the
assay and then filtered through presoaked Whatman GF/C glass filters
with Tris buffer supplemented with 1% BSA, followed by two 3-mL washes
with Tris buffer using a 24-well Brandel cell harvester (model M-24R).
The filters were dried and counted in Beckman Ready Safe scintillation
fluid with 60% efficiency. The retained radioactivity
represents [3H]dofetilide bound to myocytes.
Total and nonspecific binding was determined in the absence and presence of an excess of unlabeled dofetilide (10 µmol/L). Specific binding was determined by subtracting the nonspecific binding from the total binding. The rationale for the incubation and filtration conditions has been described previously.10 The conditions provide a maximal reduction in background (nonspecific binding) and scatter with a minimal reduction in specific binding. Under these conditions, a yield of specific [3H]dofetilide binding is 69±8% (n=43, mean±SD) of the total radioactivity retained in the filters.
Scatchard Binding Isotherm
Myocytes or crude membrane homogenate was incubated
with 10 nmol/L [3H]dofetilide in the absence or presence
of a range of unlabeled dofetilide at concentrations from
1x10-9 to 1x10-5 mol/L. The specific
binding of the ligand, [3H]dofetilide, to its binding
sites was examined by Scatchard analysis. To test the
possibility of multiple binding sites for [3H]dofetilide,
the equilibrium binding parameters were determined by using
the nonlinear least-squares curve-fitting program
LIGAND (Ligand, Elsevier, Biosoft).
Statistics
Each experiment was run in duplicate and repeated a minimum of
five times each for Scatchard analysis and drug competition
curves and a minimum of three times for the kinetic data. All the data
were analyzed by the program LIGAND (EBDA,
LIGAND, KINETIC, and DRUG, Elsevier Biosoft);
nonlinear regression analyses were used. The LIGAND
program also allows modeling of drug competition curves to establish
estimates of IC50 values for interaction with the high- and
low-affinity sites. Data were fit to one– and two–binding
site models. The fit having the lowest squares of sum of residuals was
chosen as the best fit of the data. The null hypothesis was rejected at
a level of P<.05. All data are reported as mean ±SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myocyte Preparation
Myocytes were prepared daily from adult guinea pig ventricles with a yield of 20x106 myocytes per heart, of which 80±5% were viable and rod-shaped. Similarly, myocytes were prepared from adult rat ventricles with a yield of 20x106 myocytes per heart, of which 92±3% were viable and rod-shaped. Virtually all of the rod-shaped cells excluded 0.05% trypan blue.
Conditions of [3H]Dofetilide Binding
Fig 1
, top, shows the dependence of
[3H]dofetilide binding on the number of guinea pig
myocytes. Specific binding increases with an increasing amount of
myocytes and reaches an asymptotic value with 
3x105
cells. Subsequently, binding assays were carried out with
2x105 myocytes. These concentrations were chosen because
they were below the asymptotic values of the specific
binding–myocyte concentration response relation. Association of
[3H]dofetilide with its binding site on guinea pig
myocytes is time dependent (Fig 1, bottom).
[3H]Dofetilide binding reaches equilibrium by 25 minutes.
The observed k+1 value is equally well fit to a
single- or two-site model. In the association study, the
single-site association rate constant (kobs)
was 0.241±0.02 min-1.
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) and nonspecific (
)
binding was determined with 10 nmol/L [3H]dofetilide in
the absence or presence of 3x10-5 mol/L unlabeled
dofetilide, respectively. Specific binding (
)
represented the difference between the total and
nonspecific binding. Bottom, Association of
[3H]dofetilide with myocytes. Time dependence of
[3H]dofetilide association with myocytes is shown.
Incubation of myocytes with 10 mmol/L [3H]dofetilide in
the absence or presence of 3x10-5 mol/L unlabeled
dofetilide was terminated at each time point. Specific binding (
Dissociation of [3H]dofetilide from its binding site on
guinea pig myocytes is time dependent. Fig 2 compares
the time course of dissociation of [3H]dofetilide when
fit to a two-site versus a one-site model. Dissociation was
significantly better fit to a two-site model (P<.01).
The mean k-1 values were 0.02±0.005 and
0.22±0.06 min-1 for the two binding sites,
respectively.
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Two Binding Sites for [3H]Dofetilide Binding in
Guinea Pigs
To characterize whether dofetilide binds to a single or multiple
binding sites, the equilibrium binding isotherms were subjected to
Scatchard analysis using a nonlinear least-squares program.
A representative Scatchard rearrangement of the binding
isotherm when fit with a one-site model (Fig 3, top)
or two-site model (Fig 3, middle) was compared. The data were
significantly better fit by a two-site model (P<.05),
indicating both high- and low-affinity binding sites for
[3H]dofetilide. The mean Kd and
Bmax values of the individual experiments for the two
dofetilide binding sites are shown in Table 1. The
high-affinity binding site has a Kd of 28±3
nmol/L and Bmax of 76±15 fmol/106
cells, and the low-affinity site has a Kd of
1.63±0.38 µmol/L and Bmax of 1.62±0.26
pmol/106 cells (n=11). Models with more than two
sites did not improve the fit. The normalized pooled data from 11
separate experiments are shown in Fig 3, bottom, with a sufficient
number of points to quantify the binding parameters of the
individual sites. The results obtained from the pooled data were
similar to the mean parameters obtained by fitting the data
from the individual experiments.
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To assess the relation between the presence of
[3H]dofetilide binding and the presence of the delayed
rectifying K+ channel, we characterized binding of
[3H]dofetilide to cardiac myocytes of rat, a species
without IKr. Under experimental conditions identical
to those used for guinea pigs (including an identical range of
unlabeled dofetilide concentrations), [3H]dofetilide
bound to rat ventricular myocytes (Fig 4).
However, Scatchard analysis yielded only a single
low-affinity binding site with a mean Kd of
290±80 nmol/L and Bmax of 248±75
fmol/106 cells. The density of the low-affinity
site in rat myocytes is substantially less than the density of the
low-affinity site in guinea pig myocytes.
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Drug Inhibition of [3H]Dofetilide Binding in
Guinea Pigs
The relation between the inhibition of
[3H]dofetilide binding and blockade of IKr by
class III antiarrhythmic drugs was assessed. First, we assessed whether
drugs known to block this channel
electrophysiologically also inhibit
[3H]dofetilide binding. The drugs studied were
dofetilide,2 3 4 5 6 E4031,11
clofilium,7 12 quinidine,13 14 15
sotalol,7 16 17 NAPA,16
disopyramide,18 and almokalant.11
Fig 5 shows that three representative
class III antiarrhythmic drugs (dofetilide, E4031, and sotalol) inhibit
[3H]dofetilide binding in myocytes in a
concentration-dependent manner. Table 2 shows the
mean IC50 values for the range of class III drugs
evaluated. The inhibition of [3H]dofetilide binding by
most class III antiarrhythmic drugs was significantly better fit by
interactions at a two-site model than at a single-site model.
However, disopyramide and NAPA only inhibited binding at
relatively high concentrations, and the IC50 curves fit to
a single-site model.
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), and sotalol
(
) (see also Table 2
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The second method used to assess the relation of drug-dependent
inhibition of [3H]dofetilide binding to
drug-dependent inhibition of IKr was to assess whether
the IC50 values for inhibition of high-affinity binding
correlated with the concentration of drugs required to inhibit 50% of
IKr (Fig 6). Linear regression of the
log-log plot yields a correlation slope factor of 1.12 and a
correlation coefficient r of .93 (log y=1.12 log
x+0.035). There was also a linear correlation of the mean
IC50 values of drugs inhibiting binding to the
low-affinity site and the concentrations required to block 50% of
IKr electrophysiologically with
a correlation slope factor of 1.19 and an r value of .99
(log y=1.19 log x-1.65). However, the intercept
value was significantly shifted to the right. Therefore, much higher
and suprapharmacological concentrations of the class III drugs were
required to inhibit binding to the low-affinity site.
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To further characterize whether sotalol competes with
[3H]dofetilide for binding to the low- or
high-affinity site, myocytes were incubated with 50 µmol/L
sotalol and 10 nmol/L [3H]dofetilide in the absence or
presence of a range of unlabeled dofetilide at concentrations from
1x10-9 to 1x10-5 mol/L. The equilibrium
binding isotherms were subjected to Scatchard analysis using a
nonlinear least-squares program. Fig 7 illustrates
the Scatchard plots of inhibition of [3H]dofetilide
binding in the presence or absence of sotalol. Sotalol inhibits the
interaction of [3H]dofetilide binding to the
high-affinity site, leaving binding only to the low-affinity
site (Table 1
).
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We next assessed whether drugs not known to block the delayed rectifier
K+ channel by
electrophysiological evaluation inhibited
[3H]dofetilide binding to guinea pig myocytes. The drugs
assessed included lidocaine, propranolol, isoproterenol,
norepinephrine, phenoxybenzamine, carbachol, atropine,
adenosine, verapamil, and
4-aminopyridine. These drugs did not inhibit the
binding of [3H]dofetilide at pharmacologically relevant
concentrations (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated the existence of both high- and low-affinity binding sites for [3H]dofetilide binding on guinea pig myocytes, whereas only a single low-affinity site could be demonstrated to exist in rat ventricular myocytes. Since guinea pig ventricle expresses IKr electrophysiologically but rat ventricle does not, our data suggest that the high-affinity site is associated with IKr in guinea pig myocytes. We have also reported that drugs with class III electrophysiological activity inhibit high-affinity [3H]dofetilide binding at similar concentrations and with a rank order of potency similar to those that block IKr. Scatchard analysis shows that pharmacologically relevant concentrations of sotalol interact predominantly with the high-affinity binding site, supporting the notion that the high-affinity site is associated with IKr. Drugs that are not known to block this channel do not inhibit [3H]dofetilide binding at pharmacologically relevant concentrations. The relevance of this data is that incorrect modeling of binding isotherms to a one-site model overestimated the measured IC50 values by almost one order of magnitude (Table 4
). Moreover, in contrast to the study of
Chadwick et al,7 we observed specific, saturable, and
reversible binding of [3H]dofetilide to a
low-affinity binding site on adult Sprague-Dawley rat
ventricular myocytes. Thus, care must be taken in the
design and interpretation of experiments using
[3H]dofetilide binding to species and/or tissues with the
low-affinity binding site. For example, inhibition of binding to
the low-affinity site could be misinterpreted as interaction of
class III drugs with the IKr channel.
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High-Affinity Binding Site for [3H]Dofetilide Is
Associated With IKr
We provide evidence that the high-affinity site for
[3H]dofetilide binding is related to IKr.
First, the membrane surface binding density is similar to that of this
channel. The binding density can be estimated, since it is known that
there are 50x103 [3H]dofetilide
high-affinity sites per guinea pig myocyte and that the myocyte
surface area is 13.8x103
µm2.19 If no assumptions are made about the
distribution of sites in surface membranes, this suggests that there
are about three high-affinity [3H]dofetilide sites
per square micron. By comparison, Chadwick et al7
estimated 0.2 delayed rectifier sites per square micron on the basis of
electrophysiological data. Second, the
close correlation and similar rank order of potencies between the
IC50 values required to inhibit high-affinity
[3H]dofetilide binding in the radioligand
assay and the concentrations in the in vitro
electrophysiological studies required to
block IKr strongly suggest that the radioligand
assay identifies IKr. Taken together, these data lead to
the conclusion that [3H]dofetilide binds to
IKr and that binding to this site is relevant to block of
the channel by selected class III antiarrhythmic drugs. Third, a panel
of compounds not known to bind to the delayed rectifier does not
inhibit [3H]dofetilide binding to myocytes at clinically
relevant concentrations. On the basis of membrane surface binding
density, the pharmacologically relevant binding of class III
antiarrhythmic drugs, and the lack of binding by other drugs, we
conclude that the high-affinity binding site of
[3H]dofetilide is associated with IKr in
guinea pig cardiac myocytes.
The regressions correlating affinity (Kd) measured biochemically and the electrophysiological sensitivity are limited. Electrophysiological data may reflect only functional channels, whereas binding assays may be influenced by both functional and nonfunctional channels, as has been reported for another K+ channel, minK.20 Even so, the correlation (r value) between biochemical affinity and electrophysiological sensitivity in this study was high (.93).
Low-Affinity Site for [3H]Dofetilide Is Not
Associated With IKr
We provide evidence that the low-affinity site for
[3H]dofetilide binding is not related to IKr.
First, the membrane surface binding density is substantially greater
than the estimates of the density of this channel. The binding density
can be estimated, since it is known that there are
5x104 [3H]dofetilide low-affinity
sites per guinea pig myocyte and that the myocyte surface area is
13.8x103 µm2.10 If no
assumptions are made about the distribution of sites on the surface
membranes, we estimate that there are 100 low-affinity
[3H]dofetilide sites per square micron. By comparison,
Chadwick et al7 estimated 0.2 delayed rectifier sites per
square micron on the basis of
electrophysiological data. Therefore, the
surface density of the low-affinity site is much greater than the
estimates for the density of the IKr channel. Second,
although a correlation exists between the IC50 values
required to block the low-affinity site and the concentrations
required in the in vitro
electrophysiological studies, the intercept
value of the regression line is shifted significantly to the right.
Higher concentrations are required to inhibit binding to the
low-affinity site than are required to inhibit IKr.
Third, the low-affinity binding occurs in the rat, a species that
does not express IKr at
electrophysiological studies. Taken
together, these data suggest that the low-affinity site for
[3H]dofetilide binding is not directly related to
IKr.
The low-affinity site observed in rat myocytes is not identical to the low-affinity site in guinea pig myocytes. The affinity of the binding site in rat myocytes is midway between the high- and low-affinity sites in guinea pig myocytes. It is possible that different proteins are responsible for the low-affinity binding sites in rat and guinea pig myocytes. Alternatively, the low-affinity site in the rat may have a different allosteric configuration in that species. Although we do not yet know the identity of the protein responsible for the low-affinity binding site, we provide evidence that the low-affinity site is not directly related to IKr. This is an important observation, since binding to the low-affinity site, for instance in the rat, could be misinterpreted as binding to IKr.
Two Specific Binding Sites for
[3H]Dofetilide
The heterogeneity in binding sites observed
in the present study is similar to that reported by Thomsen et
al,21 who assessed the binding of the
radioligand PD85639 to the 
-subunit of the
Na+ channel. In that study, the ligand bound to two sites
on the -subunit when transfected into Chinese hamster
ovarian cells, indicating that both the high- and
low-affinity [3H]PD85639 binding sites reside on the
-subunit. In addition, much like our studies, there were
>10-fold as many low-affinity sites as high-affinity sites.
Thomsen et al argued that the different binding sites may reflect
binding to two distinct states of the Na+ channel. Our data
do not directly address the issue of state-specific binding of
[3H]dofetilide.
In the experiments designed to measure association of the ligand
with its receptor, there is ongoing dissociation. To calculate
k+1, the kobs
values must be corrected for ongoing dissociation. Since the
dissociation process is biexponential, two k+1
values are necessarily calculated, as shown in Equation 3. The mean
calculated k+1 values (n=5) were 0.0196±0.0034
min-1 · (nmol/L)-1 for the fast
dissociating site and 0.0045±0.0021
min-1 · (nmol/L)-1 for the slow site. The
raw kobs data are equally well fit to
monoexponential or biexponential models. Even so, the
two k+1 values are calculated rather than
directly measured and are based on the assumptions underlying the
modeling process. Therefore the two k+1 values
must be considered to be only estimates.
Comparison With Previous Literature
Our observations of two binding sites of
[3H]dofetilide on guinea pig myocytes contrast with those
of Chadwick et al,7 who identified a single binding site
with an IC50 of 100 nmol/L. The presence of a second
low-affinity high-density binding site was not reported in that
study. If we model our IC50 data to a single site, we also
observe an IC50 of 150±23 nmol/L. However, when the
two-site binding is considered, the correct high-affinity
IC50 was found to be 19±5 nmol/L, very similar to the
Kd for the high-affinity site. Indeed, in
Table 4 we compare the IC50 values of a range of class III
antiarrhythmic drugs when fit to a one- or two-site model. As is
shown, the IC50 values are overestimated by almost one
order of magnitude when modeled to one site. This probably explains
some of the discrepancies between the results of the present study
and that of Chadwick et al. In addition, differences in the viability
of the myocytes in the present study (80%) versus the earlier
study (35%) may contribute to the differences. Alternatively, in the
Scatchard figure presented in the study of Chadwick et al, the
linear fit was extrapolated from a bound value of 0.15 pmol/mg
protein. Data obtained at higher concentrations were not reported,
which may explain why only the single high-affinity binding site
was identified. In contrast to the study of Chadwick et al, we also
observed specific, saturable, and reversible binding of
[3H]dofetilide to a low-affinity binding site on
adult Sprague-Dawley rat ventricular myocytes.
Thus, care must be taken in the design and interpretation of
experiments using [3H]dofetilide binding to species
and/or tissues with both binding sites. Inhibition of binding to the
low-affinity site could be misinterpreted as interaction of class
III drugs with IKr.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by Pfizer Central Research, Sandwich, UK and the Medical Research Council of Canada. Dr Duff is a Medical Scientist of the Alberta Heritage Foundation for Medical Research.
Received November 21, 1994; accepted June 6, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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