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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
JPET 284:75–82, 1998
Vol. 284, No. 1
Printed in U.S.A.
Amiodarone Inhibits the Na1-K1 Pump in Rabbit Cardiac
Myocytes after Acute and Chronic Treatment1
DAVID F. GRAY,2 ANASTASIA S. MIHAILIDOU, PETER S. HANSEN,3 KERRIE A. BUHAGIAR, NERIDA L. BEWICK,
HELGE H. RASMUSSEN and DAVID W. WHALLEY
Cardiology Department, Royal North Shore Hospital, St. Leonards, 2065 NSW, and University of Sydney, 2006 NSW, Australia
Accepted for publication September 16, 1997
This paper is available online at http://www.jpet.org
The mechanism of action of the widely used antiarrhythmic agent amiodarone is thought to involve use-dependent
block of Na1, Ca11, and K1 channels (see Nattel et al., 1992,
for review). However, when studying electrophysiological effects of amiodarone, the Na1-K1 pump should also be considered. The pump directly, or indirectly through the operation of secondary active ion transport mechanisms,
maintains the transmembrane ionic gradients. These gradients in turn drive the channel currents that are modulated
by amiodarone. In addition, the electrogenic Na1-K1 pump
current, arising from the 3Na1:2K1 exchange ratio, contributes to repolarization of the cardiac action potential (Gadsby
et al., 1985).
Treatment with amiodarone influences physicochemical
properties of the cell membrane, including fluidity and cholesterol content (Chatelain et al., 1986). Because the Na1-K1
pump is affected by such changes in membrane properties
Received for publication April 1, 1997.
1
This study was supported by the National Heart Foundation of Australia
(Grant 94S4055) and by the North Shore Heart Research Foundation.
2
D.F.G. was the recipient of a National Health and Medical Research
Council of Australia Medical Postgraduate Research Scholarship.
3
P.S.H. was the recipient of a Postgraduate Medical Research Scholarship
from the National Heart Foundation of Australia.
10 mM Na1 but had no effect on Ip at 80 mM Na1. Amiodarone
had no effect on the voltage dependence of the pump or the
affinity of the pump for extracellular K1 either after chronic
treatment or during acute exposure. We conclude that chronic
amiodarone treatment reduces overall Na1-K1 pump capacity
in cardiac ventricular myocytes. In contrast, acute exposure of
myocytes to amiodarone reduces the apparent Na1 affinity of
the Na1-K1 pump. An amiodarone-induced inhibition of the
hyperpolarizing Na1-K1 pump current may contribute to the
action potential prolongation observed during treatment with
this drug.
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ABSTRACT
Amiodarone has been shown to affect cell membrane physicochemical properties, and it may produce a state of cellular
hypothyroidism. Because the sarcolemmal Na1-K1 pump is
sensitive to changes in cell membrane properties and thyroid
status, we examined whether amiodarone affected Na1-K1
pump function. We measured Na1-K1 pump current (Ip) using
the whole-cell patch-clamp technique in single ventricular myocytes isolated from rabbits. Chronic treatment with oral amiodarone for 4 weeks reduced Ip when myocytes were dialyzed
with patch-pipettes containing either 10 mM Na1 or 80 mM
Na1. In myocytes from untreated rabbits, acute exposure to
amiodarone in vitro reduced Ip when patch pipettes contained
(Cornelius, 1991), it seems reasonable to speculate that amiodarone may alter pump function. Amiodarone might also
alter pump function through an alternative mechanism. The
drug is believed to induce a state of cellular hypothyroidism
with chronic use (Patterson et al., 1986; Singh et al., 1983).
Thyroid status in turn is an important determinant of synthesis of Na1-K1 pump units (Doohan et al., 1995; Kjeldsen
et al., 1986).
Several previous studies using isolated myocardial membrane or microsomal preparations (Broekhuysen et al., 1972;
Dzimiri and Almotrefi, 1991) have suggested that acute exposure to amiodarone decreases Na1-K1/ATPase activity,
the enzymatic equivalent of the Na1-K1 pump. Although
demonstrating that amiodarone has the potential to affect
pump activity, the physiological relevance of such studies is
unclear. The drug concentrations used were much higher
than those encountered clinically, and the studies were performed using K1 and Na1 concentrations expected to saturate binding sites rather than at physiologically relevant
concentrations. In addition, Na1-K1/ATPase studies require
the pump molecule to be isolated from the native lipid membrane and purified. As the membrane lipid environment
ABBREVIATIONS: Ip, Na1-K1 pump current; QTc, corrected QT interval; T4, thyroxine; T3, triiodothyronine; HEPES, N-2-hydroxyethylpiperazineN9-2-ethanesulfonic acid; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; TMA, tetramethylammonium; aiNa, intracellular Na1 activity; [Na]pip, pipette Na1 concentration; [K]o, extracellular K1 concentration; K0.5, concentration for half-maximal pump activation; Vm,
membrane potential; Ip-Vm, pump current-voltage relationship.
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Gray et al.
Vol. 284
modulates pump activity (Cornelius, 1991), it is unclear how
these results relate to the activity of in situ pumps.
In the present study, we examined the effects of both acute
and chronic amiodarone exposure on the Na1-K1 pump in
intact cardiac myocytes. We used the whole-cell patch-clamp
technique to measure Ip. This approach allows independent
control of ligands for the Na1-K1 pump on both sides of the
intact native membrane, as well as control of membrane
potential, variables that are important determinants of
pump function.
Methods
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Treatment protocols. Male New Zealand White rabbits, weighing 2.5 to 3.0 kg, were used in the study. Amiodarone was supplied
in its hydrochloride powder form. In experiments examining the
effects of chronic treatment, gelatin capsules containing appropriate
quantities of amiodarone were made up at two weekly intervals. A
single daily amiodarone capsule was administered to rabbits orally
in a dose of 80 mg/kg/day for 4 weeks. This protocol was based on a
previous study in rabbits demonstrating clinically relevant changes
in electrophysiological and electrocardiographic parameters with 50
to 100 mg/kg oral amiodarone daily (Kodama et al., 1992). Control
rabbits received empty gelatin capsules in the same manner.
A surface electrocardiogram was obtained before treatment and at
the end of the 4-week treatment protocol to assess the effects of
amiodarone on the QT interval. This parameter has been shown to
correlate well with myocardial concentrations of amiodarone (Debbas et al., 1984). Bazett’s formula was used to correct the QT interval
(QTc) for heart rate (QTc 5 QT/=RR interval). The ECG electrodes
were placed on the shaved precordium of conscious rabbits in positions that provided ECG deflections of adequate amplitude to make
accurate determinations of QT interval. Recordings were performed
using a single-channel ECG machine (Nihon Kohden Cardiofax,
model SC-513E) at a paper speed of 50 mm/sec. Recordings were
obtained from at least two lead orientations. Blood was taken from a
marginal ear vein at base line and after 4 weeks of treatment to
assess the effects of amiodarone on thyroid function. Serum total T4
and T3 were measured quantitatively using the Ciba Corning Automated Chemiluminescence System (ACS:180 system, Ciba Corning
Diagnostics Corp, Medfield, MA). The serum amiodarone level was
determined in the blood sample collected after 4-week treatment.
The level was measured using an HPLC method adapted from Law
et al. (1984).
In experiments examining the acute effects of amiodarone on the
Na1-K1 pump, myocytes isolated from untreated rabbits were exposed to the drug in-vitro. A 1 mM stock solution of amiodarone in
100% ethanol was prepared and used within 2 weeks. On the day of
experimentation, the stock solution was diluted 100-fold in solutions
used to superfuse the myocytes; thus, the superfusates had a nominal amiodarone concentration of 10 mM in 1% ethanol, but these
solutions appeared to be supersaturated. We therefore measured
amiodarone content in the superfusate using HPLC. The mean final
amiodarone concentration in the superfusate was 0.61 6 0.13 mM
(n 5 6; range, 0.30 –1.20 mM). Control myocytes in these experiments
were exposed to superfusates containing 1% ethanol only.
Measurement of Ip. Single ventricular myocytes were isolated as
described previously(Hool et al., 1995). After isolation, myocytes
were stored at room temperature until used for experimentation.
Myocytes were used on the day of isolation only, and pump currents
were measured 2 to 10 hours after the heart was excised. Myocytes
were voltage clamped with wide-tipped (4 –5 mm) borosilicate glass
pipettes made as described previously (Whalley et al., 1993). In
experiments measuring Ip at a fixed membrane potential of 240 mV,
pipettes were filled with a solution containing (in mM) 70 potassium
glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP and sodium
glutamate plus TMAzCl 90. The Na1 concentration in the solution
was either 10 mM, which is near the physiological intracellular level,
or 80 mM, a level expected to nearly saturate intracellular pump
sites. The solution was titrated to pH 7.05 6 0.01 at 35°C with 1 M
KOH. In experiments designed to examine the relationship between
Ip and membrane voltage pipettes were filled with a solution containing (in mM) 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5
EGTA, 2 MgATP, 60 TMAzCl, 20 tetraethylammonium chloride, 70
CsCl and 50 aspartic acid. To examine the Ip-Vm relationship at a
high intracellular Na1 level, the solution was similar except that
sodium glutamate was 80 mM, CsCl was 65 mM, aspartic acid was
45 mM, and TMAzCl was omitted. The solution was titrated to pH
7.05 6 0.01 at 35°C with 1 M HCl. When filled with the above
solutions, patch pipettes had resistances of 0.8 to 1.2 MV.
Myocytes were suspended in a tissue bath mounted on an inverted
microscope. They were superfused with modified Tyrode’s solution
that contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44
NaH2PO4, 10 glucose and 10 HEPES. It was titrated to pH 7.4 at
35°C with 1 M NaOH. This solution was used while the whole-cell
configuration was established and cell membrane capacitance was
measured. In most experiments, the superfusate was then changed
to a solution that was similar except that it was nominally Ca11 free
and in addition contained 2 mM BaCl2 and 0.2 mM CdCl2. Superfusates that were Ca11 free and contained CdCl2 were used to prevent
Ca11 overloading of myocytes when cells were patch-clamped with
pipettes containing high Na1 concentrations. In experiments examining the effect of extracellular K1 on pump activity, we varied the
K1 concentration of this Ca11-free superfusate between 0 and 15
mM. TMAzCl was used to maintain constant osmolality in these
experiments.
Dose of ouabain. A previous study in noncardiac tissue suggested that amiodarone may competitively inhibit ouabain binding
to the Na1-K1 pump (Prasada Rao et al., 1986). To ensure that the
concentration of ouabain used in experimental protocols (100 mM)
was sufficient to completely block the Na1-K1 pump in the presence
of amiodarone, we performed a series of preliminary experiments on
control myocytes and myocytes exposed to amiodarone in vitro. We
found that after the pump had been inhibited by 100 mM ouabain,
there was no additional shift in holding current on increasing the
ouabain concentration to 500 mM, indicating that 100 mM ouabain
caused complete pump inhibition. Unless otherwise indicated, Ip was
identified by the shift in holding current induced by exposure of
myocytes to 100 mM ouabain (Sigma Chemical, St. Louis, MO).
Membrane currents were recorded using the continuous singleelectrode voltage-clamp mode of an Axoclamp-2A amplifier and Axotape or pCLAMP software (Axon Instruments, Foster City, CA).
Voltage-clamp protocols were generated with pCLAMP. Details of
the experimental protocols used to measure membrane capacitance
and Ip and details of the electronic recording system have been
described previously (Whalley et al., 1993). Ip is reported normalized
for membrane capacitance unless otherwise indicated.
Statistics. Results are expressed as mean 6 S.E.M. Statistical
comparisons are made using Student’s t test for paired or unpaired
observations. Dunnett’s test was used when the same control group
was used for more than one comparison. P , .05 is regarded as
significant in all comparisons. Nonlinear regression was used to fit
the Hill equation to data.
Results
Effects of chronic amiodarone on ECG and thyroid
function. Amiodarone is known to prolong the QT interval
on the surface ECG and affects thyroid hormone levels by
inhibiting peripheral conversion of T4 to T3. The effects of
chronic treatment on thyroid function, heart rate, and QTc
are summarized in table 1. There was no significant difference in any of these parameters between the control and
treated groups of rabbits at base line. Amiodarone treatment
Amiodarone Inhibits the Na1-K1 Pump
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TABLE 1
Effects of chronic amiodarone treatment on thyroid function, heart rate and QTc
Placebo
Amiodarone
Pb
T3 (nmol/liter)
T4 (nmol/liter)
Heart rate
QTc (sec)
a
b
a
Base line
4 weeks
P
1.9 6 0.2
61.4 6 4.4
226 6 10
0.28 6 0.003
2.3 6 0.2
66.8 6 5.7
227 6 15
0.27 6 0.003
NS
NS
NS
.037
Base line
4 weeks
Pa
2.2 6 .1
69.1 6 2.1
218 6 7
0.27 6 .003
2.2 6 .2
138.8 6 10.2
209 6 7
0.31 6 0.006
NS
,.001
NS
,.001
NS
,.001
NS
,.01
Level of significance for differences in variables at 4 weeks compared with base line in each group of rabbits.
Level of significance for differences between control rabbits and amiodarone-treated rabbits after 4-week treatment.
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resulted in a significant increase in serum T4 and a significant prolongation of QTc. The treated rabbits had a serum
amiodarone level of 1.14 6 0.15 mmol/liter, which is similar to
the concentration found during chronic treatment in humans
(Debbas et al, 1984; Ikeda et al., 1984; Weinberg et al., 1993).
The treatment protocol was well tolerated. One rabbit lost
weight during the treatment period and was found to have a
toxic serum amiodarone level of 3.9 mmol/liter. Its surface
ECG showed slow (80 bpm) broad QRS complexes. Results
from this rabbit were not included in the analysis.
Effect of chronic amiodarone treatment on Ip. Myocytes from 5 control rabbits and 5 rabbits treated with amiodarone were voltage-clamped at 240 mV, and Ip was measured using pipettes with Na1 concentrations ([Na]pip) of 10
or 80 mM. Results from these experiments are summarized
in figure 1A. When [Na]pip was 10 mM, mean Ip of 14 myocytes from rabbits treated with amiodarone was 0.24 6 0.03
pA/pF, whereas mean Ip of 10 myocytes from rabbits given
placebo was 0.36 6 0.03 pA/pF. This 33% difference was
statistically significant. Mean Ip in myocytes from the rabbits
given placebo was similar to mean Ip in myocytes from untreated rabbits reported previously (Gray et al., 1997). When
[Na]pip was 80 mM, amiodarone treatment resulted in a
significant, 25% reduction in mean Ip (1.46 6 0.07 pA/pF, n 5
7, vs. 1.94 6 0.05 pA/pF, n 5 6). We conclude that chronic
amiodarone treatment causes a reduction in overall Na1-K1
pump capacity.
Effect of chronic amiodarone on the affinity of the
Na1-K1 pump for extracellular K1. Because it has been
suggested that the effect of amiodarone on Na1-K1/ATPase
is K1 dependent (Almotrefi and Dzimiri, 1991), we examined
the effect of chronic amiodarone treatment on the apparent
affinity of the pump for extracellular K1 concentrations
([K]o). We measured changes in Ip in response to changes in
[K]o. To facilitate the detection of small pump currents at low
[K]o we used [Na]pip of 80 mM to induce near-maximal activation of the pump at intracellular sites.
After establishing the whole-cell configuration, we inactivated the pump by switching to a K1-free superfusate. This
superfusate was nominally Ca11 free and contained 2 mM
CdCl2 to eliminate Ca11 channel currents and Na1-Ca11
exchange current. Myocytes were voltage clamped at 240
mV, and Ip was identified as the membrane current resulting
from reactivation of the pump on exposure to different [K]os.
Each myocyte was exposed to a random sequence of at least
three of the following [K]o concentrations (in mM): 0.5, 1, 2, 3,
5.6 and 15. The resulting currents were normalized relative
to the current at 5.6 mM [K]o (Ip%). This concentration was
therefore used in every series of exposures. Each exposure to
a K1 concentration was bracketed by reexposure to the K1free superfusate until Ip had returned to its base-line level.
Fig. 1. A, Effect of chronic amiodarone (amio) treatment on Na1-K1
pump current (Ip), measured when the Na1 concentration in the patch
pipette ([Na]pip) was 10 mM (open bars) and 80 mM (solid bars). Numbers
in parentheses indicate the number of myocytes studied in each group.
Amiodarone treatment significantly reduced Ip when [Na]pip was 10 mM
and when [Na]pip was 80 mM. B, Effect of extracellular K1 ([K]o) on Ip in
myocytes from rabbits chronically treated with amiodarone (F, dashed
line) and from control rabbits (‚, solid line). Ip has been normalized to the
current measured when [K]o is 5.6 to eliminate variability of data arising
from measurement of cell membrane capacitance.
We previously published an illustration of the experimental
protocol and representative traces of membrane currents
(Gray et al., 1997). To examine whether pump run-down
occurred, we concluded the protocol by reexposing a number
of myocytes to the first [K]o concentration used. There was no
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Gray et al.
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evidence of pump run-down in the time taken to conclude the
experimental protocol ('18 –20 min). We previously determined that K1-induced shifts in holding currents attributed
to the activation of the Na1-K1 pump were not contaminated
by other K1-sensitive currents (Gray et al., 1997).
Experiments were performed on 19 myocytes from 5 amiodarone-treated rabbits and 17 myocytes from 4 control rabbits. The [K]o/Ip% relationships are summarized in figure 1B.
When the Hill equation was fitted to the data, we obtained a
K1 concentration for half-maximal pump activation (K0.5) of
2.3 mM for myocytes from amiodarone-treated rabbits and
2.7 mM for control rabbits. The Hill coefficients were 1.58
and 1.59, respectively. To allow statistical comparisons, we
repeatedly fitted the Hill equation to randomly selected series of values for Ip at all six [K]o concentrations tested. This
allowed us to derive mean K0.5 values and mean Hill coefficients for 10 derived [K]o activation curves for myocytes from
the amiodarone-treated rabbits and 11 derived [K]o activation curves for myocytes from control rabbits. There was no
significant difference between the mean values. We conclude
that chronic amiodarone treatment does not affect the apparent K1 affinity of the Na1-K1 pump.
Effect of chronic amiodarone on the Ip-Vm relationship. Because amiodarone is an amphiphile and amphiphiles
can alter the voltage dependence of the Na1-K1 pump
(Läuger, 1991), we examined whether chronic amiodarone
treatment affects the Ip-Vm relationship. After establishing
the whole-cell configuration, we superfused myocytes with
Ca11-free modified Tyrode’s solution, and we voltageclamped the myocytes at 240 mV. We then applied 320-msec
voltage steps in 20-mV increments to test potentials ranging
from 2100 to 160 mV. Averaged membrane currents at each
test potential after superfusion of ouabain were subtracted
from the respective averaged membrane currents before
ouabain exposure to derive Ip at each test potential. Experiments were performed at [Na]pip of both 10 and 80 mM.
Figure 2A illustrates the voltage step protocol and an example of the resulting membrane currents for a myocyte studied
using [Na]pip of 10 mM. Details of the experimental protocol
and data analysis have been published previously (Gray et
al., 1997).
The mean Ip-Vm relationships for 13 myocytes from 5 amiodarone-treated rabbits and 17 myocytes from 8 control rabbits at [Na]pip of 10 mM are illustrated in figure 2B. In this
and all other Ip-Vm relationships, we normalized Ip to the
current measured at 0 mV to facilitate comparisons between
experiments. The Ip-Vm relationships in myocytes from both
groups of rabbits are near-linear and have a positive slope
over the range of voltages examined. There were no major
differences in the relationships for myocytes from the two
groups. The mean Ip-Vm relationships for 7 myocytes from 3
amiodarone-treated rabbits and 8 myocytes from 3 control
rabbits at [Na]pip of 80 mM are shown in figure 2C. The
relationships in myocytes from both groups of rabbits are
virtually superimposable, exhibiting a positive slope at negative potentials and a negative slope at positive potentials.
We conclude that chronic amiodarone treatment does not
affect the Ip-Vm relationship, at either physiological levels of
intracellular Na1 or levels of intracellular Na1 expected to
cause near-saturation of Na1 binding sites.
Effect of chronic amiodarone on intracellular Na1. If
chronic amiodarone treatment inhibits Na1-K1 pump activ-
Vol. 284
Fig. 2. A, Voltage-clamp protocol and holding currents before and after
exposure of a myocyte to 100 mM ouabain. The myocyte was from a rabbit
treated with amiodarone. The current traces shown are the averaged
currents from three applications of the voltage-clamp protocol. [Na]pip
was 10 mM. B, Normalized Ip-Vm relationships for 13 myocytes isolated
from rabbits treated with amiodarone (F) and 8 myocytes from control
rabbits (‚). [Na]pip was 10 mM. C, Ip-Vm relationships for 7 myocytes from
amiodarone-treated rabbits and 8 myocytes from control rabbits when
[Na]pip was 80 mM. Amiodarone had no significant effect on the Ip-Vm
relationships.
ity, it may be expected to alter steady-state intracellular Na1
activity (aiNa). However, the direction and magnitude of any
such change in aiNa cannot be assumed because amiodarone
may also influence Na1 influx. We therefore examined the
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effect of chronic amiodarone treatment on aiNa. We determined aiNa in papillary muscles from 11 control rabbits and
11 rabbits treated with amiodarone for 4 weeks. We used
ion-sensitive microelectrodes to measure aiNa as described
previously (Hool et al., 1995). One or two determinations of
aiNa were made with separate microelectrode impalements in
tissue from each rabbit. Where two measurements were
made, the mean value was used for statistical comparisons.
Determinations of aiNa were made only after microelectrode
recordings had been stable for $20 minutes. The aiNa in
control rabbits was 8.1 6 0.4 mM. In amiodarone-treated
rabbits, aiNa was 9.8 6 0.8 mM. This difference was not
statistically significant (P 5 .07). Amiodarone is expected to
reduce Na1 influx via Na1 channels (Mason et al., 1984);
because amiodarone reduces cardiac metabolic rate (Charlier
et al., 1968), treatment may have reduced Na1 influx via the
Na1-H1 exchanger. One may speculate that such decreases
in Na1 influx partially offset the rise in aiNa expected from
pump inhibition.
Acute effect of amiodarone on Ip. We next examined
whether acute exposure of myocytes to amiodarone affected
the Na1-K1 pump. For these experiments, myocytes were
isolated from untreated rabbits and exposed to amiodarone
in the tissue bath. After establishing the whole-cell configuration, we superfused myocytes with Ca11-free Tyrode’s solution that contained 2 mM BaCl2 and either amiodarone in
1% ethanol or 1% ethanol alone. The cells were exposed to
this superfusate for a minimum of 6 minutes before changing
to a superfusate that was similar except that it contained 100
mM ouabain. The duration of exposure was adopted from
previous studies on Na1 channels (Follmer et al., 1987) and
Ca11 channels (Nishimura et al., 1989) in cardiac myocytes.
These studies indicated that steady state effects were
achieved after '6-min exposure to amiodarone in vitro.
When [Na]pip was 10 mM, mean Ip of 10 myocytes exposed
to (nominally) 10 mM amiodarone in 1% ethanol was 0.19 6
0.02 pA/pF, whereas mean Ip of 10 myocytes exposed to
ethanol alone was 0.31 6 0.03 pA/pF. This 39% reduction was
statistically significant. Mean Ip of myocytes exposed to 1%
ethanol was similar to mean Ip of myocytes from untreated
rabbits not exposed to ethanol (Gray et al., 1997; Whalley et
al., 1993), indicating that 1% ethanol does not affect Ip when
membrane potential is held constant at 240 mV. We performed an additional four experiments to determine that the
duration of exposure was adequate to achieve steady state
effects. In these experiments, myocytes were exposed to amiodarone for $20 min (range, 20 to 28 min) before Ip was
measured. Mean Ip (0.21 6 0.01 pA/pF) was similar to the
mean Ip of myocytes exposed to amiodarone for '6 min only
(0.19 6 0.02 pA/pF). This indicates that steady state was
achieved using 6-min exposure.
To examine whether acute exposure of myocytes to amiodarone in vitro also causes pump inhibition when intracellular Na1 is at high levels, we measured Ip using a [Na]pip of 80
mM. Mean Ip of 6 myocytes was 1.90 6 0.13 pA/pF, which is
not significantly different from mean Ip in 6 control myocytes
(1.94 6 0.05 pA/pF). The mean Ip values measured using a
[Na]pip of either 10 and 80 mM in myocytes exposed to amiodarone and control myocytes are summarized in figure 3A.
We conclude from these experiments that acute exposure to
amiodarone inhibits Na1-K1 pump function when intracellular Na1 is near physiological levels, whereas it has no
Amiodarone Inhibits the Na1-K1 Pump
Fig. 3. A, Effect of acute exposure to amiodarone on Ip, measured using
[Na]pip of 10 mM (open bars) and 80 mM (solid bars). Numbers in parentheses indicate how many myocytes were studied in each group. Exposure
of myocytes to amiodarone significantly reduced Ip when [Na]pip was 10
mM. 1% ethanol alone did not affect Ip. Amiodarone had no effect on Ip
when [Na]pip was 80 mM. B, Effect of [K]o on Ip in myocytes exposed to
amiodarone in 1% ethanol in vitro (F, dashed lines) and in myocytes
exposed to 1% ethanol alone (‚, solid line). Ip has been normalized to the
current measured when [K]o is 5.6 mM. C, Normalized Ip-Vm relationships for 9 myocytes exposed to amiodarone in 1% ethanol (F) and for 8
myocytes exposed to 1% ethanol alone (‚) in vitro.
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Gray et al.
Discussion
The major finding of our study is that both chronic and
acute exposure to amiodarone decreased Na1-K1 pump activity in intact cardiac myocytes. After chronic treatment,
pump activity was reduced at both low and high [Na]pip. This
pattern suggests a decrease in overall pump capacity. Acute
exposure to amiodarone in vitro decreased pump activity only
when [Na]pip was near the physiological level for intracellular Na1 and had no significant effect at levels expected to
saturate intracellular pump sites. This pattern is consistent
with a decrease in the apparent affinity of the pump for
intracellular Na1. A difference in effects between acute and
chronic exposure is also well recognized for the clinical use of
the drug (Ikeda et al., 1984) and for its effects on ion channel
function (Varró et al., 1996). The [Na]pip-dependent difference in effects between chronic and acute exposure suggests
that amiodarone affects the pump via two different mechanisms. With chronic exposure, amiodarone might affect the
pump via both mechanisms: one related to a decrease in the
abundance of Na1-K1 pumps and one due to a direct effect of
the drug in the membrane. However, amiodarone induced a
similar degree of inhibition with chronic and acute exposure
when [Na]pip was 10 mM. This similarity might be explained
by wash-out of amiodarone while cells from treated animals
were maintained in amiodarone-free solutions for 2 to 10 hr
before Ip was measured, with a loss of a direct effect of the
drug bound to the membrane.
Two previous studies have examined the effect of chronic
amiodarone treatment on the Na1-K1 pump. Prasada Rao et
al. (1986) administered amiodarone parenterally to rats for 6
weeks. They found that amiodarone had no effect on activity
of brain synaptosome Na1-K1/ATPase activity. To our
knowledge, a study by Hensley et al. (1994) provides the only
previous data on cardiac tissue. They examined the effects of
3- to 6-week parenteral amiodarone therapy on protein expression and mRNA levels for the alpha and beta subunits of
the Na1-K1 pump in rat ventricular muscle. At 3 weeks,
abundance of the alpha-1, alpha-2 and beta-1 subunits was
significantly decreased. This was accompanied by a 30% to
33% decrease in serum T3 and T4 levels consistent with a
systemic amiodarone-induced hypothyroid state. Because hypothyroidism reduces synthesis of Na1-K1 pump subunits in
the heart (Horowitz et al., 1990, Kamitani et al., 1992), the
decrease in subunit abundance at 3 weeks might be due to
the effect of amiodarone on thyroid status. After 6-week
therapy, T3 and T4 levels and alpha-1 and beta-1 subunit
abundance had returned to control values, whereas the abundance of the alpha-2 subunit remained significantly depressed. Alpha-2 subunits do not appear to be quantitatively
important in the rat, and Hensley et al. (1994) emphasized
that the functional significance of altered alpha-2 expression
was uncertain. The present study does not identify effects of
amiodarone on specific subunits; however, it does indicate
that chronic treatment has a functionally significant inhibitory effect on the sarcolemmal Na1-K1 pump.
Our study indicates that chronic treatment with amiodarone induces a decrease in Ip when the pump is maximally
stimulated. Maximal Ip is highly correlated with the number
of Na1-K1 pump units expressed in the plasmalemma of
voltage-clamped oocytes (Jaunin et al., 1992). If the same
applies to pump units in the sarcolemmal membrane, our
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effect when intracellular Na1 is at levels expected to nearly
saturate intracellular Na1 binding sites. This suggests that
acute exposure to amiodarone reduces the apparent affinity
of the pump for intracellular Na1.
Effect of acute amiodarone on the affinity of the
Na1-K1 pump for extracellular K1. To examine the effect
of acute amiodarone exposure on K1 affinity, we measured Ip
at different levels of [K]o. We used a [Na]pip of 80 mM. After
establishing the whole-cell configuration, we inactivated the
Na1-K1 pump by superfusing myocytes with K1-free Tyrode’s solution. This superfusate also contained amiodarone.
Myocytes were then voltage-clamped at 240 mV, and after a
minimum of 6-min exposure to the amiodarone-containing
superfusate, the pump was reactivated by exposing myocytes
to a superfusate that was similar except that it contained
different [K]o values. In contrast to experiments examining
the effects of chronic amiodarone treatment, each myocyte
was exposed in a random sequence to all of the following [K]o
values (in mM):1, 2, 3, 5.6 and 15. Results were normalized
relative to 5.6 mM [K]o. Each exposure to a [K]o concentration was bracketed by return to the K1-free superfusate until
Ip had returned to its base-line level. Control myocytes were
maintained in the whole-cell configuration for the same duration as amiodarone-exposed myocytes before Ip was measured.
Nine myocytes were acutely exposed to amiodarone in
vitro. When the Hill equation was fitted to the data, we
obtained a K0.5 value of 2.50 mM and a Hill coefficient of 1.84.
To allow statistical comparisons, we fitted data from each
experiment to the Hill equation to derive the respective mean
values for K0.5 and Hill coefficient. There was no significant
difference between the mean values of either for myocytes
exposed to amiodarone or control myocytes. The [K]o/Ip%
relationship for myocytes exposed to amiodarone has been
plotted in figure 3B along with the relationship for control
myocytes. We conclude from these experiments that acute
exposure of myocytes to amiodarone has no effect on the
apparent [K]o affinity of the Na1-K1 pump.
Effect of acute amiodarone on the Ip-Vm relationship. We next examined whether acute exposure of myocytes
to amiodarone affects the Ip-Vm relationship. After establishing the whole-cell configuration, the superfusate was
changed to Ca11-free Tyrode’s solution that contained either
amiodarone in 1% ethanol or 1% ethanol alone. The myocytes
were voltage-clamped at 240 mV; after a minimum exposure
time of 6 min to this superfusate, we applied the voltage step
protocol illustrated in figure 2A before and after exposure to
100 mM ouabain as outlined previously. Because in vitro
exposure had no effect on Ip when [Na]pip was 80 mM, these
experiments were only performed at [Na]pip of 10 mM.
The mean Ip-Vm relationships for 9 myocytes exposed to
amiodarone and 8 myocytes exposed to ethanol have been
plotted in figure 3C. The Ip-Vm relationships in myocytes
exposed to amiodarone and to ethanol demonstrate a positive
slope at negative potentials and a negative slope at positive
potentials. The negative slope at positive potentials is not
seen in control myocytes not exposed to amiodarone or ethanol (Gray et al., 1997). It would thus appear that the negative
slope at positive potentials is due to an effect of the vehicle,
1% ethanol, rather than an effect of amiodarone. We conclude
that acute exposure to amiodarone has no effect on the Ip-Vm
relationship.
Vol. 284
Amiodarone Inhibits the Na1-K1 Pump
1998
may speculate that the effect of amiodarone could be due to
modification of the physicochemical properties of the lipid
membrane. Amiodarone is known to insert into the hydrocarbon core of the lipid bilayer and decrease membrane lipid
mobility and membrane fluidity (Chatelain et al., 1986).
Changes in membrane fluidity, in turn, may affect pump
activity by impairing lateral movement of the pump molecule
and hence conformational changes during the pump cycle
(see Cornelius, 1991, for review). An alternative explanation
may be provided by the cationic amphiphilic nature of amiodarone. Cationic amphiphiles have been shown to decrease
the apparent Na1 affinity of Na1-K1/ATPase reconstituted
into liposomes (Cornelius, 1995).
Chronic treatment with amiodarone reduced Ip by 33%
when [Na]pip was 10 mM. This inhibitory effect is similar to
that estimated for clinical use of digoxin (Rasmussen et al.,
1990; Schmidt et al., 1991). This finding may have implications for our understanding of the effect of the drug when
used clinically. It prolongs action potential duration and
myocardial refractoriness, effects that are believed to be
largely mediated via block of voltage-gated K1 channels
(Balser et al., 1991). The Na1-K1 pump generates a hyperpolarizing membrane current that contributes to repolarization. This effect becomes more significant as intracellular
Na1 increases, for example, during tachyarrhythmias
(Gadsby, 1982). The inhibitory effect of amiodarone on the
pump is therefore expected to contribute, at least in part, to
the class III effects of the drug.
Acknowledgments
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results suggest that amiodarone induces a decrease in the
number of functional Na1-K1 pump units. The amiodaroneinduced decrease in pump function occurred in the absence of
systemic hypothyroidism. However, this does not rule out the
possibility that amiodarone exerted its effect on the pump via
interference with thyroid hormone action at the intracellular
level because amiodarone inhibits the binding of T3 to its
nuclear receptors (Drvota et al., 1994; Latham et al., 1987).
The pattern of pump inhibition is similar to that reported by
Doohan et al. (1995) in rabbits with experimentally induced
hypothyroidism. It is also of interest to note that some of the
electrophysiological effects of amiodarone, including prolongation of action potential duration, QT interval and sinus
cycle length, are at least partly dependent on thyroid hormone activity and are greatly diminished or abolished by
concurrent hypothyroidism (Polikar et al., 1986; Talajic et al.,
1989).
Several previous studies have reported inhibition of purified cardiac Na1-K1/ATPase, the enzymatic equivalent of the
Na1-K1 pump, during acute exposure to amiodarone in vitro
(Broekhuysen et al., 1972; Dzimiri and Almotrefi., 1991).
Although these studies suggest that acute amiodarone exposure may inhibit the pump, their significance is unclear for
several reasons. Isolation of Na1-K1/ATPase from the sarcolemmal membrane necessarily involves removing the
pump molecule from its native lipid environment. Because
the lipid environment is an important regulator of pump
activity (Cornelius, 1991) and may itself be influenced by
amiodarone (Chatelain et al., 1985, 1986), the response of
isolated Na1-K1/ATPase to amiodarone may not be representative of the behavior of the pump in the intact myocyte.
In the studies by Broekhuysen et al. (1972) and Dzimiri and
Almotrefi (1991), Na1-K1/ATPase was examined using saturating concentrations of Na1 and K1 to produce maximal
pump activity. They therefore provide no information about
the effect of amiodarone on the activity of the pump at physiological ligand concentrations. Finally, the concentrations of
amiodarone necessary to induce significant ATPase inhibition were substantially higher than those encountered clinically.
In the only published study on intact cardiac muscle, Aomine (1989) indirectly determined the acute effects of amiodarone on pump activity in guinea pig papillary muscles
using conventional microelectrode techniques. He found that
exposure to 44 mM amiodarone decreased the postoverdrive
hyperpolarization attributed to pump activity. Somewhat
paradoxically, no effect was observed at the higher concentration of 440 mM.
Our in vitro study differs from previous studies in several
aspects. We examined the effect of 0.3 to 1.2 mM amiodarone,
a concentration range similar to that encountered clinically,
and we examined the pump in its intact membrane while
varying extracellular and intracellular ligand concentrations. In contrast to the studies on isolated ATPase, we found
no effect on Ip measured under conditions expected to maximally stimulate the pump. However, pump inhibition could
be demonstrated when we used a [Na]pip near physiological
levels for intracellular Na1. This is consistent with a decrease in the apparent affinity of the pump for intracellular
Na1 without an influence on overall pump capacity.
Although the mechanism for modulation of the Na1 affinity of the pump cannot be determined from our study, one
81
Amiodarone was a gift from Sanofi Winthrop Australia. The assistance of Dr. Annette Gross in performing serum amiodarone measurements is gratefully acknowledged.
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Vol. 284
Send reprint requests to: Dr. David Whalley, Cardiology Department, Royal
North Shore Hospital, Pacific Highway, St. Leonards, 2065, Sydney, Australia.