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Introduction
Papaverine (6,7-dimethoxy-1-veratrylisoquinoline), a
substituted alkaloid from Papaver
somniferum, has been known to relax many kinds of smooth muscles. Because of its
relaxant effects on smooth muscle, papaverine has been used as
a vasodilator agent[1_3] and a therapeutic agent for renal
colic[4] and penile
impotence[5]. It was also proposed as an "ideal
coronary vasodilator"[1]. Additionally, intra-arterial
papa-verine infusion has been used for the prevention and
treatment of vasospasm following subarachnoid
hemorrhage[6,7]. However, under certain clinical settings, such as cases of
overdose, papaverine induced cardiac arrhythmias
including prolonged QT intervals that can lead to the
life-threatening ventricular arrhythmia, torsades de
points[8_11].
One means by which drugs can prolong QT intervals is
through the inhibition of one or more repolarizing
K+ channel currents in the
myocardium[12,13]. In humans, the most
common channel linked to drug-induced QT interval
prolongation is the rapid component of the delayed rectifier
K+ channel
(IKr)[14]. The human ether-a-go-go-related gene,
HERG, encodes the pore-forming subunit of
IKr[15,16]. Naturally, many drugs associated with QT interval
prolongation have been found to block HERG
channels[17_21]. It can be assumed therefore that papaverine may cause LQT
by inhibiting HERG/IKr. To examine this possibility, we
investigated the effect of papaverine on HERG currents
expressed in Xenopus oocytes. We found that papaverine
effectively inhibited HERG currents in a state-independent
manner in Xenopus oocytes.
Materials and methods
Recording of the action potential in rabbit cardiac
myocytes Rabbits (Male, New Zealand white rabbits,
weighing 1.8_2.0 kg each) were anesthetized by injections of
thiopental sodium (10 mg/kg) into a marginal ear vein. Their
hearts were rapidly excised and transferred to Tyrode's
solution (137 mmol/L NaCl, 5.4 mmol/L KCl, 1.05 mmol/L
MgCl2, 1.8 mmol/L CaCl2, 5 mmol/L glucose, and 10 mmol/L HEPES
at pH 7.4), and the hearts were oxygenated with 100%
O2 at room temperature. The ventricular muscles (1.5_2 mm in
width and 2_4 mm in length) were carefully dissected from
the left ventricular wall. All tissues were less than 1 mm in
depth.
Each specimen of the dissected tissue was mounted
horizontally in a recording chamber, and continuously
super-fused with oxygenated Tyrode's solution at a rate of 5
mL/min. One end of each tissue was fixed by an insect pin to
the bottom of the chamber coated with Sylgard. The tissue
next to the insect pin was pressed against the chamber floor
by stimulation electrodes, which were used to elicit the
action potentials. The cardiac tissues were stimulated with
square pulses (2 Hz, 1 ms duration, 20%_30% above the
threshold voltage) using a stimulator with a stimulus
isolation unit (WPI, Sarasota, FL, USA). After allowing 2 h for
stabiliza-tion, the action potentials were recorded with a
3 mol/L KCl-filled conventional microelectrode (20_30
MΩ) that was connected to an amplifier (KS-700, WPI, USA), and
these action potentials were displayed on an oscilloscope
(Dual beam storage 5113, Tektronix, Beaverton, OR, USA).
After 1 h of stabilization of recording, the drugs were applied.
Oocyte preparation Oocytes were prepared as described
previously[22]. Briefly, ovarian lobes excised from the
anesthetized Xenopus laevis (Xenopus I, USA) were treated with
0.2% collagenase (type II, Sigma Co, St Louis, MO, USA) for
1_2 h in Ca2+-free Barth's solution. The composition of
Ca2+-free Barth's solution is as follows: 88.7 mmol/L NaCl, 1.0
mmol/L KCl, 2.4 mmol/L NaHCO3, 0.8 mmol/L
MgSO4 7H2O, and 5 mmol/L HEPES (pH=7.5). cRNA (50 nL, 0.3_1.0 ng/nL)
of HERG synthesized from the linearized cDNA using
in vitro transcription kit (Ambion Inc, Austin, TX, USA) was injected
in stage V or VI oocytes with glass capillaries connected
with a microdispensor (VWR Scientific Co, Grove, IL, USA).
After the injection, oocytes were cultured at 18 ºC in Barth's
solution containing 88.0 mmol/L NaCl, 1.0 mmol/L KCl, 2.4
mmol/L NaHCO3, 0.8 mmol/L
MgSO4 7H2O, 0.3 mmol/L
Ca(NO3)2 4H2O, 0.4 mmol/L
CaCl2, and 5 mmol/L HEPES (pH=
7.5), supplemented with 2 mmol/L pyruvate and 50 g/mL
gentamicin sulfate. The culture medium was changed daily.
Currents were recorded from 2 to 7 d after injection.
Whole cell current recording in
Xenopus oocytes HERG currents were recorded using a 2-electrode voltage-clamp
amplifier (OC-725C, Warner Instruments, Hamden, CT, USA)
from the oocytes placed in the recording chamber (2.0 mL)
superfused with Oocyte-Ringer solution containing 96.0
mmol/L NaCl, 2.0 mmol/L KCl, 1.0 mmol/L
MgCl2, 1.8 mmol/L CaCl2
2H2O, and 5.0 mmol/L HEPES (pH=7.5). Stimulation
and data acquisition were controlled with Digidata 1200
(Axon Instruments) and pClamp 6.04 (Axon Instruments,
USA). Electrodes were fabricated from glass capillaries
containing an inner filament (OD 1.5 mm, ID 1.12 mm; WPI, USA).
Electrodes filled with 3 mol/L KCl had a resistance of 1_2 for
current-passing electrodes and 2_4 for voltage-recording
electrodes.
Data analysis The voltage dependence of HERG current
activation was determined for each oocyte by fitting peak
values of the tail current
(Itail) versus test potential
(Vt) to a Boltzmann function:
Where Itailmax is the maximum tail current,
V1/2 is the voltage at which 50% of the channels are activated, and
k is the slope factor. To examine steady-state inactivation,
conditioning pulses between -130 and +20 mV in 10 mV increments
for 60 ms were applied after a depolarizing pulse to +20 mV
for 900 ms, followed by a common test pulse to +20 mV. The
peak current amplitudes during the test pulses were plotted
as a function of the previous conditioning pulses.
Normalized steady-state inactivation as a function of prepulse of
test potential was also fitted to a Boltzmann function. The
data were expressed as the mean±SEM. Paired Student's
t-test was used to compare the drug effect on HERG currents.
ANOVA repeated measures, followed by Tukey's
post-hoc tests were used to study concentration-dependent drug
effects.
Drugs All drugs were purchased from Sigma Co (St Louis,
USA). Papaverine was dissolved in 10_3 mol/L stock
solution and stored at 4 ℃ until dilution within the perfusion
solution immediately before use.
Results
To identify the general electrophysiological effect of
papaverine on cardiac action potential, we examined the
effects of papaverine on cardiac action potential in isolated
rabbit ventricular myocytes stimulated at a frequency of
2 Hz (Figure 1). After 20 min of exposure, the action potential
duration at 90% repolarization (APD90) in the ventricular
myocytes increased from 219±5 ms to 267±6 ms and 355±18
ms (n=4) by 10 and 30 µmol/L papaverine, respectively.
Resting membrane potential was depolarized from -81±3 mV to
-77±4 mV (n=3) by 30 µmol/L papaverine, but not by 10
µmol/L papaverine (-81± 5 mV, n=4).
The effect of papaverine on HERG currents was studied
in Xenopus oocytes injected with HERG mRNA. Figure 2A
shows voltage-clamp data obtained 10 min after application
of 100 µmol/L of papaverine. Families of current traces from
1 cell are shown for control conditions and after exposure to
papaverine with the voltage protocol shown in Figure 2A.
Cells were clamped at a holding potential of -80 mV.
Depolarizing steps were applied for 4 s to voltages between -70
and +40 mV in 10 mV increments. Papaverine (100 μmol/L)
suppressed both the outward and tail currents (Figure 2A),
which was only partially (72%±8% of the
control, n=4) recovered in the 30 min drug wash-out. Current-voltage plots
of outward currents present at the end of the depolarizing
step and peak tail current are depicted in Figure 2B and 2C,
respectively. Current amplitudes were normalized to peak
values obtained from each cell. For control conditions, the
threshold for activating HERG currents was close to -40 mV,
and full activation was obtained at a voltage close to +10
mV. In the presence of 100 μmol/L of papaverine, outward
currents at the depolarizing step and tail current amplitude
were reduced, compared with control condition, to ~40% at
all tested potentials.
Papaverine blocked HERG currents in a
concentration-dependent manner Steady-state block was obtained by
applying depolarizing steps from -80 to 0 mV for the 10 s
period every 30 s, and peak tail current was measured after
the repolarizing steps to -40 mV for 6 s at different drug
concentrations. Analysis of the data with the Hill equation
gave a half-maximal inhibitory concentration
(IC50) value of 71.0 μmol/L and Hill coefficient of 0.81 (Figure 3).
Papaverine is known as a phosphodiesterase inhibitor.
To determine whether the effect of papaverine on HERG was
related to phosphodiesterase inhibition, the effects of
theophylline, another phosphodiesterase inhibitor, on HERG
currents was investigated. Theophylline (500 μmol/L) did
not affect HERG currents (Figure 4A), which were completely
blocked by dofetilide (Figure 4B), known to be a selective
IKr blocker, suggesting that the papaverine block on HERG is
independent from the phosphodiesterase inhibition.
Drugs that block ion channels often alter the voltage
dependence of channel kinetics. Therefore, we also
analyzed the voltage dependence of the activation of the peak
amplitude of the decaying tail currents in the absence or
presence of papaverine (100 μmol/L). The voltage
dependence of the HERG activation was determined using 4 s test
pulses to potentials ranging from -70 to +40 mV. Peak
amplitudes of tail currents were measured at -40 mV, plotted as a
function of test potential, and were fitted with a Boltzmann
function (Figure 2C). In the control experiment, the
activation curve had a midpoint of -16.4±1.1 mV and a slope factor
of 6.8±0.4 mV (n=6). There was no significant change in
voltage dependence of the activation curve
(V1/2 -16.5±2.4 mV and a slope factor of 5.8±1.5 mV,
n=6, P>0.05 vs the control in both) by papaverine.
We also investigated the effects of papaverine at a
concentration of 100 µmol/L on the inactivation of HERG currents.
Steady-state inactivation currents were studied before and
after the application of papaverine using a dual-pulse
protocol as described in Materials and methods. The
steady-state inactivation curve was shifted to a negative potential
(V1/2 -68.7±3.2 mV in the control and -77.4±1.6 mV after
papaverine, P<0.05, n=5; Figure 5). The slope factor of
steady-state inactivation decreased slightly from 24.3±2.9
mV in the control to 17.2_1.3 mV in the presence of
papaverine.
To directly test the effect of papaverine on HERG
inactivation, we examined the time course of inactivation in
the absence and presence of papaverine. The inactivation
time course of expressed currents was analyzed by applying
brief hyperpolarizing pulses to allow the HERG channel to
recover from inactivation after an initial long depolarizing
pulse. Depolarizing test pulses were then applied to record
inactivating currents (Figure 5C). The time course of fast,
inactivating currents could be fitted by a single-exponential
function. In the presence of papaverine, the time constants
for inactivation significantly decreased at all tested
potentials (Figure 5D). Recovery from inactivation was also
measured using the same pulse protocol shown in Figure 4. Tail
currents could be fitted by a double-exponential function,
and the fast component was defined as the time constant of
recovery from inactivation. In the presence of papaverine,
the time constants for inactivation and recovery from
inactivation significantly decreased at all potentials (Figure 5D).
To further delineate the underlying mechanism for
papaverine inhibition of HERG currents, we examined the fully
activated I-V relationships by applying various test
potentials after a depolarizing conditioning pulse (Figure 6). The
slope conductance measured as a slope of the
current-voltage relationship curve between -130 and -110 mV decreased
from 78.03±4.25 µS of the control to 56.84±5.33, 36.06±6.53,
and 27.09±5.50 µS (n=6) in the presence of 30, 100, and 300
µmol/L papaverine, respectively (P<0.05
vs the control at each concentration).
On the basis of single-channel studies, a 5-state model
has been suggested for HERG channels summarizing 1 open,
1 inactivated, and 3 closed states[23]. Therefore, we
investigated whether papaverine block is state dependent (Figure
7). After the control currents were measured, the membrane
potential was held at -80 mV to keep the channel in the closed
conformation during wash-in of 100 µmol/L of papaverine.
The first depolarization to 0 mV after 10 min at -80 mV yielded
most of the blocking effect. The initial current
inhibition (61.0%±4.6%, n=4) reflects that papaverine bound HERG
without channel opening and/or activation, whereas the
additional time-dependent inhibition (4.8%±2.2%,
n=4) observed after channel activation may represent additional
papaverine inhibition on HERG channels during the open state.
Discussion
In the present study, we show that a clinical vasodilator,
papaverine, increases cardiac action potential duration and
inhibits cardiac K+ channel HERG expressed in
Xenopus oocytes. Papaverine has been shown to prolong QT
intervals and cause ventricular
arrhythmia[11]. Many commonly used drugs, including antiarrhythmic, antihistamine,
antipsychotic, and antibiotic agents are associated with
drug-induced LQTS. Most of these drugs either block
HERG-dependent K current (IKr) in ventricular myocytes or inhibit
liver enzymes that are important for metabolic degradation
of other drugs that block IKr. It is well known that
heterologously-expressed HERG currents share pharmacological and
biophysical properties with
IKr[15,16]. The characteristics of
the currents recorded in the present study correspond to
HERG currents: slow current activation at negative potentials,
large long-lasting tail currents on repolarization, strong
inward rectification, and sensitivity to class III antiarrhythmic
methanesulfonanilide such as dofetilide (Figure 4).
Considering all of these, papaverine may cause LQT through the
inhibition of HERG/IKr in cardiac myocytes. The present
study is the first to characterize the interaction between
papaverine and HERG showing an ionic mechanism
underlying papaverine-induced ventricular arrhythmia.
The major finding of the present study is that papaverine
blocks HERG channels in a concentration-dependent, but
voltage- and state-independent manner in
Xenopus oocytes. Taken into account that
in vitro data is influenced by several artificial factors such as expression system properties,
temperature, and lack of subunits, the degree of HERG
blocked by papaverine seems appropriate. The affinity of
ion channel blockers is reduced in Xenopus oocytes due to
follicular tissues and the yolk[24]. Consistent with this,
papaverine inhibited HERG currents with an
IC50 of ~70 µmol/L in Xenopus
oocytes (Figure 3), and significantly increased
action potential duration in rabbit myocytes at 10 µmol/L of
papaverine (Figure 1). This suggests the different
pharmacokinetics of papaverine in the 2 different cells. However,
we cannot exclude the possibility that papaverine may affect
other ionic currents[25,26] in addition to the blockage of
IKr, for it depolarized resting membrane potential in ventricular
myocytes. Further evaluation of IC50 of papaverine on
IKr in ventricular myocytes will be helpful to address it.
The state-dependence of HERG channel block has been
described for numerous substances, such as
bertosamil[27],
fluvoxamine[28], and
KCB-328[29] with the majority of those binding to the channel in the open state. Choe
et al[25] provide evidence for open channel blockade by papaverine in
their studies of Kv1.5 stably expressed in Ltk- cells by
demonstrating the accelerated decline of the current during
depolarization, as well as steeper blockade in the voltage
range of the channel opening. The Kv1.5 current is a rapidly
activating and relatively slow inactivating delayed rectifier
current, and this differs significantly from the HERG channel.
In the present study, papaverine did not modify the time
course of channel activation. If present, a tail current
"crossover", due to transient channel unblocking during
repolarization[30], provides evidence for open channel block.
This was not seen in the present study. Depolarization of
membrane potential did not increase HERG current
inhibition by papaverine at which most channels would be in the
inactivated state. Consistent with the tonic block achieved
without channel opening or activation (Figure 7), these
results suggest that papaverine mainly bind closed HERG
channels.
Various drugs inhibit HERG currents binding to the
inactivated HERG channel[32]. Sites in the outer pore region such
as G628, G631, S620, A614, and V630 have been known to
involve HERG channel inactivation, which also postulated
as a binding site(s) of the drug acting with the inactivated
HERG channel[33]. H587 of the S5-P
loop[34] and M651 of the S6
sites[35] also involved HERG inactivation kinetics.
Papaverine markedly accelerated inactivation at most potentials,
consistent with the observed 9 mV hyperpolarizing shift in
the voltage-dependence of HERG channel inactivation, which
implies a decrease in channel
availability[31]. Papaverine also markedly increased the rate of recovery from inactivation
(Figure 5D). These data imply that the drug acts on
inactivated HERG. Therefore, we may speculate that these sites
involved in HERG inactivation at least partly contribute to
HERG inhibition by papaverine.
There is no consensus about confirming the threshold
for the predication of clinically important HERG channel
blockade based on an in vitro model. One hypotheses is
that drugs will be free of liability for the ventricular
arrhythmia torsades de pointes if they fail to produce a 20%
blockade at the highest achievable free plasma
concentration[36]. Another hypothesis is that drugs for which the
IC50 is at least 30-fold greater than the highest achievable free or total
plasma concentration will be free of liability for torsades de
pointes[18,36]. Although limited data are available with regard
to expected maximum plasma concentrations after
administration of papaverine, it reached to 49.4_56.6 µmol/L with
various carriers in rabbits[37]. The values are similar to
papaverine IC50 on HERG block even in
Xenopus oocytes. Although maximum
Cmax values would be useful in estimating a
worst-case clinical relevance of HERG channel blockade, it is
notable that papaverine did cause arrhythmia in
humans[8_11].
In summary, this report is the first to detail the effects of
papaverine on HERG, explaining cellular mechanism for QT
prolongation. We found that papaverine blocked HERG
currents via binding closed, open, and inactivated channels.
These blocking properties may contribute to the
arrhythmo-genic properties of papaverine.
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