Departments of Pharmacology (C.I.S., J.J.B., R.Z.K.), Physiology,
and Cardiovascular Research Laboratories (K.H.Y., J.C.H.), School of
Medical Sciences, University of Bristol, University Walk, Bristol,
United Kingdom
Pyrethroid insecticides are known to modify neuronal sodium channels,
inducing persistent, steady-state sodium current at depolarized
membrane potentials. Cardiac myocytes are also rich in sodium
channels but comparatively little is known about the effect of
pyrethroids on the heart, or on the cardiac sodium channel isoform. In
the present study therefore, we determined the actions of type I and
type II pyrethroids against rat and guinea pig ventricular myocytes
under current and voltage clamp, and on isolated perfused rat hearts.
In myocytes, tefluthrin (type I) and fenpropathrin and 
-cypermethrin
(type II) prolonged action potentials and evoked afterdepolarizations.
The time course of sodium current (INa) was also prolonged
by these compounds. Pyrethroids delayed INa inactivation,
when measured under selective conditions as current sensitive to 30 µM tetrodotoxin, by increasing the proportion of slowly inactivating
current at the expense of fast inactivating current. Further
experiments, focusing on fenpropathrin, revealed that its effects on
INa inactivation time course were dose-dependent, and the
Na+ "window-current" was increased in its presence. In
unstimulated, isolated hearts perfused with the same pyrethroids, the
variability in contraction amplitude increased due to variations in the
intervals between heartbeats. These potentially arrhythmogenic changes
are consistent with the effects observed at the cellular level. The type I pyrethroid tetramethrin had little effect in any of the preparations. These findings suggest that some pyrethroids possess considerable mammalian cardiac arrhythmogenic potential, the
manifestation of which in vivo may depend on the route of exposure.
 |
Introduction |
Sodium
channels in excitable tissues can be modified by lipid-soluble toxins
produced by a variety of venomous animals and poisonous plants
(Honerjäger, 1982
; Hille, 1992; Wang and Wang, 1998). Included
within this group are pyrethrins, which are insecticidal esters
isolated from Chrysanthemum species, and pyrethroids, their synthetic derivatives. The synthetic compounds are divided into two
groups on the basis of differences in chemical structure (Gammon et
al., 1981; Valentine, 1990). Type I pyrethroids have no
-cyano-3-phenoxybenzyl group and appear to act principally on
sensory nerves; the type II pyrethroids have an
-cyano-3-phenoxybenzyl group and appear to preferentially affect
motor nerves. Pyrethroid modification of insect neuronal
Na+ channels evokes prominent
afterdepolarizations that prolong the action potential duration (Song
and Narahashi, 1996; Lee et al., 1999). These actions cause paralysis,
leading ultimately to insecticidal activity (Gammon et al., 1981).
It has been calculated that pyrethroids account for roughly 25% of
global insecticide sales (Williamson et al., 1996), and environmental
exposure to these widely used compounds is quite common. It is also
known that pyrethroid toxicity encompasses aquatic vertebrates and
mammals that may be accidentally exposed. As in insects, the primary
mode of pyrethroid action in vertebrates involves the modification of
neuronal Na+ channels. During depolarizations,
pyrethroid-modified neuronal Na+ channels carry a
slowly inactivating Na+ current
(INa) that increases in amplitude during
pyrethroid exposure. Pyrethroid modification also gives rise to a
slowly declining inward Na+ tail current upon
membrane repolarization (Chinn and Narahashi, 1986; deWeille and
Leinders, 1989; Holloway et al., 1989; Song and Narahashi, 1996; Vais
et al., 2000). In addition, both voltage-dependent activation and
inactivation of Na+ channels are shifted to
hyperpolarized potentials by pyrethroids (Narahashi et al., 1995;
Trainer et al., 1997). By these mechanisms, pyrethroid-modified
Na+ channels underlie the prominent neuronal
afterdepolarizations that produce typical toxic effects (Song and
Narahashi, 1996; Lee et al., 1999).
The selectivity and relative toxicity of pyrethroids in neuronal tissue
from different species has been rather well researched (Valentine,
1990; Narahashi, 1996; Tabarean and Narahashi, 1998; Motomura and
Narahashi, 2000), but the potential cardiotoxicity of this group of
compounds appears to have been little studied. It is known that brain
tissue contains multiple Na+ channel isoforms
encoded by different genes (e.g., SCN1A-3A) with still other isoforms
present in the peripheral nervous system (for review, see Clare et al.,
2000). There is also a specific cardiac Na+
channel isoform (encoded by SCN5A), which exhibits distinct
voltage-dependent kinetic properties and pharmacology (Krafte et al.,
1991; Fozzard and Hanck, 1996; Balser, 1999). The cardiac channel has
important clinical significance in that its mutations are implicated in malignant cardiac arrhythmias (Wang et al., 1995; Chen et al., 1998).
However, despite the importance of this cardiac
Na+ channel, research into its interactions with
pyrethroids has been almost entirely lacking. Studies in whole cardiac
tissue showed that pyrethroids are positively inotropic, possibly as a
result of depolarization of myocardial sympathetic nerve terminals and
the resulting effects of released catecholamines (Forshaw and Bradbury,
1983; Berlin et al., 1984). However, an additional inotropic effect was
attributed to Na+ loading of the cardiac tissue,
possibly resulting from pyrethroid modification of native cardiac
Na+ channels. In support of this finding are
limited data suggesting that single cardiac Na+
channels are susceptible to modification by the type II pyrethroid deltamethrin (Grant et al., 1993). However, no detailed
electrophysiological characterization of the effects of pyrethroids on
macroscopic cardiac Na+ current has so-far been
carried out. In the present study therefore, we have evaluated the
actions of pyrethroids on isolated mammalian ventricular cells and
whole hearts. Our results reveal that these agents can prolong action
potentials in ventricular myocytes and, under voltage clamp, prolong
the duration of INa. In spontaneously beating
isolated hearts, pyrethroids increased the contractile variability,
consistent with considerable arrhythmogenic potential for this class of compounds.
| |
Materials and Methods |
Isolated Rat Ventricular Myocytes.
Ventricular myocytes were
isolated from hearts of male Wistar rats (~200 g) killed by
intraperitoneal injection of pentobarbitone sodium (200 mg/kg). The
isolated heart was perfused at 37°C on Langendorff apparatus and
myocytes were enzymatically isolated (Spencer et al., 2000). Briefly,
this involved 5 min of perfusion with a nominally
Ca2+-free solution (see below) containing 0.25 mg/ml collagenase (Worthington type II, 215 units/mg), 0.05 mg/ml
protease (Pronase), and 0.3 mg/ml bovine serum albumin. Chopped
ventricular fragments were gently agitated in perfusion solution for
approximately 30 min. Dissociated myocytes were decanted at 5-min
intervals and refrigerated at 4°C until use (<8 h).
Electrophysiology of Rat Ventricular Myocytes.
All myocytes
were whole cell voltage-clamped at 
40 mV using an Axopatch 200A
amplifier and single patch electrodes with resistances of 2.5 to 5 M
. Coupling to pCLAMP software (version 6.0) was used to observe
"signature currents" evoked by a voltage-clamp step to 90 mV for
10 ms followed by a linear membrane potential ramp to +70 mV (Spencer
et al., 2000). In current-clamp mode, action potentials were evoked by
brief suprathreshold current pulses determined individually for each
myocyte. All electrophysiological experiments were performed at room
temperature (20-25°C) and a basic stimulation frequency of 0.33 Hz
was used throughout. Continuous data recordings were made during 3 to 5 min of superfusion with pyrethroids followed by a comparable washout
period (unless voltage control was lost). Bath solutions were
completely exchanged within 2 min. All signals were initially digitized
at 10 kHz and stored on digital audiotape. In off-line analysis,
current and voltage were sampled at 1 kHz and low-pass filtered at 500 Hz.
Solutions for Rat Myocytes.
Unless stated, all chemicals
were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Cell
isolation solution contained the following: 135.0 mM NaCl, 5.4 mM KCl,
1.0 mM MgCl2, 0.3 mM NaH2PO4, and 10.0 mM HEPES
(neutralized to pH 7.2 with NaOH). For initial removal of blood from
the heart and for storage of the myocytes, 0.2 mM
Ca2+ was added to this solution. Isolated
myocytes were superfused with modified Tyrode's solutions containing
145.0 mM NaCl, 4.0 mM KCl, 1.0 mM MgCl2, 2.0 mM
CaCl2, 10.0 mM D-glucose, and 10.0 mM
HEPES (neutralized to pH 7.4 with NaOH). Pyrethroids (Reidel-de Haen,
St. Louis, MO) were dissolved in dimethyl sulfoxide to
make 50 mM stock solutions. Aliquots (<0.05% by volume) were added to
superfusion solutions immediately before use to give a final concentration of 10 µM. The effects of two type I pyrethroids (tefluthrin and tetramethrin) and two type II pyrethroids
(fenpropathrin and -cypermethrin) were studied. Readers are referred
to the Sigma-Aldrich on-line catalog (www.sigma-aldrich.com) for the full chemical names of these compounds. Cells were dialyzed with pipette solution that had the following composition: 75.0 mM
K-glutamate; 30.0 mM
K-piperazine-N,N'-bis[2-ethanesulfonic acid]
(PIPES), neutralized to pH 7.1 with KOH; 20.0 mM KCl, 0.5 mM
MgCl2, 0.05 mM K2-EGTA,
10.0 mM Mg-ATP, 5.0 mM Tris-phosphocreatine, 0.1 mM Tris-GTP, and 5.0 mM pyruvic acid.
Isolated Guinea Pig Myocytes.
Myocytes were also isolated
from male guinea pigs (~400 g). Hearts were digested using
collagenase and protease enzymes in an enzymatic dispersion method
broadly similar to that described above (Levi and Issberner, 1996). The
guinea pig cells were stored at room temperature in solution containing
1 mM Ca2+ until use. Cells remained viable for up
to 8 h after isolation.
Guinea Pig Myocyte Electrophysiology.
Isolated ventricular
myocytes were superfused at 37°C in ramp experiments; for selective
INa recordings, experiments were conducted at
room temperature (20-22°C). Patch pipettes (Corning 7052 glass; A-M
Systems, Everett, WA) were pulled (P-87; Sutter Instrument Co., Novato,
CA) and polished (Narishige MF-83 microforge) to resistances of between
2 and 3 M for voltage-ramp experiments. Whole cell voltage-clamp
recordings were made using an Axopatch 200B amplifier and cell
capacitance was measured by either analyzing the charging transients
elicited by a 5-mV voltage step or reading the capacitance value from
the dial on the amplifier after compensating for series resistance and
cell capacitance. These methods have been shown previously to give
similar values for cell capacitance (Hancox et al., 1993). Series
resistance values were in the range of 2 to 4 M. Typically, 75 to
80% of series resistance could be compensated.
For the selective recording of INa, pipettes had
resistances of between 1 and 2 M to facilitate intracellular
dialysis and to minimize voltage errors due to uncompensated series
resistance. These experiments were also conducted at room temperature
to slow the kinetics of the current. To further facilitate quantitative recordings of INa, cesium-based internal and
external solutions were used to block contamination from
K+ currents. Also, to reduce the transmembrane
gradient for Na+ entry (thereby reducing the size
of the current), these solutions had similar levels of sodium.
Nitrendipine (20 µM) was included in the external solutions to block
any contamination from L-type Ca2+ current, and
BAPTA was included in the internal solution to prevent contamination
due to the internal calcium transient. The liquid junction potential
between the pipette and external solutions was <2 mV in all cases and
no corrections were made. External superfusate was changed using a
rapid solution-switching device (Levi et al., 1996). For most
experiments, INa was measured selectively as the
difference current following application of tetrodotoxin (TTX). This
minimized the likelihood that current records were contaminated by
residual uncompensated capacitative current. Moreover, recent
experiments in our laboratory demonstrated that parameters of
INa recorded from guinea pig ventricular myocytes
with these solutions were similar between "control" and
"partially inhibited" INa. This suggests that
voltage clamp is adequately controlled during flow of
INa using this method (Yuill et al., 2000).
Solutions for Guinea Pig Myocytes.
Myocytes were initially
superfused with modified Tyrode's solution containing 140.0 mM
NaCl, 4.0 mM KCl, 2.5 mM CaCl2, 1.0 mM
MgCl2, 10.0 mM D-glucose, and 5 mM
HEPES (adjusted to pH 7.45 with NaOH). Signature currents were
evoked as described above using a K+-based
pipette (internal) solution, which contained 10.0 mM NaCl, 113.0 mM
KCl, 0.4 mM MgCl2, 5.0 mM
K2ATP, 5.0 mM D-glucose, 10.0 mM
HEPES, and 5.0 mM BAPTA (adjusted to pH 7.2 with KOH). For quantitation
of INa, the pipette solution contained 130.0 mM
CsCl, 10.0 mM NaCl, 0.4 mM MgCl2, 5.0 mM Mg-ATP,
5.0 mM glucose, 10.0 mM HEPES, and 5.0 mM BAPTA (adjusted to pH 7.3 with CsOH). The external solution for these experiments contained 130.0 mM CsCl, 10.0 mM NaCl, 1.2 mM MgCl2, 1.0 CaCl2 mM, 11.0 mM D-glucose, and 20.0 mM HEPES (adjusted to pH 7.3 with CsOH). Pyrethroids were treated as
described earlier. TTX (Tocris, Bristol, UK) was dissolved in deionized
water (3 mM) and added directly to superfusion solutions as necessary
to give a final concentration of 30 µM.
Data Analysis and Statistics.
Voltage- and current-clamp
protocols were generated using the program Winwcp (version 1.7a;
written and supplied by John Dempster, Strathclyde University, Glasgow,
UK) via a Digidata 1200B interface (Axon Instruments, Foster City, CA).
Data were recorded on-line at 2 kHz, except INa
data for which a recording frequency of 10 kHz was used. Data were
stored on the hard disk of an IBM compatible PC, and analyzed using
Winwcp. Figures were constructed using FigP (Biosoft, Cambridge,
UK), and statistical analysis performed using EXCEL (Microsoft,
Redmond, WA).
Data from both rat and guinea pig myocytes are shown as mean ± S.E.M. and statistical comparisons were made using Student's t test. A p value of <0.05 was taken as
statistically significant.
Isolated Perfused Rat Hearts.
A limited series of
experiments was performed to examine the effects of pyrethroids on
Langendorff-perfused isolated rat heart preparations. In these
experiments, the hearts were retrogradely perfused at 38°C with a
modified Krebs-Henseleit solution, equilibrated with
O2, CO2 (95%, 5%),
containing 118.5 mM NaCl, 25.0 mM NaHCO3, 1.2 mM
MgSO4, 1.2 mM
KH2PO4, 3.0 mM KCl, 2.5 mM
CaCl2, 11.1 mM D-glucose, pH 7.4. Pyrethroids were added to this perfusion solution at 10 µM as
described for the myocyte experiments. Control experiments were also
performed in which 10 µM propranolol was included in the perfusion
solution to eliminate catecholamine release from myocardial sympathetic
terminals. In all experiments, each heart was allowed to stabilize for
a 10-min period before pyrethroid perfusion was started. Ventricular
contractions, recorded via a strain gauge, were digitized at 400 Hz
using proprietary software (Chart; AD Instruments, NSW, Australia).
Peak contractile force was output in ASCII files and analyzed using
in-house software (Borg et al., 2001).
| |
Results |
Rat Ventricular Myocytes.
Initial characterization of
pyrethroid effects on rat ventricular cells was achieved by examining
the profiles of action potentials (APs) before and after adding
pyrethroids (at 10 µM) to the superfusion solution. Each panel of
Fig. 1 shows two examples of superimposed APs for each of four pyrethroids studied. Single APs recorded under
control conditions and in the presence of fenpropathrin (Fig. 1a),
-cypermethrin (Fig. 1b), tefluthrin (Fig. 1c), or tetramethrin (Fig.
1d) are compared. With the exception of tetramethrin, all pyrethroids
markedly prolonged the late phase of AP repolarization. APs often had a
secondary upward voltage deflection after the initial rapid
repolarization phase (Fig. 1b, right; and c). In addition, early
afterdepolarizations frequently occurred (Fig. 1a, left; b; and c,
right). The timing of single or multiple afterdepolarizations with
respect to the AP upstroke appeared to vary randomly. Consistent changes in resting membrane potential were absent, and occasional APs
without afterdepolarizations were still observed during pyrethroid superfusion.
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Fig. 1.
Effects of superfused pyrethroids on rat ventricular
action potential morphology. Superimposed representative APs recorded
in control, prior to superfusion with pyrethroids, and in the presence
of fenpropathrin (a), -cypermethrin (b), tefluthrin (c), and
tetramethrin (d). Results from separate individual cells are shown in
left and right panels. In all panels, APs recorded in the presence of
the pyrethroids are indicated with asterisks (*). Note the variation
in control AP morphology from cell to cell, but that this variation had
little influence over the AP changes attributable to the effects of
pyrethroids.
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The effects of pyrethroids were quantified in terms of AP duration at
90% repolarization (APD90) measured between the
peak of the AP overshoot and the point at which repolarization reached 90% of the overshoot voltage. This action potential duration is plotted versus time of exposure to the four pyrethroids in Fig. 2. The upper panels show typical effects
(on APD90) of type II pyrethroids, fenpropathrin
and -cypermethrin. Effects of type I pyrethroids, tefluthrin and
tetramethrin, are shown in the lower panels. In all cases, data were
taken from representative cells. Pyrethroids were superfused for a
period of 5 min, with 2 min of washout also shown. In fenpropathrin
(Fig. 2a), APD90 was essentially unchanged for
the first 1 to 2 min (the time taken for exchange of bath solutions),
after which it continuously increased. In all myocytes exposed to this
compound (n = 7), the increase in APD90 was accompanied by a large increase in the
variability of this parameter. Individual APs often exceeded 1 s
in duration and some reached 3 s. Any further AP prolongation was
obscured by the following stimulus (interstimulus interval 3 s).
Figure 2b shows that during superfusion with -cypermethrin,
APD90 increased smoothly after the first 1 to 2 min, but the maximal APD90 never exceeded 1 s (n = 6). Figure 2c shows that similar progressive increases in APD90 were observed during
superfusion with the type I pyrethroid tefluthrin (n = 8). However, in two tefluthrin-treated myocytes a slight, but not
significant, increase in APD variation was also seen. Tetramethrin on
the other hand (Fig. 2d) was without appreciable effects in six of
eight myocytes. In two cells (data not shown),
APD90 did increase toward the end of the 5-min
superfusion period. Due to this significant delay however, this late
increase in APD90 was not necessarily
attributable to the action of the pyrethroid. In most cells,
APD90 slightly decreased in the presence of
tetramethrin, as shown in Fig. 1d. The effects of the three active
pyrethroids were only poorly reversible. Association with lipids in the
cell membrane accounts for this slow reversibility and also the general
observation that pyrethroid actions in diverse cell types develop
rather slowly (Berlin et al., 1984; Chinn and Narahashi, 1986; Tabarean
and Narahashi, 1998).
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Fig. 2.
Plots showing the time course of the effects of
pyrethroids on action potential duration. Data from representative rat
ventricular cells are presented. Action potential duration is expressed
as APD90 (see text). a, APD90 after adding 10 µM fenpropathrin to the superfusion solution, at time = 0, monitored for 5 min followed by 2 min of washout as indicated above the
plot; b-d, identical plots to a for the other pyrethroids as
indicated.
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A summary of the data on the effects of pyrethroids on action potential
duration is displayed in Table 1.
Considerable inter-cell variability was observed in the control
APD90 determined at the time of switching to
pyrethroid-containing solution. Values ranged from approximately 60 to
400 ms across all cells studied. This variation may be explained by the
fact that we used undivided rat ventricles for cell isolation,
producing a mixture of epicardial and endocardial cells. By visual
inspection, these cells are indistinguishable; however, epicardial
cells in this species have a considerably shorter APD than endocardial
cells (Komukai et al., 2000). Furthermore, APD variability at a
constant cycle length is not uncommon even in more homogeneous
populations of isolated myocytes (Coronel et al., 1997). Nevertheless,
the mean values listed in Table 1 are useful for comparative purposes.
Statistically significant increases in APD90
between the start and end of 3 min of superfusion were observed with
fenpropathrin and tefluthrin. In the case of -cypermethrin, a
significant prolongation of APD was observed 5 min after the start of
superfusion. Tetramethrin was without significant effects. Taken
together, these results indicate that both AP prolongation and
afterdepolarizations occur in rat ventricular myocytes during
pyrethroid exposure. Although cardiac APs are well known to be much
longer than those from neurons, making a direct comparison between data
from the two tissues difficult, the pyrethroid effects that we observed
were consistent, in broad terms, with those made for other cell types,
e.g., cerebellar neurons (Song and Narahashi, 1996).
To clarify further the origins of changes in cardiac myocyte APs during
pyrethroid superfusion, we performed voltage-clamp experiments to
observe ionic currents during membrane potential ramps. Our previous
results have shown the considerable utility of ascending voltage ramps,
over the range of potentials encountered in the AP, for qualitatively
determining which ionic currents are modified by experimental compounds
(Spencer et al., 2000). These signature currents were therefore
continuously recorded before and during superfusion with pyrethroids.
An indication of the relative potency of INa
modification can be derived by comparing the profile of the current
signatures. Figure 3 shows that
fenpropathrin (Fig. 3a), -cypermethrin (Fig. 3b), and tefluthrin (Fig. 3c) all profoundly prolonged the component of signature current
identified previously as INa. These effects
consisted of either the addition of a very slow trailing edge to the
otherwise crisp spike of current, as with the more potent pyrethroids
fenpropathrin and tefluthrin, or the addition of the late current
"hump" as seen with -cypermethrin. Such responses were observed
in at least four myocytes for each pyrethroid and correlated well with
the effects on APD90 shown in Fig. 1.
Tetramethrin produced only slight effects on INa
(Fig. 1d), in agreement with its lack of effects on
APD90 (Fig. 3d). Therefore, the consistency of
pyrethroid effects on INa in rat signature
currents shown in Fig. 3 and on the cellular AP (Fig. 1) suggests that
increased Na+ influx during
INa was responsible, directly or indirectly, for producing the observed changes in APD.
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Fig. 3.
Signature currents recorded during 500-ms linear
voltage ramps from 90 to +70 mV, before and during superfusion with
pyrethroids. In each panel, the control response prior to pyrethroid
superfusion is displayed on the left. On the right is a typical
signature current obtained after 3 or more minutes of superfusion with
the compound indicated above. Asterisks indicate that pyrethroids
prolonged the component previously identified as INa
(Spencer et al., 2000).
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Guinea Pig Ventricular Myocytes.
To elucidate the effects of
pyrethroids on cardiac INa, a series of
experiments was performed to quantify the voltage- and time-dependent
properties of this current. Selective recordings of
INa were made using conventional "square
pulse" voltage commands using recording conditions recently validated
for guinea pig ventricular myocytes (Yuill et al., 2000). Selective
INa measurements in the present study therefore
focused on myocytes isolated from this species. Initial experiments
used voltage ramps (at velocities between 0.32 and 0.8 mV/ms) to obtain
signature currents in guinea pig cells for comparison with the results
from rat myocytes presented above. In these experiments, performed at
37°C to confirm that pyrethroids retained their efficacy at mammalian
body temperature, standard external solution was used and then one of
three pyrethroid agents (fenpropathrin, tefluthrin, or tetramethrin)
was added. For each condition, net currents were measured and then 30 µM TTX was applied. This allowed us to measure the TTX-sensitive (TTX-S) current during the ramp before and during exposure to each
compound. Sample data for fenpropathrin are shown in Fig. 4. Similar to our observations from rat
myocytes, INa was visible as a large and fast
downward deflection during the ramp (Fig. 4a), which was blocked by 30 µM TTX (Fig. 4b). In the presence of 10 µM fenpropathrin, this
current component became wider (Fig. 4c), while retaining its
sensitivity to TTX (Fig. 4d). Recordings displayed on a faster time
scale show the TTX-S current for standard extracellular solution and
for fenpropathrin (Fig. 5a). TTX-S current was prolonged by fenpropathrin. Similar results were observed in nine cells to which ramp protocols were applied. In 10 other cells,
10 µM tefluthrin also prolonged the TTX-S current component during
applied voltage ramps (Fig. 5b), while in five cells, tetramethrin exerted little effect (Fig. 5c). In summary therefore, pyrethroids produced qualitatively similar effects on INa in
guinea pig and rat ventricular myocytes.
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Fig. 4.
Effects of 10 µM fenpropathrin on guinea pig whole
cell signature current during an applied voltage ramp. a, current under
control conditions, elicited from a holding potential of 50 mV by a
ramp from 100 mV to +60 mV. b, 30 µM TTX abolished the sodium
current component. c, application of 10 µM fenpropathrin prolonged
the sodium current component during the ramp. d, shows that 30 µM TTX
also inhibited the sodium current in the presence of fenpropathrin. The
time scale bar refers to all panels.
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Fig. 5.
Effects of fenpropathrin, tefluthrin, and
tetramethrin on TTX-sensitive currents elicited by voltage ramps. a,
left, shows a TTX-sensitive current under control conditions, elicited
by a voltage-ramp protocol. Fenpropathrin (10 µM) prolonged the
TTX-sensitive current (right). b, 10 µM tefluthrin also prolonged the
TTX-sensitive current. c, left, shows a TTX-sensitive current elicited
under control conditions. Tetramethrin (10 µM) had comparatively
little effect on the current (c, right). Time scale bar refers to all
panels.
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While voltage-ramp protocols revealed that pyrethroids can alter
cardiac ventricular INa in more than one species,
such protocols are not suitable for quantitative analysis of drug
effects. This necessitates the conventional approach of applying
depolarizing test pulses to maintained voltages. These square pulse
experiments were performed at room temperature (see Materials and
Methods) to slow down the kinetics of INa.
Pulses were applied from highly negative membrane potentials to avoid
partial inactivation of INa due to a negative
shift in its voltage-dependent inactivation under conditions of
internal fiber/cell dialysis (Makielski et al., 1987; Feng et al.,
1996; Yuill et al., 2000). Thus, similar to previous
INa experiments, cell membrane potential was held at 80 mV and then a 2-s prepulse to hyperpolarize to 140 mV preceded depolarizing test pulses. Figure
6 shows INa (as
TTX-S current) elicited by test pulses from 140 to 30 mV in control conditions and in the presence of fenpropathrin (Fig. 6a), tefluthrin (Fig. 6b), and tetramethrin (Fig. 6c). In control solution,
INa activated rapidly, and also showed rapid
time-dependent inactivation during the applied pulse. In the presence
of either fenpropathrin or tefluthrin, INa was
prolonged. Tetramethrin had little or no observable effect. As reported
previously (Yuill et al., 2000), the time course of inactivation of
guinea pig ventricular INa under our control
conditions was best described by a double exponential process. We
therefore fitted INa decline in typical currents
with the following equation:
|
(1)
|
where It is the current at time t,
Af is the current described by a fast
time-constant (
f) and As
the current described by a slow time constant
(s).
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Fig. 6.
Effect of fenpropathrin, tefluthrin, and tetramethrin
on sodium currents recorded selectively. a, left, shows a rapidly
activating and inactivating, TTX-sensitive INa, elicited
under control conditions by a step depolarization (top) from 140 to
30 mV. a, right, current amplitude shows a slight increase and a
slowing of inactivation time course after application of 10 µM
fenpropathrin. b, left, shows a TTX-sensitive INa under
control conditions. b, right, 10 µM tefluthrin produced an increase
in current amplitude and slowed inactivation time course. Tetramethrin
(10 µM) had no visible effect on INa (c, left and right).
Note, time bases shown in the top panels (voltage protocols) also refer
to the bottom panels.
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For eight cells to which 10 µM fenpropathrin was applied, 96.5 ± 0.56% of control INa inactivation was
described by a f of 1.68 ± 0.15 ms,
while 3.5 ± 0.56% was described by a s
of 12.85 ± 1.12 ms. In the presence of fenpropathrin the absolute
values of f and s
were unchanged (f = 1.47 ± 0.25 ms,
s= 14.89 ± 1.24 ms; p > 0.25 for both). However, the relative proportions of fast and slowly
inactivating current were significantly altered (Af = 63.58 ± 1.97%,
As = 36.42 ± 1.97%; p < 0.0001 for both). For seven cells to which 10 µM tefluthrin was
applied, the control parameters were 97.86 ± 0.65% of
INa described by a f of
1.63 ± 0.12, and 2.14 ± 0.65% was described by a
s of 13.62 ± 0.92 ms. Once again, the
values of f and s
were not significantly changed by the pyrethroid
(f = 1.56 ± 0.34 ms,
s= 13.63 ± 0.79 ms; p > 0.8 for both), while the relative proportions of fast and slowly
inactivating current were altered (Af = 35.2 ± 5.80%, As = 64.80 ± 5.80%;
p < 0.0001 for both). For five cells treated with
tetramethrin, control and drug parameters were similar (control: Af = 98.4 ± 0.25%,
f = 1.48 ± 0.09 ms,
As = 1.6 ± 0.25%,
s = 13.75 ± 1.56 ms; tetramethrin:
Af = 97.06 ± 0.61%,
f = 1.62 ± 0.13 ms,
As = 2.94 ± 0.61%,
s = 13.92 ± 2.07 ms; p > 0.1 for all). Thus, in agreement with the voltage-ramp protocols,
tetramethrin exerted little effect on INa
elicited by a voltage step. In contrast, both fenpropathrin and
tefluthrin prolonged INa duration, by increasing the proportion of slowly inactivating current at the expense of more
rapidly inactivating current.
To focus further on the action of pyrethroids on cardiac
INa, we selected fenpropathrin for more detailed
study. First, the concentration dependence of the effects on the
relative proportions of fast and slowly inactivating current was
studied by applying a lower (1 µM) and a higher (50 µM)
concentration of this agent. At 1 µM, fenpropathrin only slightly
affected the distribution of INa inactivation
rates in seven cells. In control, 96.51 ± 0.58% of
INa (Af) had
f of 1.54 ± 0.07 ms and 3.49 ± 0.58% (As) had s = 12.02 ± 0.74 ms. In 1 µM fenpropathrin, the values of f and s were
unchanged (f = 1.46 ± 0.08 ms,
s= 13.7 ± 0.72 ms; p > 0.25 for both) although the relative proportions of the current
described by f and s
were significantly altered (Af = 89.22 ± 1.09%, As = 10.77 ± 1.09%;
p < 0.05 for both). A fenpropathrin concentration of
50 µM produced comparatively larger alterations in
INa (applied to seven cells) than had been
observed in experiments with 10 µM fenpropathrin. Under control
conditions Af = 96.0 ± 1.02%,
f = 1.58 ± 0.09 ms,
As = 4.0 ± 1.02%,
s = 11.86 ± 0.59 ms; in 50 µM
fenpropathrin Af = 37.82 ± 4.98%,
f = 1.36 ± 0.11 ms;
As = 62.17 ± 4.98%,
s = 18.54 ± 0.81 ms. Thus, at 50 µM
the majority of INa inactivation was described by
the slower time constant (p < 0.05). An additional
effect at this concentration was that magnitude of the slow time
constant was modestly, but significantly increased (p < 0.05). To summarize, these results indicated that fenpropathrin
produced a clear concentration-dependent increase in the proportion of
slow compared with fast-inactivating INa. At 50 µM, s was also increased to a modest extent.
Considered collectively, the data suggest that the major effect of this
agent on INa time course was to alter the
partitioning of inactivation between fast and slow components.
We also investigated the effects of 10 µM fenpropathrin on
voltage-dependent properties of INa. Figure
7a shows a "family" of
INa recordings (TTX-S) at a range of test
potentials between 60 and 20 mV. At each test potential,
fenpropathrin prolonged INa (Fig. 7b). Table
2 summarizes the detailed effects of
fenpropathrin on the time course of current inactivation during a
single voltage-clamp step. The mean control amplitude (A) and values for the five cells examined in this study are similar to those
obtained in previous experiments (Yuill et al., 2000). Across the range
of potentials examined, the dominant effect of fenpropathrin was to
modify the relative proportions of rapidly and slowly inactivating INa. INa prolongation
produced by the pyrethroid correlated with an increased contribution of
slower inactivation to the overall time-dependent inactivation process.
For each of five cells, the current-voltage (I-V) relationship for
INa was also constructed and data were fitted by
a modified Boltzmann equation of the form:
![<UP>I<SUB>Na</SUB></UP>=<UP>G<SUB>max</SUB></UP>(<UP>V<SUB>m</SUB></UP>−<UP>V<SUB>rev</SUB></UP>)/(1+<UP>exp</UP>[(<UP>V<SUB>0.5</SUB></UP>−<UP>V<SUB>m</SUB></UP>)/<UP>k</UP>])](/content/vol298/issue3/fulltext/1067/img002.gif)
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(2)
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where INa represents current density at test
potential Vm and Gmax is
maximal INa conductance.
Vrev is the reversal potential, V0.5 is the membrane potential exhibiting
half-maximal current activation, and k is the slope factor that
describes the steepness of activation for the current.
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Fig. 7.
Effect of fenpropathrin on the current-voltage
relationship of INa recorded selectively. a, families of
INa under control conditions, elicited by step
depolarizations (below) from a prepulse potential of 140 mV to a
range of more positive test potentials, and b, in the presence of 10 µM fenpropathrin. c, the mean I-V relationships for peak
INa (open symbols: control, data fitted by eq. 2 using
V0.5 = 37.55 ± 0.33 mV, k = 5.14 ± 0.29 mV; closed symbols: fenpropathrin, V0.5 = 43.67 ± 0.18 mV, k = 6.57 ± 0.15 mV). d, the
fractional increase in peak INa after application of
fenpropathrin plotted against test potential. Although the largest
increase was observed at 60 mV, this could not be plotted because
control INa was close to zero, making the proportionate
increase in current with fenpropathrin extremely high.
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TABLE 2
Voltage dependence of inactivation timecourse parameters from guinea
pig ventricular myocytes in the presence and absence of 10 µM
fenpropathrin
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For the fits corresponding to each cell, control data showed a mean
V0.5 value of 38.1 ± 1.39 mV; while k was
4.78 ± 0.41 mV. This V0.5 value is similar
to that recently reported for guinea pig ventricular
INa (Yuill et al., 2000) and compares well with values reported previously for human atrial INa
(38.6 mV; Feng et al., 1996) and cat atrial INa
(41.8 mV; Follmer et al., 1987). In the presence of fenpropathrin,
V0.5 was significantly shifted to 43.14 ± 2.53 mV (p < 0.05) and k was 6.6 ± 0.09 mV,
representing an approximately 5-mV negative shift in half-maximal
activation potential for INa. Figure 7c shows I-V
plots of mean INa normalized to cell capacitance,
which were fitted by eq. 2 (V0.5 and k values are
given in the legend). Clearly, the I-V relationship shows a leftward
shift, at potentials negative to 20 mV, consistent with a negative
shift in the voltage dependence of INa
activation. Moreover, at potentials between 60 and 40 mV, the
amplitude of peak current was significantly increased
(p < 0.05). At potentials positive to 30 mV, current
amplitude was not enhanced. To better highlight this altered voltage
dependence, at each test potential (and for each cell), the peak
amplitude of INa in the presence of fenpropathrin
was expressed as a fractional increase above the corresponding control
current (Ifen/Icontrol 1). Mean fractional increases are plotted against test potential in
Fig. 7d. A clear trend appears in the voltage dependence of the
observed effect: the increase in current amplitude with fenpropathrin
was greatest at the most negative potentials in the range tested.
Figure 8 shows that we also determined
the effects of fenpropathrin on the voltage dependence of steady-state
INa inactivation. The experimental protocol used
is shown in Fig. 8b. Prepulses of 1-s duration to a range of test
potentials preceded a 50-ms test pulse to 30 mV. Sample current
records are shown in Fig. 8a. For both control and fenpropathrin
measurements, INa magnitude after each prepulse
potential was normalized to the maximal INa value
elicited by the protocol. The resultant values were used to construct
inactivation plots fitted by a Boltzmann equation of the form:
![<UP>Inactivation variable</UP>=1−(1/(1+<UP>exp</UP>[(<UP>V<SUB>0.5</SUB></UP>−<UP>V<SUB>m</SUB></UP>)/<UP>k</UP>]))](/content/vol298/issue3/fulltext/1067/img003.gif)
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(3)
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where Vm has the meaning in eq. 2,
V0.5 is the potential at which
INa is half maximally inactivated and k is the
slope factor for the inactivation curve.
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Fig. 8.
Effect of fenpropathrin on the voltage dependence of
INa inactivation. a, left, INa records elicited
under control conditions, by step depolarizations to 30 mV from a
range of more negative prepulse potentials (b). a, right, the effect of
10 µM fenpropathrin. c, the inactivation curve under control
conditions (data fitted by eq. 3;  , V0.5 = 92.07 ± 0.33 mV, k = 7.94 ± 0.29) and after
application of 10 µM fenpropathrin ( , V0.5 = 97.11 ± 0.40 mV, k = 7.23 ± 0.35).
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The inactivation parameters of INa were
investigated for a sample of eight cells; control parameters varied
within a wide range: the most negative value for
V0.5 was 103.68 mV and the most positive was
79.71 mV. There was comparatively little variation in k values. The
mean V0.5 value obtained by pooling control data was 92.06 ± 3.19 mV, and the mean k value was 5.2 ± 0.31 mV. Fenpropathrin produced a leftward shift of the inactivation curve in individual cells. Pooling calculated V0.5 and
k values gave a mean V0.5 of 97.02 ± 2.65 mV and k of 5.75 ± 0.18 mV. Figure 8c shows a plot of pooled
control and fenpropathrin inactivation variables from each cell
(mean ± S.E.M.); each plot was then fitted by eq. 3
(V0.5 and k values given in the figure legend).
For the fit to the mean data, fenpropathrin produced a negative shift of the V0.5 for voltage-dependent inactivation by
~5 mV. Due to the large variation in V0.5
values between cells (even in control), this shift was not found to be
statistically significant at the p < 0.05 level
(p = 0.07).
Pyrethroids Affect the Cardiac INa Window.
The
V0.5 and k values obtained from the I-V fits
(Fig. 7c) and inactivation curves (Fig. 8c) for
INa were used to estimate the
INa window as shown in Fig.
9. The inactivation parameters were
simulated at 2-mV intervals using eq. 3 and activation parameters were
simulated using the following equation:
![<UP>Activation variable</UP>=1/(1+<UP>exp</UP>[(<UP>V</UP><SUB>0.5</SUB>−<UP>V<SUB>m</SUB></UP>)/<UP>k</UP>])](/content/vol298/issue3/fulltext/1067/img004.gif)
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(4)
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where "activation variable" at any test potential
(Vm) occurs within the range 0 to 1;
V0.5 and k have similar meanings to those in eq.
2. For the control situation, the small area of overlap in Fig. 9
denotes the INa window region: a potential range
over which a small persistent Na+ entry would be
anticipated. In the simulated presence of fenpropathrin, in which both
activation and inactivation curves were shifted 5 mV to negative
potentials, the INa window was larger than under control conditions. This suggests that over the window voltage range,
steady-state Na+ entry via
INa could have been increased by fenpropathrin.
Due to alterations in the time and voltage dependence of
INa, it is therefore likely that an increased
window current contributes to toxic effects in the presence of
pyrethroids. In total, these changes in INa
appear to be involved in AP prolongation and the generation of
afterdepolarizations in cardiac cells.
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Fig. 9.
Effects of fenpropathrin on the simulated
INa window. Experimental data from Figs. 7 and 8 were used
together with eqs. 3 and 4 (see Results) to calculate
activation and inactivation variables at 2-mV intervals between 140
mV and +40 mV. At each voltage (Vm), control activation
variable = 1/(1 + exp [(37.55 Vm)/5.14]),
fenpropathrin activation variable = 1/(1 + exp [(43.67 Vm)/6.57 ]), control inactivation variable = 1 (1/(1 + exp [(92.07 Vm)/7.94])),
fenpropathrin inactivation variable = 1 (1/(1 + exp
[(97.11 Vm)/7.23])). The resulting simulated
activation and inactivation curves were then overlaid and the area of
overlap selected and shown at a high magnification to illustrate the
INa window in control () and in fenpropathrin (). The
potential of the peak of the INa window shifted to a more
negative potential in the presence of the pyrethroid and the integrated
window current increased by ~43%.
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Isolated Perfused Rat Hearts.
To examine the possible effects
of circulating pyrethroids at the whole heart level, we determined the
force of contraction (FOC) of isolated perfused rat hearts beating
spontaneously. The resting heart rate of these preparations was
325 ± 10 beats per min (n = 22). A continuous
recording from a one such heart is shown in Fig.
10a. Data were recorded for an
approximately 10-min control period followed by an equivalent period of
perfusion by 10 µM tefluthrin. Peak FOC, displayed in arbitrary
units, was fairly stable throughout the control period. A switching
artifact, appearing as a transient dip in FOC, invariably accompanied
the exchange of control and pyrethroid-containing perfusion solutions. In the example shown in Fig. 10a, after approximately 5 min of pyrethroid perfusion, noise in the peak FOC trace dramatically increased. This was found to be attributable to periodical episodes of
intense variation in contractile amplitude. Figure 10b shows individual
contractions during one of these episodes. The variations in
contractile amplitude were associated with irregularities in the
intervals between heartbeats. Periods in which beats were absent became
randomly interspersed in the recordings, and therefore, it appears that
periods of contractile variability involved episodic cardiac asystoly.
However, no significant differences in overall heart rate were observed
between control and pyrethroid perfusion periods for any of the
compounds studied. The appearance of irregular activity during
perfusion with pyrethroids suggests that these compounds have
considerable proarrhythmic potential.
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Fig. 10.
Effects of pyrethroids on contractility in isolated
Langendorff-perfused rat heart preparations. a, continuous plot derived
from a recording of peak FOC before and during perfusion of a
representative preparation with 10 µM tefluthrin. The composition of
the perfusion solution is indicated above the double-headed arrows. A
dip in FOC after changing solutions was probably an artifact of the
fact that the pyrethroid-containing solution had been static in the
perfusion line prior to switching. b, individual contractions of the
same unstimulated preparation recorded during the control period (left)
and during pyrethroid perfusion (right). c, histograms showing mean
coefficients of variation in contractile amplitude during control and
pyrethroid perfusions for each of the compounds indicated below the
horizontal axis. Asterisks indicate statistically significant
differences compared with control values.
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Quantification of the proarrhythmic activity of pyrethroids was
achieved by calculating the coefficient of variation (COV is standard
deviation/mean) in contractility for the whole of the control and
pyrethroid perfusion periods for each heart. Figure 10c shows
histograms of the mean COV calculated before and during perfusion with
10 µM pyrethroid for fenpropathrin (n = 7),
-cypermethrin (n = 5), tefluthrin (n = 5), and tetramethrin (n = 5). As in myocytes, tetramethrin produced no significant effects in isolated hearts. All of
the other pyrethroids caused significant increases in COV (p < 0.05) consistent with proarrhythmic activity. To
determine whether this proarrhythmic behavior resulted from a direct
effect on the myocardium or from pyrethroid-induced depolarization of sympathetic terminals in the heart (cf. Berlin et al., 1984), control
experiments were performed in which 10 µM propranolol was added to
the perfusion solution. In five hearts perfused with this
-adrenoreceptor blocker (data not shown), contractile variability increased significantly during perfusion with 10 µM tefluthrin (p < 0.05). Therefore, pyrethroid-induced arrhythmia
is likely to have originated from direct effects of these insecticides
on the myocardium, consistent with the electrophysiological effects on
isolated myocytes described above. Parallels between the observable effects of pyrethroids on cardiac INa and APD in
myocytes, and the contractility of isolated hearts, therefore indicate
that this class of compounds can cause substantial disruption of the regular cardiac rhythm.
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Discussion |
The principal findings of the present study at the cellular level
are that the type I pyrethroid tefluthrin and the type II pyrethroids
fenpropathrin and -cypermethrin 1) prolonged ventricular action
potentials and evoked afterdepolarizations; 2) modified the time course
of INa by altering the relative proportions of fast and slowly inactivating current; and 3) altered the voltage dependence of INa. At the whole heart level,
these effects corresponded with a pyrethroid-induced increase in the
variability of contractile force, suggestive of proarrhythmic activity.
Several aspects of these findings merit detailed consideration.
Na+ Channel Targets of Pyrethroid Modification.
Previous molecular studies have established conclusively that the
insecticidal activity of pyrethroids is uniquely determined by the
locus coding for the para neuronal Na+
channel (Miyazaki et al., 1996; Williamson et al., 1996; Lee et al.,
1999). Indeed, point mutations in para
Na+ channels suffice to confer pyrethroid
resistance (and cross-resistance to DDT). In both vertebrate and
invertebrate neurons, Na+ channel modification by
pyrethroids produces a steady-state Na+ current
and a large, slowly declining tail current after repolarization. These
effects appear to be attributable to the suppression of both
voltage-dependent inactivation and deactivation of
Na+ channels (Vais et al., 2000). The
steady-state current that is produced appears to underlie the prominent
neuronal afterdepolarizations that characterize pyrethroid poisoning.
In the present study in cardiac cells, we observed close parallels
between prolongation of cardiac Na+ current by
pyrethroids, AP changes, and proarrhythmia. Widening of the
INa peak in signature currents from both rat and
guinea pig ventricular myocytes resulted from apparent modifications in
the time- and voltage-dependent properties of the underlying cardiac
Na+ channels. In the presence of fenpropathrin,
which may be considered stereotypical for active pyrethroids,
INa both activated and inactivated at potentials
~5 mV more negative than in control. This small shift in voltage
dependence, consistent with effects of active pyrethroids in rat
cerebellar neurons (Song and Narahashi, 1996), was sufficient to
increase the calculated Na+ window current (Fig.
9). More prominent still was the effect of fenpropathrin on fast,
voltage-dependent inactivation of cardiac Na+
channels. A number of groups have reported this inactivation process to
be biexponential (Brown et al., 1981; Sakakibara et al., 1992; Yuill et
al., 2000). In the present experiments, fenpropathrin prolonged the
time course of INa by increasing the proportion of current described by a slower inactivation time constant. Such changes in time course were almost certainly involved in prolonging the
ventricular myocyte APD, the generation of early afterdepolarizations, and possibly arrhythmogenesis in the whole heart. Indeed, in the present studies, the degree of slowing of inactivation was found to
depend upon the fenpropathrin concentration. However, the appearance, at a fenpropathrin concentration of 50 µM, of an additional effect on
the magnitude of the slow time constant, precluded the determination of
a simple dose-response relationship for this slowing of inactivation.
Our findings may also reveal unique biochemical features in the actions
of pyrethroids against mammalian cardiac INa. In
particular, although pyrethroids delayed whole cell
INa inactivation, we observed little steady-state
current during depolarizing pulses, nor were large, slowly deactivating
tail currents seen upon repolarization from step or ramp protocols.
This observation contrasts strongly with almost all pyrethroid data
from neuronal studies (Chinn and Narahashi, 1986; deWeille and
Leinders, 1989; Song and Narahashi, 1996; Tabarean and Narahashi,
1998), but is perhaps consistent with known structural differences
between cardiac and central nervous system Na+
channels (Krafte et al., 1991; Balser, 1999). This observation is,
however, likely to represent a genuine characteristic of the pyrethroids we tested. In additional experiments (data not shown) we
examined the effects of veratrine alkaloids on
INa in rat ventricular signature currents (cf.
Chattou et al., 2000). Those effects, which closely resembled
pyrethroid modification of neuronal INa (Chinn
and Narahashi, 1986), included the prolongation of
INa leading to a large, slow tail current after
repolarization. The absence of similar tail currents in both rat and
guinea pig signature currents during superfusion with pyrethroids may
suggest that pyrethroid binding sites and actions may differ
significantly between neuronal and cardiac Na+
channels. However, in a single previous study using type II pyrethroids to prolong INa in rabbit ventricular cells,
effects on tail Na+ currents were reported (Grant
et al., 1993). Species differences or experimental factors such as the
fact that different pyrethroids (fenvalerate and deltamethrin) were
used in cell attached recordings by Grant et al. (1993) may account for
the discrepancy between our findings and that study in this respect.
Nevertheless, our central findings of INa
prolongation, leftward shift in activation curves and an increased
window INa do appear to be consistent with
facilitated Na+ channel opening as seen by Grant
et al. (1993).
Like pyrethroids, another class of Na+
channel-modifying drugs based on the parent structure of DPI 201-106 (i.e., BDF 9148/9198) also inhibits cardiac Na+
channel inactivation (Ravens et al., 1991; Yuill et al., 2000). Using
similar methods to those described in the present report, Yuill et al.
(2000) showed that profound APD prolongation, afterdepolarizations, and
considerable steady-state INa were elicited by
BDF 9198 in guinea pig ventricular myocytes. Despite large steady-state
currents, repolarization of voltage-clamp steps in the presence of BDF
9198 was not associated with large, slowly decaying tail currents. Such
data imply that despite slowing INa inactivation,
this compound did not significantly alter the time course of current deactivation.
Modifiers of Inactivation in Cardiac Na+ Channels.
Fast inactivation gating in mammalian Na+
channels is particularly prone to interference by various lethal toxins
and mutations. Hydrophobic toxins such as veratridine or batrachotoxin
bind via the channel pore and, as well as negatively shifting the
threshold voltage for INa activation by up to 50 mV, eliminate almost all fast inactivation (Honerjäger, 1982;
Wang and Wang, 1998, 1999; Chattou et al., 2000). Neuronal
Na+ channels also appear to be stabilized in the
open state by pyrethroids that eliminate fast inactivation (Chinn and
Narahashi, 1986; deWeille and Leinders, 1989; Motomura and Narahashi,
2000). The present findings indicate a subtler mode of pyrethroid
action for the agents we applied to cardiac cells. The partitioning of
rapidly inactivating INa into components
described by fast and slow time constants (Yuill et al., 2000) was
clearly altered in our experiments by both fenpropathrin and
tefluthrin. During superfusion with control solutions, more than 95%
of fast-inactivating current was described by an 
1-ms time constant
(), the remaining 5% by of 10 ms. The proportion of current
described by the faster time constant fell to about 64% in the
presence of 10 µM fenpropathrin, and as low as 35% in the presence
of 10 µM tefluthrin or 50 µM fenpropathrin. Although the origin of
these two phases of inactivation is at present unresolved, they
probably reflect pyrethroid-sensitive conformational influences on
charge movements within the channel protein.
Underlying the fast inactivation of cardiac Na+
channels appears to be a mechanism similar to N-type inactivation of
Shaker K+ channels (Hoshi et al.,
1990). The region between channel domains III and IV (III-IV linker)
seems to act as an inactivation particle that swings over the open
inner pore mouth. Interestingly, a mutation in the III-IV linker region
(
KPQ) is associated with a severe form of human long-QT syndrome
(LQT3; Wang et al., 1995). This three amino acid deletion has no
effect, however, on the kinetics of fast inactivation (Bennett et al.,
1995; Dumaine et al., 1996). Rather, a persistent
Na+ current is observed, suggesting the presence
of a population of noninactivating Na+ channels.
The macroscopic current in KPQ resembles the current carried by
pyrethroid-modified mammalian neuronal Na+
channels (Chinn and Narahashi, 1986) and on the surface, this observation suggests that pyrethroids could inhibit the inactivation particle mechanism. Consistent with this idea, in insect neuronal Na+ channels, pyrethroid binding sites have been
localized to S6 segments of domains 1 and 2 and may correspond to a
docking region for the inactivation particle (Williamson et al., 1996;
Miyazaki et al., 1996; Balser, 1999). In contrast, in another human
long-QT mutation, R1623Q, Na+ channels exhibit a
markedly slowed time course of inactivation without generating
steady-state INa (Kambouris et al., 1998; Makita et al., 1998). The phenotype of heterologously expressed R1623Q produces a very similar current to fenpropathrin- or
tefluthrin-modified native guinea pig cardiac INa
observed in the present study (Fig. 7). The structural location of
R1623Q appears to be in the outer part of the domain 4 S4 helix that is
close to the voltage sensor region and may be associated with
activation-inactivation coupling. By analogy, the pyrethroid binding
site in the guinea pig (and rat) cardiac Na+
channel could therefore plausibly be located well away from channel regions specifically concerned with the III-IV linker mechanism. Further site-directed mutagenesis studies aimed at the pyrethroid binding site of SCN5A channels might clarify this issue.
Na+ Current and Action Potential Prolongation in
Heart.
Toxins that prolong the open state of cardiac sodium
channels elicit both AP prolongation and afterdepolarizations
(Honerjäger, 1982). The underlying mechanisms appear to be
distinct for each, but ultimately stem from imbalances in the normal
sequence of inward and outward currents that maintain the AP plateau.
An exceedingly delicate balance seems to exist between inward and
outward ionic currents during the AP plateau such that the net
transmembrane current is close to zero. Toxins and mutations that
increase Na+ currents appear to upset this
balance in favor of prolonged net inward current with concomitant AP
prolongation (Bennett et al., 1995; Wang et al., 1995). AP
prolongation, in turn, may directly or indirectly provoke
Ca2+ influx into the cell. First, reactivation of
sarcolemmal L-type Ca2+ currents may occur during
the prolonged AP, directly increasing cytosolic
Ca2+ concentration (Makielski and January, 1998).
Second, cytosolic Na+ loading during prolonged
INa almost certainly leads to increased Ca2+ influx via the
Na+-Ca2+ exchanger
mechanism. Most lipid-soluble Na+ channel toxins
appear to be positively inotropic via this mechanism (Honerjäger,
1982; Berlin et al., 1984). Thus, Ca2+ overload
of the sarcoplasmic reticulum could prime the cell for mistimed
Ca2+ releases, evoking afterdepolarizations.
However, it is worth considering that in the present experiments,
prolongation of APD often exceeded 1 s (Fig. 1) yet the duration
of INa was more modestly prolonged (Fig. 3).
Therefore, if this AP prolongation involved Ca2+-dependent currents, the underlying SR
Ca2+ overload should have been indicated by
positive inotropic effects. Surprisingly, the contractions of isolated
spontaneously beating rat hearts were not increased by pyrethroids.
One explanation for this discrepancy may lie in the arrhythmic activity
we observed. In the absence of pyrethroids, the rate of spontaneous
beating in our preparations corresponded to a frequency of about 5 Hz.
In this frequency domain, rat ventricular muscle exhibits a positive
force-frequency relationship due to the direct activation of
contractions by Ca2+ influx during the AP (Tang
et al., 1996). Therefore, an asystolic pause, acting as an
instantaneous reduction in heart rate, could reduce the amplitude of
subsequent contractions by shifting the myocardium out of the region of
positive force-frequency relationship. This appears to be the case in
Fig. 10b. Although APs from ventricular myocytes (Fig. 1) often
contained multiple afterdepolarizations in the presence of pyrethroids
consistent with the contractile variations we observed in the
Langendorff hearts, determining the precise origin of arrhythmias
(supraventricular versus ventricular) in the whole heart preparations
was beyond the scope of the present study. Comparative experimentation
on cardiac tissues from different regions of the heart (e.g.,
sinoatrial node, atrial and ventricular muscle) might reveal which of
these are most susceptible to pyrethroids.
Mammalian Pyrethroid Toxicity.
Pyrethroid poisoning after
accidental exposure of humans and domestic animals is characterized
mostly by symptoms related to neuronal hyperexcitability (Valentine,
1990; Narahashi et al., 1995; Motomura and Narahashi, 2000
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Abstract
Introduction
