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Block of deltamethrin-modified sodium current in cultured mouse neuroblastoma cells: local anesthetics as potential antidotes
Brain Research, 518 (1990) 11-18 Elsevier
11
BRES 15507
Block of deltamethrin-modified sodium current in cultured mouse neuroblastoma cells: local anesthetics as potential antidotes Marga Oortgiesen, Regina G.D.M. van Kleef and Henk P.M. Vijverberg Research Institute of Toxicology, University of Utrecht, Utrecht (The Netherlands) (Accepted 7 November 1989) Key words: Neuroblastoma; Voltage clamp; Sodium current; Pyrethroid; Anticonvulsant; Local anesthetic
Effects of local anesthetics and anticonvulsants on the pyrethroid-modified sodium current in cultured mouse neuroblastoma cells have been investigated using the suction pipette voltage clamp technique. In the presence of 10/~M of the pyrethroid deltamethrin the sodium current consists of an enhanced peak current during membrane depolarization and a slowly decaying, deltamethrin-induced tail current remaining after repolarization. At the onset of block the local anesthetics tetracaine, lidocaine and QX 314 reduced the deltamethrin-induced tail current more effectively than the peak current. Lidocaine, but not phenytoin, caused a time-dependent block of tail currents evoked by membrane depolarizations lasting 10-1000 ms. Both lidocaine- and phenytoin-induced blocks were independent of the membrane potential during the tail current. The anticonvulsants phenytoin, phenobarbital and valproate blocked the tail and the peak sodium current to the same extent, but diazepam, mephenesin and urethane blocked the peak current more effectively. Vitamin E, which suppresses pyrethroid-induced paresthesia of the skin, had no effect on the voltage-dependent sodium current. It is concluded that indirect effects of anticonvulsants on pyrethroid-induced toxic symptoms predominate, whereas local anesthetics preferentially block the pyrethroid-induced tail current. Therefore, local anesthetics are potentially useful pyrethroid antidotes. INTRODUCTION Pyrethroids constitute a class of highly effective insecticides that act on the nervous system. Although pyrethroids exhibit low oral toxicity to mammals, they cause severe neurologic symptoms when the nervous system is directly exposed 3°. On the basis of intravenous poisoning symptoms in the rat pyrethroids have been divided into two classes: one that induces hypersensitivity and tremors of gradually increasing intensity, and another that causes profuse salivation, choreoathetosis and clonic seizures 3°. The latter class comprises most of the highly active a-cyano-3-phenoxybenzyl pyrethroids. A c o m m o n adverse effect of these a-cyano pyrethroids consists of cutaneous paresthesia upon dermal exposure to relatively low doses 11'2°. In man massive exposure may result in muscular fasciculations, disturbance of consciousness and convulsive attacks 13. Although the prognosis of occupational acute pyrethroid poisoning is generally good 13, treatment has been symptomatic and not very effective thus far. Results from studies into the treatment of pyrethroid-poisoned animals with anti-convulsant benzodiazepines and with phenobarbital have been variable and in several cases a delay in the onset of poisoning symptoms and an increase in LDso values of pyrethroids
was observed 8'1°'12'27'28. With mephenesin derivatives and urethane not only prophylactic but also therapeutic effects have been obtained 2'3'9'14'18. The in vivo occurrence of skin sensations and muscular fasciculations correlate well with structure-related excitatory effects of pyrethroids on the in vitro electrical activity of sense organs and sensory nerves, motor nerve endings and muscle fibres 3z. Pyrethroids induce repetitive activity and membrane depolarization in various parts of the peripheral as welt as in the central nervous system by prolonging the m e m b r a n e sodium current 28'33. From this major mechanism of action it is to be expected that sodium channel blockers antagonize pyrethroid effects. Patch clamp experiments have shown that pyrethroidmodified sodium channels remain in the open state much longer than normal sodium channels 7,34,38. Ideally, a pyrethroid antidote should selectively block pyrethroidmodified sodium channels. Therefore, use-dependent sodium channel blockers and compounds that selectively block open sodium channels are potential pyrethroid antidotes. Several compounds used in the symptomatic treatment of pyrethroid poisoning, e.g., diazepam, phenytoin and phenobarbital, have also been reported to block sodium channels 15,21,36. However, it is not clear whether these compounds antagonize pyrethroid effects
Correspondence: H.P.M. Vijverberg, Research Institute of Toxicology, University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
12 by directly interacting with sodium channels, by a selective suppression of secondary effects of pyrethroids or by a general depressive effect on nervous system activity. Cultured cells of the mouse neuroblastoma clone N1E-115 contain voltage-dependent sodium channels22. Effects of pyrethroids on sodium channels in these cells have already been described in detail 26"38. In the present study effects of experimental antidotes and of various known sodium channel blockers have been investigated in voltage-clamped neuroblastoma cells treated with the a-cyano pyrethroid deltamethrin.
period starting just before addition of the antidote and lasting till steady effects were reached. During this period a slow onset of the antidote effects was observed in parallel with the increasing concentration of the antidote at the cell membrane.
Chemicals Deltamethrin [a-S-cyano-3-phenoxybenzyl-(1R,cis)-3-(2,2-dibromovinyl)-2,2-dimethyl-cyclopropanecarboxylate] was donated by Roussel Uclaf. Tetrodotoxin, lidocaine, tetracaine, mephenesin, urethane, phenytoin (sodium salt), sodium valproate and vitamin E (Dc-a-tocopherol acetate) were obtained from Sigma. Diazepam and phenobarbital were obtained from Holland Pharmaceutical Supply, Alphen a/d Rijn, The Netherlands. The lidocaine derivatives QX 314 [2-(triethylamino)-N-(2,6-dimethylphenyl)-acetamide] and QX 222 [2-(trimethylamino)-N-(2,6-dimethylphenyl)-acetamide] were kindly supplied by Astra Pharmaceutica, Rijswijk, The Netherlands.
MATERIALS AND METHODS RESULTS
Cell culture Mouse neuroblastoma cells of the clone NIE-1151 were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 7.5% fetal calf serum and the following amino acids (in mM): L-cysteine.HCl 0.3, c-alanine 0.4, c-asparagine 0.45, L-aspartic acid 0.4, c-proline 0.4, and c-glutamic acid 0.4. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO 2. Cells of passage 30-45 were subcultured in 35 mm plastic tissue culture dishes. Cell differentiation was initiated 2-3 days after plating cells in DMEM supplemented with 1.5% fetal calf serum and 1% DMSO. This medium was refreshed every 2-3 days. The cells were used in the experiments 5-9 days after induction of differentiation.
Experimental procedure Experiments were carried out using the suction pipette technique for combined voltage clamp and internal perfusion of the neuroblastoma cells19. Fire-polished suction pipettes had an internal tip diameter of approximately 5 pm and a resistance of 400-600 KO. The liquid junction potential at the tip of the electrode was compensated before each experiment and remained constant within 1 mV. After obtaining seal resistances of 50-150 MI2, the membrane inside the pipette was ruptured and the cell was voltage clamped at -80 mV. The series resistance of 61)0-800 KI2 was compensated for 70-90%. Voltage-dependent sodium currents were evoked by step depolarizations of the membrane to 0 mV, preceded by a conditioning hyperpolarization to -120 mV for 100 ms to remove resting sodium channel inactivation. The holding current was recorded continuously. All responses were low-pass filtered (-3 dB at 2.5 kHz; 12 dB/octave), digitized by a transient recorder (8 bits; 1024 points/record) and stored on magnetic disc for off-line computer analysis. The external solution contained (in mM): NaCI 120, tetraethylammonium (TEA) chloride 25, tetramethylammonium chloride 20, CaCI2 1.8, MgCI2 0.4 and HEPES 25. The pH was adjusted to 7.4 with approximately 10 mM TEA-OH. The suction pipettes were perfused with an internal solution containing (in raM): CsF 120, NaOH 20, HEPES 25, and sucrose 10. The pH was adjusted to 7.1 with c-glutamic acid. Experiments were carried out at room temperature (20-24 °C). Deltamethrin, dissolved in dimethylsulphoxide (DMSO), was used to prepare a suspension in external solution with a final concentration of 10/~M. DMSO at its final concentration of 0.1% (v/v) did not affect the sodium current. The culture medium was replaced by a freshly prepared external solution containing deltamethrin at least 40 min in advance of the experiment. A small quantity of a concentrated stock solution of an antidote was added to the external solution in the culture dish at a location distant from the cell and was left to equilibrate by diffusion. Final concentrations are indicated and effects became steady 15-30 min after addition of the antidotes. The data were obtained in the
Control sodium currents were recorded in voltageclamped N1E-115 cells in the presence of 10 /~M deltamethrin and consisted of a slowly decaying peak inward current during m e m b r a n e depolarization to 0 mV, followed by a large, long-lasting sodium tail current after repolarization to the holding potential o f - 8 0 mV. This is very similar to previously recorded deltamethrin-modified sodium current in n e u r o b l a s t o m a cells 25. At the start of experiments the amplitude of both the peak current and the deltamethrin-induced tail current varied between cells and a m o u n t e d to -15.5 + 10 n A and -12.5 + 9 n A (mean + S.D.; n = 26), respectively. The ratio of the amplitude of tail and peak current changed in the beginning of the experiment. A t the first depolarization the tail-to-peak ratio ranged from 0.25 to 1.0 and on average a m o u n t e d to 0.73 + 0.23 (n = 15). After about 7 depolarizations at a frequency of 0.01-0.02 Hz the tail current had increased significantly (Wilcoxon's signed rank test; P < 0.01), whereas the peak current remained constant (P > 0.1). The holding current r e m a i n e d stable as well. A paired comparison of the tail to peak ratio at the first and the seventh depolarization for each cell revealed a significant increase ( P < 0.01) of the ratio at the last depolarization to 0.88 + 0.30 (n = 15; range = 0.28-1.33). The value of the initial tail-to-peak ratio appeared not correlated with the period of exposure to deltamethrin, which varied b e t w e e n 40 min and 6 h (Spearman's rank correlation test; P = 0.57) and the increase in the tail-to-peak ratio appeared i n d e p e n d e n t of the initial value and of the period of exposure (P = 0.71 and P = 0.47, respectively). Antidotes were administered after stabilization of the deltamethrin-modified sodium currents. The sodium channel blocker tetrodotoxin ( q T X ) reduced the peak as well as the tail current. Fig. l a shows deltamethrin-modified sodium currents before as well as 5 and 7 min after the addition of 0.5 p M T T X to the
13 external solution. A t equilibrium the p e a k and the tail current were c o m p l e t e l y blocked, confirming that both current c o m p o n e n t s are m e d i a t e d by v o l t a g e - d e p e n d e n t sodium channels. Sodium currents were r e c o r d e d at regular intervals from the time of the addition of T T X until steady block was achieved. A two-dimensional plot o f the a m p l i t u d e of the tail current against the amplitude of the p e a k current, both normalized to their respective initial values, is p r e s e n t e d in Fig. l b . The d a t a showed no deviation from linearity ( P = 0.24). In another experi-
a 80 mV
-120 " ~ - -
TTX
TTX 7 rain
control
1hA I
ment similar results were o b t a i n e d (regression lines identical; P = 0.22). T h e slope of the regression line through the results from both cells a m o u n t e d to 0.99 + 0.04 ( + 9 5 % confidence limit). T h e linear relationship demonstrates that the tail current, which is carried entirely by d e l t a m e t h r i n - m o d i f i e d sodium channels, and the p e a k current were equally affected by T T X as the concentration gradually increased to its final value of 0.5 /~M. Effects of lidocaine and its derivatives Q X 314 and Q X 222 on the sodium current in the presence of 10 p M deltamethrin are d e p i c t e d in Fig. 2. T h e steady effect of 0.6 m M lidocaine (Fig. 2a) consisted of a reduction of the p e a k amplitude to 66% of its initial value, while at the same time the tail a m p l i t u d e was r e d u c e d to 52%. The time course of the sodium current was not significantly altered by lidocaine. A f t e r raising the lidocaine concentration to 1.4 m M the p e a k and the tail a m p l i t u d e further r e d u c e d to 37% and 23% (Fig. 2a) of the initial values, respectively. External application of the lidocaine derivatives Q X
50 ms
/
/
// i/
8 0 _120 ~
"~
.80 mV
/
/// // •
t.4
mM
1hA[ 50 m s
0//~//
0
I 15 min
QX 222 30 min
2 ,^{ / / / /
0.0 0.0
~
50 ms
/ /
/ /
'
~
'
'
'
0.5
'
'
'
'
1.0
relative peak current
Fig. 1. Effects of "I"TX on the sodium current after exposure to 10 /~M deltamethrin. Deltamethrin-modified sodium currents were evoked by the step depolarization indicated, a: deltamethrinmodified sodium current (left), and sodium currents 5 min (middle) and 7 min (right) after subsequent addition of a small amount of I T X to a final concentration of 0.5 /~M. b: two-dimensional representation of the onset of the blocking effect of 0.5/~M TTX on the peak and the tail current. The amplitude of the peak current (abscissa) and that of the deitamethrin-induced tail current have both been normalized to the respective values before addition of I"TX. Data marked with asterisks were obtained from the records in (a). Linear regression analysis showed no deviation of the tail-to-peak relationship from linsarity (P = 0.24). The slope was 1.03 + 0.07 (_+95% confidence limits).
3 nAL~ 5O ms
Fig. 2. Effects of the local anesthetics lidocaine, QX 222 and QX 314 on the sodium current after exposure to 10 /~M deltamethrin. Deltamethrin-modified sodium currents were evoked by the step depolarization indicated, a: deltamethrin-modified sodium current (left), and subsequent steady effects of 0.6 mM (middle) and 1.4 mM lidocaine (right). b: deltamethrin-modified sodium current (left), and sodium currents 15 min (middle) and 30 min (right) after internal perfusion of the cell with 1 mM QX 222. c: deltamethrinmodified sodium current (left), and sodium currents 14 min (middle) and 25 min (right) after internal perfusion of the cell with 1 mM QX 314.
14 314 and Q X 222 did not affect the sodium current in the presence of deltamethrin. H o w e v e r , internal perfusion with 1 m M of Q X 222 and of O X 314 blocked both the p e a k and the tail current. With O X 222 the peak and the tail current were r e d u c e d in parallel to 66% of their initial values after 15 min and to 5% 30 min after switching the pipette solution to one containing 1 m M of the local anesthetic (Fig. 2b). Similar to lidocaine, O X 314 blocked the tail current to a greater extent than the peak current. A f t e r 14 min of internal perfusion with a pipette solution containing 1 m M Q X 314 the amplitude of the peak current was reduced to 74% of its initial value, while at the same time the amplitude of the tail current was reduced to 50%. A f t e r 25 min of internal perfusion with 1 m M O X 314 the peak and the tail current were r e d u c e d to 16% and 12%, respectively (Fig. 2c). In contrast to the other local anesthetics O X 314 caused an acceleration of the decay of the slow c o m p o n e n t of the sodium current during m e m b r a n e depolarization. The gradual block of the peak and the tail current after adding 1.6 m M lidocaine to the external solution was investigated in two experiments. In contrast to TTX, lidocaine initially blocked the tail current more than the p e a k current resulting in the non-linear plot of the tail against the p e a k amplitude presented in Fig. 3. The slope of the curve at the onset of block, i.e., in the concen-
tration range in which lidocaine r e d u c e d the p e a k as well as the tail current less than 35%, a m o u n t e d to 1.5 -4-_(1.3 ( + 9 5 % confidence limit). The initial slopes of the tail-to-peak relationships o b t a i n e d during the onset of block by internal Q X 222 and Q X 314 were 1.2 + 0.4 and 1.8 + 0 .6, respectively. External application of 0.1 m M of the local anesthetic tetracaine caused nearly c o m p l e t e block of the sodium
a
1.0 mephenesin
o ~poo~ 0 0 0 0
o
c
-120 ~
-80 mV
I
= 0.5
>o o
1 nA J__._ 100 m~
I
0.0 q 0,0
i
i
i
i
i 0.5
l
i
i
i 1.0
1.0
o phenyloin
1,0 lidocaine
{ o
O
a) o 0.5
O
E
0 0 0
0
oo
•®
- 120~-J
I
-80 mV
0
0.5
8 0.0 0,0
o.5 relative
0.0
,
0.0
,
i
,
i
,
~
0.5
,
~
,
1.0
relative peak current
Fig. 3. The tail-to-peak relationship during the onset of the blocking effect of 1.6 mM lidocaine in a 10 /~M deltamethrin-treated neuroblastoma cell. The amplitude of the peak current {abscissa) and that of the deltamethrin-induced tail current have both been normalized to the respective values before addition of lidocaine. The initial slope of the tail-to-peak relationship was 1.5 + 0.3 (+95% confidence limits) and was obtained by least-squares linear regression of datapoints with values >0.65.
peak
1.0
current
Fig. 4. The tail-to-peak relationship during the onset of the blocking effect of 1 mM mephenesin and 1 mM phenytoin in 10 pM deltamethrin-treated neuroblastoma cells. The amplitude of the peak current (abscissa) and that of the deltamethrin-induced tail current have both been normalized to the respective values before addition of mephenesin or phenytoin. The initial slopes of the tail-to-peak relationship have been fitted by least-squares linear regression of datapoints with values >0.65. a: the inset shows the deltamethrin-modified sodium current and the steady effect of 1 mM mephenesin. The initial slope of the tail-to-peak relationship during the onset of block by mephenesin was 0.5 + 0.1 (+95% confidence limits), b: the inset shows the deltamethrin-modified sodium current and the steady effect of 0.5 mM phenytoin. The initial slope of the tail-to-peak relationship during the onset of block by phenytoin was 1.3 _+ 0.4.
15
current in the presence o f deltamethrin. During the onset
o b t a i n e d in the presence of external lidocaine and
of this effect the tail current was blocked to a greater extent than the p e a k current. T h e initial slope of the tail-to-peak relationship at the onset of block by tetracaine a m o u n t e d to 1.4 + 0.3. T h e effects of various e x p e r i m e n t a l pyrethroid antidotes and some c o m m o n anticonvulsants have also been investigated. M e p h e n e s i n caused only partial block of the v o l t a g e - d e p e n d e n t sodium current in the presence of deltamethrin. T h e inset of Fig. 4a shows two superimp o s e d sodium current records o b t a i n e d before and at equilibrium after adding 1 m M mephenesin to the external solution. T h e p e a k current was r e d u c e d to 57% of its initial value and the tail current to 68%. The slope of the tail-to-peak relationship during the onset of block by m e p h e n e s i n in two cells a m o u n t e d to 0.5 + 0.2 (Fig. 4a). P h e n y t o i n at 0.5 m M r e d u c e d the p e a k current to 60% and the tail current to 54% of the initial values in 13 min (Fig. 4b, inset). A t 1 m M phenytoin both current c o m p o n e n t s were almost completely blocked. The slope of the tail-to-peak relationship during the onset of block by 1 m M p h e n y t o i n in two cells a m o u n t e d to 1.3 + 0.4 (Fig. 4b). T h e effects of 12 c o m p o u n d s on the sodium current in the presence of 10 # M deltamethrin are summarized in Table I. The slopes of the tail-to-peak relationships
tetracaine and internal Q X 314 were significantly greater than 1.0 (two-tailed Student's t-test; P < 0.05), indicating that low concentrations o f these local anesthetics blocked the tail current to a greater extent than the p e a k current. In contrast, mephenesin, d i a z e p a m and urethane m o r e effectively blocked the p e a k than the tail current as the slopes of the tail-to-peak relationships were significantly smaller than 1.0 ( P < 0.05). Internal Q X 222, phenobarbital, phenytoin and v a l p r o a t e a p p e a r e d to block the p e a k and the tail current to the same extent. Vitamin E, which has been r e p o r t e d to suppress cutaneous paresthesia caused by d e r m a l exposure to a - c y a n o pyrethroids in man 29, did not affect the d e l t a m e t h r i n - m o d i f i e d sodium current when present in the external solution at a concentration of 1 m M for a p e r i o d of up to 2 h.
1.0
o--o control H lidocaine g ,~ 0.s
TABLE I
Effects o f various sodium channel blockers and experimental antidotes on the voltage-dependent sodium current o f cultured mouse neuroblastoma cells in the presence o[ lO ttM deltamethrin
Compound
Final concentration (M)
TTX Lidocalne QX 222 (int) QX314 (int) Tetracalne
1.6.10-3 10-3 10-3 10-~
Urethane Mephenesin
10 -3 10-3
5'10 -7
Diazepam
5.104
Phenobarbital
5.10 -3
Phenytoin Valproate Vitamin E
10-3 10-3 10-3
0.0
Initial bloc~ Steady effec~ slope tail vs peak peak tail 0.9 + 0.2 1.5 + 0.3* 1.2 + 0.4 1.8 + 0.6* 1.4 _+0.3* 0.6 + 0.3* 0.5 + 0.1" 0.4 + 0.3* 1.2 + 0.3 1.3 + 0.4 1.0 + 0.3 -
0.00 0.19 0.04 0.14 0.19 0.75 0.54 0.21 0.32 0.09 0.59 1.0
0.00 0.13 0.03 0.11 0.14 0.79 0.77 0.24 0.18 0.04 0.53 1.0
Slope of the two-dimensional plot of tail vs. peak amplitude (+95% confidence limit) determined by linear regression of datapoints in the concentration range in which the compounds reduced the amplitudes of the peak and tail current to 65-100% of the initial values. Slope values < 1 indicates a selective block of peak current at low concentrations and slope values > 1 indicate a selective block of tail current. z Fraction of the initial amplitude remaining at steady effect, i.e., at final concentration. * Slope significantly different from 1.0 (two tailed Student's t-test, P < 0.05).
'
,
'
0
b
,
,
500
1.0 ~
,
,
1000 ms
1
o--o control H
phenytoin
g
o 0.5 >=
:
°
o
m
0.0
,
0
,
,
,
,
,
500
1000 ms t
Fig. 5. Relation between the period of depolarization and the
deltamethrin-induced tail current before and after exposure to lidocalne (a) and phenytoin (b). Tail currents were evoked by depolarizations to 0 mV for 10-1000 ms. The tail current amplitudes have been normalized to the values obtained with 10 ms depolarizations, a: at equilibrium 0.8 mM lidocaine reduced the tail current evoked by a 20 ms depolarization to 41% of its initial value. With increasing the duration of depolarization a time-dependent increase of block is observed, which declined again after depolarizations >200 ms. b: the steady block of the tail current by 0.6 mM phenytoin remained virtually constant for the range of depolarizations investigated.
16 Vm -80 mV
-4O 0.0
0.8 mM lidocaine and 0.6 external solution, except This result indicates that lidocaine and phenytoin potential.
mM phenytoin (Fig. 6) to the for a constant scaling factor. block of the tail current by is independent of membrane
DISCUSSION
o
control
•
phonyloin 1.0
Fig. 6. I- V relationship of the 10 pM deltamethrin-induced tail current before and after exposure to 0.6 mM phenytoin. Tail currents were measured at different repolarization potentials (Vm) after conditioning membrane depolarizations to 0 mV for 20 ms. The tail current amplitudes have been normalized to the values obtained at -80 mV. The block of the tail current by phenytoin remained virtually constant at membrane potentials between 0 and -80 inV. As pyrethroids induce long-lasting repetitive activity and nerve membrane depolarization, the time dependence and the potential dependence of the effects of lidocaine and phenytoin on the deltamethrin-induced tail current have been investigated. In the presence of 10 ktM deltamethrin maximum tail current amplitudes were obtained with depolarizations of 10-20 ms. The tail current amplitude decreased when the depolarization period was further increased to 1 s. The peak current amplitude was reduced by 0.8 mM lidocaine to 41% and by 0.6 mM phenytoin to 69% of the initial value, whereas the amplitudes of tail currents after 20 ms depolarizations were reduced to 27% and 56%, respectively. Fig. 5, in which normalized values of the deltamethrin-induced tail currents are presented, shows the dependence of block of the tail current by lidocaine and phenytoin on the duration of the membrane depolarization. Although the control curves obtained in the presence of deltamethrin varied between experiments, possibly because of several degrees temperature differences, similar effects were obtained in 3 experiments with each blocker. The effect of lidocaine increased with depolarizations lasting up to 200 ms, but after longer depolarizations this increase gradually vanished (Fig. 5a). The effect of phenytoin remained virtually the same for depolarizations ranging from 10 ms to 1 s (Fig. 5b). In the same cells the dependence of the effects of |idocaine and phenytoin on membrane potential was examined. The membrane was repolarized to potentials between -80 and 0 mV after depolarization to 0 mV for 20 ms. I - V curves obtained for the deltamethrin-induced tail current were identical to those obtained after adding
Deltamethrin greatly affects the voltage-dependent sodium current in voltage-clamped mouse neuroblastoma cells. The peak sodium current during depolarization is enhanced and a slowly decaying sodium tail current appears after repolarization of the membrane. Patch clamp experiments have shown that normal as well as pyrethroid-modified sodium channels contribute to the peak current during membrane depolarization TM. However, it is likely that at the time of peak, the sodium current is mainly carried by normal sodium channels, as after deltamethrin only a small increase of the peak amplitude is observed (R.G.D.M. van Kleef and H.P.M. Vijverberg, unpublished result) and the opening of pyrethroid-modified sodium channels is delayed TM. On the other hand, the deltamethrin-induced slow tail current is entirely carried by modified sodium channels. The experiments, in which TTX gradually caused a complete block of the sodium inward current in the presence of deltamethrin without changing the tailto-peak ratio, indicate that pyrethroid-modified and normal sodium channels are equally TTX-sensitive. In addition, the results are consistent with the notion that the effect of TTX is restricted to a reduction of the maximum sodium conductance. At the start of the experiments the tail-to-peak ratio varied. When the membrane was depolarized for the first time after voltage clamping neuroblastoma cells exposed to 10 p M deltamethrin for 40 min to 6 h, the tail-to-peak ratio showed significant variation irrespective of the period of exposure. This indicates that the fraction of deltamethrin-modified sodium channels varied between cells. Further, the increase of the tail current amplitude during the first membrane depolarizations appeared independent of the initial tail-to-peak ratio and the period of exposure. Without excluding the previous hypothesis that pyrethroids affect voltage-dependent sodium channels in the resting configuration 3~, this result indicates that the effect of deltamethrin is enhanced by channel opening. Voltage-dependent sodium channels are regarded as the main target site of pyrethroids. Receptor-operated ion channels, which have been postulated as potential targets of pyrethroids as well, were recently demonstrated not to be directly responsible for the toxic symptoms induced by pyrethroids 6'23'24. Therefore, se-
17 lective antidotes should block pyrethroid-modified sodium channels completely without affecting normal sodium channels. Although a highly selective block of the deltamethrin-induced tail current was not observed in the present experiments, lidocaine, QX 314 and tetracaine more effectively blocked the tail current. The selectivity of QX 222 could neither be distinguished from the other local anesthetics, nor from that of TTX (see Table I). The greater sensitivity of the tail current to local anesthetics was anticipated, since these compounds block open sodium channels 4 and the pyrethroid-modified channels remain open for prolonged periods 34'38. It has also been reported that octylguanidine, which has a similar mechanism of action as local anesthetics, selectively blocks open sodium channels in pronase-treated and in pyrethroid-treated squid giant axons 16"35. Open channel block by local anesthetics is most clearly observed as a time-dependent block of the prolonged sodium current in pronase-treated squid axons 5. In the present experiments block of the deltamethrin-induced tail current by lidocaine was time-dependent, but did not depend on the membrane potential during the tail current. Differences in use-dependent block by lidocaine, QX 222 and QX 314 have been found in voltage-clamped squid giant axons 37, but the presently observed blocking effects of these local anesthetics at low concentrations (see Table I) are not statistically different. This result and the transient character of the time-dependence of the tail current block by lidocaine (see Fig. 5a) suggest that deltamethrin affects the interaction of lidocaine with the sodium channel. Additional experiments are needed for a more detailed description of the interaction of local anesthetics with pyrethroid-modified sodium channels. The anticonvulsants phenytoin, phenobarbital and valproate blocked the sodium current without affecting the tail-to-peak ratio. Similar to local anesthetics phenytoin causes resting as well as use-dependent block of the sodium current in neuroblastoma cells21. However, unlike the present results with lidocaine, phenytoin block of the tail current is time-independent (see Fig. 5b), which REFERENCES 1 Amano, T., Richelson, E. and Nirenberg, P.G., Neurotransmitter synthesis by neuroblastoma clones, Proc. Natl. Acad. Sci. U.S.A., 6 (1972) 258-263. 2 Bradbury, J.E., Forshaw, P.J., Gray, A.J. and Ray, D.E., The action of mephenesin and other agents on the effects produced by two neurotoxic pyrethroids in the intact and spinal rat, Neuropharmacology, 22 (1983) 907-914. 3 Bradbury, J.E., Gray, A.J. and Forshaw, P., Protection against pyrethroid toxicity in rats with mephenesin, Toxicol. Appl. Pharmacol., 60 (1981) 382-384. 4 Cahalan, M., Molecular properties of sodium channels in excitable membranes. In C.W. Cotman, G. Poste and G.L. Nicolson (Eds.), The Cell Surface and Neuronal Function,
agrees with the previous finding that octylguanidine and pyrethroids independently modify voltage-dependent sodium channels in squid axons 35. Mephenesin, urethane and diazepam, which have been reported to block voltage-dependent sodium channels in different preparations 17'25'36, initially blocked the peak current to a greater extent than the tail current (see Table I). The apparent selectivity for blocking normal sodium channels with respect to deltamethrin-modified sodium channels shows that these compounds do not simply reduce the maximum sodium conductance, as has been suggested for urethane before 25. The poor block of the deltamethrin-induced tail current by most of the anticonvulsants at relatively high concentration indicates that their prophylactic and therapeutic effects in the treatment of the neuroexcitatory symptoms of pyrethroids are not mediated by a selective interaction with deltamethrin-modified sodium channels. The multiple actions of these compounds suggest that indirect effects, which generally cause depression of nervous system excitability, predominate. This conclusion is supported by the finding that the batrachotoxinand the veratridine-induced sodium influx in N18 neuroblastoma cells are inhibited by various anticonvulsants at concentrations in excess of therapeutic levels 36. The suppression of pyrethroid-induced skin sensations by vitamin E 28 also seems to be mediated by indirect effects, because the sodium current is not affected at all. In contrast, local anesthetics, which appear to block pyrethroid-modified sodium channels more effectively, are potentially useful antidotes in the treatment of accidental pyrethroid poisoning. However, the therapeutic value of local anesthetics as antidotes against pyrethroid poisoning symptoms remains to be evaluated.
Acknowledgements. The authors wish to thank Ms. P. Martens for maintaining the cell culture, Ing. A. de Groot for expert technical assistance. Prof. J. van den Bercken is thanked for his support during the experiments. This work was financially supported by Shell Internationale Research Maatschappij B.V.
Elsevier, Amsterdam, 1980, pp. 1-47. 5 Cahalan, M.D., Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons, J. Biophys., 23 (1978) 285-311. 6 Chalmers, A.E., Miller, T.A. and Olsen, R.W., Deltamethrin: a neurophysiological study of the sites of action, Pestic. Biochem. Physiol., 27 (1987) 36-41. 7 Chinn, K. and Narahashi, T., Stabilization of sodium channel states by deitamethrin in mouse neuroblastoma cells, J. Physiol. (Lond.), 380 (1986) 191-207. 8 Cole, L.M. and Casida, J.E., Pyrethroid toxicology in the frog, Pestic. Biochem. Physiol., 20 (1983) 217-224. 9 Cotonat, J., Bieys, M. and Foulhoux, P., Effet antagoniste du phenprobamate et du carbamate de mrphrnrsine sur l'intoxication ~t la deltamrthrine, J. Toxicol. Clin. Exp., 7 (1987) 5-19.
18 10 Cremer, J.E., Cunningham, V.J., Ray, D.E. and Sarna, G.S., Regional changes in brain glucose utilization in rats given a pyrethroid insecticide, Brain Research, 194 (1980) 278-282. 11 Flannigan, S.A. and Tucker, S.B., Variation in cutaneous sensation between synthetic pyrethroid insecticides, Contact Dermatitis, 13 (1985) 140-147. 12 Gammon, D.W., Lawrence, L.J. and Casida, J.E., Pyrethroid toxicology: protective effects of diazepam and phenobarbital in the mouse and the cockroach, Toxicol. Appl. Pharmacol., 66 (1982) 290-296. 13 He, F., Wang, S., Liu, L., Chen, S., Zhang, Z. and Sun, J., Clinical manifestations and diagnosis of acute pyrethroid poisoning, Arch. Toxicol., 63 (1989) 54-58. 14 Horimori, T., Nakanishi, T., Kawaguchi, S., Sako, H., Suzuki, T. and Miyamoto, J., Therapeutic effects of methocarbamol on acute intoxication by pyrethroids in rats, J. Pestic. Sci., 11 (1986) 9-14. 15 Kendig, J.J., Courtney, K.R. and Cohen, E.N., Anesthetics: molecular correlates of voltage- and frequency-dependent sodium channel block in nerve, J. Pharmacol. Exp. Ther,, 210 (1979) 446-452. 16 Kirsch, G.E., Yeh, J.Z., Farley, J.M. and Narahashi, T., Interaction of n-alkylguanidines with the sodium channels of squid axon membrane, J. Gen. Physiol., 76 (1980) 315-335. 17 Klee, M.R. and Faber, D.S., Mephenesin blocks early inward currents and strychnine-induced multiple discharges of Aplysia neurons, Pfliigers Arch., 346 (1974) 97-106. 18 Leclercq, M., Cotonat, J. and Foulhoux, P., Recherche d'un antagonisme ~ l'intoxication par la deltam6thrine, J. Toxicol. Clin. Exp., 6 (1986) 85-93. 19 Lee, K.S., Akaike, N. and Brown, A.M., The suction pipette method for internal perfusion and voltage clamp of small excitable cells, J. Neurosci. Methods, 2 (1980) 51-78. 20 Le Quesne, P.M., Maxwell, I.C. and Butterworth, S.T.G., Transient facial sensory symptoms following exposure to synthetic pyrethroids: a clinical and electrophysiological assessment, Neurotoxicology, 2 (1980) 1-11. 21 Matsuki, N., Quandt, EN., Ten Eick, R.E. and Yeh, J.Z., Characterization of the block of sodium channels by phenytoin in mouse neuroblastoma cells, J. Pharmacol. Exp. Ther.. 228 (1984) 523-530. 22 Moolenaar, W.H. and Spector, I., Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions, J. Physiol. (Lond.), 278 (1978) 265-286. 23 Ogata, N., Vogel, S.M. and Narahashi, T., Lindane but not deltamethrin blocks a component of GABA-activated chloride channels, FASEB J., 2 (1988) 2895-2900. 24 Oortgiesen, M., Van Kleef, R.G.D.M. and Vijverberg, H.P.M., Effects of pyrethroids on neurotransmitter-operated ion chan-
25 26
27
28
29
30
31
32
33
34
35
36
37
38
nels in cultured mouse neuroblastoma cells, Pestic. Biochem. Physiol., 34 (1989) 164-173. Picbon, Y., Pharmacological characterization of ionic channels in unmyelinated axons, J. Physiol. (Paris), 77 (1981) 1119-1128. Ruigt, G.S.F., Neijt, H.C., Van der Zalm, J.M. and Van den Bercken, J., Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells, Brain Research, 437 (1987) 309-322. Staatz, C.G., Bloom, A.S. and Lech, J.J., A pharmacological study of pyrethroid neurotoxicity in mice, Pestic. Biochem. Physiol., 17 (1982) 287-292. Staatz-Benson, C.G. and Hosko, M.J., Interaction of pyrethroids with mammalian spinal neurons, Pestic. Biochem. Physiol., 25 (1986) 19-30. Tucker, S.B., Flannigan, S.A. and Ross, C.E., Inhibition of cutaneous paresthesia resulting from synthetic pyrethroid exposure, lnt. J. Dermatol., 10 (1984) 686-689. Verschoyle, R.D. and Aldridge, W.N., Structure-activity relationships of some pyrethroids in rats, Arch. Toxicol., 45 (1980) 325-329. Vijverberg, H.P.M. and Van den Bercken, J., Action of pyrethroid insecticides on the vertebrate nervous system, Neuropathol. Appl. Neurobiol., 8 (1982) 421-440. Vijverberg, H.P.M., De Weille, J.R., Ruigt, G.S.E and Van den Bercken, J., The effect of pyrethroid structure on the interaction with the sodium channel in the nerve membrane. In M.G. Ford, G.G. Lunt, R.C. Reay and P.N.R. Usherwood (Eds.), Neuropharmacology and Pesticide Action, Ellis Horwood, Chichester, U.K., 1986, pp. 267-285. Vijverberg, H.P.M., Van der Zalm, J.M. and Van den Bercken J., Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves, Nature (Lond.), 295 (1982) 601-603. De Weille, J.R. and Leinders, T., The action of pyrethroids on sodium channels in myelinated nerve fibres and spinal ganglion cells of the frog, Brain Research, 482 (1989) 324-332. De Weille, J.R., Vijverberg, H.P.M. and Narahashi, T., Interactions of pyrethroids and octylguanidine with sodium channels of squid giant axons, Brain Research, 445 (1988) 1-11. Willow, M., Kuenzel, E.A. and Catteral, W.A., Inhibition of voltage-dependent sodium channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs diphenylhydantoin and carbamazepine, Mol. Pharmacol., 25 (1984) 228-234. Yeh, J.Z. and Tanguy, J., Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons, Biophys. J., 47 (1985) 685-694. Yamamoto, D., Quandt, F.N. and Narahashi, T., Modification of single sodium channels by the insecticide tetramethrin, Brain Research, 274 (1983) 344-349.
11
BRES 15507
Block of deltamethrin-modified sodium current in cultured mouse neuroblastoma cells: local anesthetics as potential antidotes Marga Oortgiesen, Regina G.D.M. van Kleef and Henk P.M. Vijverberg Research Institute of Toxicology, University of Utrecht, Utrecht (The Netherlands) (Accepted 7 November 1989) Key words: Neuroblastoma; Voltage clamp; Sodium current; Pyrethroid; Anticonvulsant; Local anesthetic
Effects of local anesthetics and anticonvulsants on the pyrethroid-modified sodium current in cultured mouse neuroblastoma cells have been investigated using the suction pipette voltage clamp technique. In the presence of 10/~M of the pyrethroid deltamethrin the sodium current consists of an enhanced peak current during membrane depolarization and a slowly decaying, deltamethrin-induced tail current remaining after repolarization. At the onset of block the local anesthetics tetracaine, lidocaine and QX 314 reduced the deltamethrin-induced tail current more effectively than the peak current. Lidocaine, but not phenytoin, caused a time-dependent block of tail currents evoked by membrane depolarizations lasting 10-1000 ms. Both lidocaine- and phenytoin-induced blocks were independent of the membrane potential during the tail current. The anticonvulsants phenytoin, phenobarbital and valproate blocked the tail and the peak sodium current to the same extent, but diazepam, mephenesin and urethane blocked the peak current more effectively. Vitamin E, which suppresses pyrethroid-induced paresthesia of the skin, had no effect on the voltage-dependent sodium current. It is concluded that indirect effects of anticonvulsants on pyrethroid-induced toxic symptoms predominate, whereas local anesthetics preferentially block the pyrethroid-induced tail current. Therefore, local anesthetics are potentially useful pyrethroid antidotes. INTRODUCTION Pyrethroids constitute a class of highly effective insecticides that act on the nervous system. Although pyrethroids exhibit low oral toxicity to mammals, they cause severe neurologic symptoms when the nervous system is directly exposed 3°. On the basis of intravenous poisoning symptoms in the rat pyrethroids have been divided into two classes: one that induces hypersensitivity and tremors of gradually increasing intensity, and another that causes profuse salivation, choreoathetosis and clonic seizures 3°. The latter class comprises most of the highly active a-cyano-3-phenoxybenzyl pyrethroids. A c o m m o n adverse effect of these a-cyano pyrethroids consists of cutaneous paresthesia upon dermal exposure to relatively low doses 11'2°. In man massive exposure may result in muscular fasciculations, disturbance of consciousness and convulsive attacks 13. Although the prognosis of occupational acute pyrethroid poisoning is generally good 13, treatment has been symptomatic and not very effective thus far. Results from studies into the treatment of pyrethroid-poisoned animals with anti-convulsant benzodiazepines and with phenobarbital have been variable and in several cases a delay in the onset of poisoning symptoms and an increase in LDso values of pyrethroids
was observed 8'1°'12'27'28. With mephenesin derivatives and urethane not only prophylactic but also therapeutic effects have been obtained 2'3'9'14'18. The in vivo occurrence of skin sensations and muscular fasciculations correlate well with structure-related excitatory effects of pyrethroids on the in vitro electrical activity of sense organs and sensory nerves, motor nerve endings and muscle fibres 3z. Pyrethroids induce repetitive activity and membrane depolarization in various parts of the peripheral as welt as in the central nervous system by prolonging the m e m b r a n e sodium current 28'33. From this major mechanism of action it is to be expected that sodium channel blockers antagonize pyrethroid effects. Patch clamp experiments have shown that pyrethroidmodified sodium channels remain in the open state much longer than normal sodium channels 7,34,38. Ideally, a pyrethroid antidote should selectively block pyrethroidmodified sodium channels. Therefore, use-dependent sodium channel blockers and compounds that selectively block open sodium channels are potential pyrethroid antidotes. Several compounds used in the symptomatic treatment of pyrethroid poisoning, e.g., diazepam, phenytoin and phenobarbital, have also been reported to block sodium channels 15,21,36. However, it is not clear whether these compounds antagonize pyrethroid effects
Correspondence: H.P.M. Vijverberg, Research Institute of Toxicology, University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
12 by directly interacting with sodium channels, by a selective suppression of secondary effects of pyrethroids or by a general depressive effect on nervous system activity. Cultured cells of the mouse neuroblastoma clone N1E-115 contain voltage-dependent sodium channels22. Effects of pyrethroids on sodium channels in these cells have already been described in detail 26"38. In the present study effects of experimental antidotes and of various known sodium channel blockers have been investigated in voltage-clamped neuroblastoma cells treated with the a-cyano pyrethroid deltamethrin.
period starting just before addition of the antidote and lasting till steady effects were reached. During this period a slow onset of the antidote effects was observed in parallel with the increasing concentration of the antidote at the cell membrane.
Chemicals Deltamethrin [a-S-cyano-3-phenoxybenzyl-(1R,cis)-3-(2,2-dibromovinyl)-2,2-dimethyl-cyclopropanecarboxylate] was donated by Roussel Uclaf. Tetrodotoxin, lidocaine, tetracaine, mephenesin, urethane, phenytoin (sodium salt), sodium valproate and vitamin E (Dc-a-tocopherol acetate) were obtained from Sigma. Diazepam and phenobarbital were obtained from Holland Pharmaceutical Supply, Alphen a/d Rijn, The Netherlands. The lidocaine derivatives QX 314 [2-(triethylamino)-N-(2,6-dimethylphenyl)-acetamide] and QX 222 [2-(trimethylamino)-N-(2,6-dimethylphenyl)-acetamide] were kindly supplied by Astra Pharmaceutica, Rijswijk, The Netherlands.
MATERIALS AND METHODS RESULTS
Cell culture Mouse neuroblastoma cells of the clone NIE-1151 were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 7.5% fetal calf serum and the following amino acids (in mM): L-cysteine.HCl 0.3, c-alanine 0.4, c-asparagine 0.45, L-aspartic acid 0.4, c-proline 0.4, and c-glutamic acid 0.4. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO 2. Cells of passage 30-45 were subcultured in 35 mm plastic tissue culture dishes. Cell differentiation was initiated 2-3 days after plating cells in DMEM supplemented with 1.5% fetal calf serum and 1% DMSO. This medium was refreshed every 2-3 days. The cells were used in the experiments 5-9 days after induction of differentiation.
Experimental procedure Experiments were carried out using the suction pipette technique for combined voltage clamp and internal perfusion of the neuroblastoma cells19. Fire-polished suction pipettes had an internal tip diameter of approximately 5 pm and a resistance of 400-600 KO. The liquid junction potential at the tip of the electrode was compensated before each experiment and remained constant within 1 mV. After obtaining seal resistances of 50-150 MI2, the membrane inside the pipette was ruptured and the cell was voltage clamped at -80 mV. The series resistance of 61)0-800 KI2 was compensated for 70-90%. Voltage-dependent sodium currents were evoked by step depolarizations of the membrane to 0 mV, preceded by a conditioning hyperpolarization to -120 mV for 100 ms to remove resting sodium channel inactivation. The holding current was recorded continuously. All responses were low-pass filtered (-3 dB at 2.5 kHz; 12 dB/octave), digitized by a transient recorder (8 bits; 1024 points/record) and stored on magnetic disc for off-line computer analysis. The external solution contained (in mM): NaCI 120, tetraethylammonium (TEA) chloride 25, tetramethylammonium chloride 20, CaCI2 1.8, MgCI2 0.4 and HEPES 25. The pH was adjusted to 7.4 with approximately 10 mM TEA-OH. The suction pipettes were perfused with an internal solution containing (in raM): CsF 120, NaOH 20, HEPES 25, and sucrose 10. The pH was adjusted to 7.1 with c-glutamic acid. Experiments were carried out at room temperature (20-24 °C). Deltamethrin, dissolved in dimethylsulphoxide (DMSO), was used to prepare a suspension in external solution with a final concentration of 10/~M. DMSO at its final concentration of 0.1% (v/v) did not affect the sodium current. The culture medium was replaced by a freshly prepared external solution containing deltamethrin at least 40 min in advance of the experiment. A small quantity of a concentrated stock solution of an antidote was added to the external solution in the culture dish at a location distant from the cell and was left to equilibrate by diffusion. Final concentrations are indicated and effects became steady 15-30 min after addition of the antidotes. The data were obtained in the
Control sodium currents were recorded in voltageclamped N1E-115 cells in the presence of 10 /~M deltamethrin and consisted of a slowly decaying peak inward current during m e m b r a n e depolarization to 0 mV, followed by a large, long-lasting sodium tail current after repolarization to the holding potential o f - 8 0 mV. This is very similar to previously recorded deltamethrin-modified sodium current in n e u r o b l a s t o m a cells 25. At the start of experiments the amplitude of both the peak current and the deltamethrin-induced tail current varied between cells and a m o u n t e d to -15.5 + 10 n A and -12.5 + 9 n A (mean + S.D.; n = 26), respectively. The ratio of the amplitude of tail and peak current changed in the beginning of the experiment. A t the first depolarization the tail-to-peak ratio ranged from 0.25 to 1.0 and on average a m o u n t e d to 0.73 + 0.23 (n = 15). After about 7 depolarizations at a frequency of 0.01-0.02 Hz the tail current had increased significantly (Wilcoxon's signed rank test; P < 0.01), whereas the peak current remained constant (P > 0.1). The holding current r e m a i n e d stable as well. A paired comparison of the tail to peak ratio at the first and the seventh depolarization for each cell revealed a significant increase ( P < 0.01) of the ratio at the last depolarization to 0.88 + 0.30 (n = 15; range = 0.28-1.33). The value of the initial tail-to-peak ratio appeared not correlated with the period of exposure to deltamethrin, which varied b e t w e e n 40 min and 6 h (Spearman's rank correlation test; P = 0.57) and the increase in the tail-to-peak ratio appeared i n d e p e n d e n t of the initial value and of the period of exposure (P = 0.71 and P = 0.47, respectively). Antidotes were administered after stabilization of the deltamethrin-modified sodium currents. The sodium channel blocker tetrodotoxin ( q T X ) reduced the peak as well as the tail current. Fig. l a shows deltamethrin-modified sodium currents before as well as 5 and 7 min after the addition of 0.5 p M T T X to the
13 external solution. A t equilibrium the p e a k and the tail current were c o m p l e t e l y blocked, confirming that both current c o m p o n e n t s are m e d i a t e d by v o l t a g e - d e p e n d e n t sodium channels. Sodium currents were r e c o r d e d at regular intervals from the time of the addition of T T X until steady block was achieved. A two-dimensional plot o f the a m p l i t u d e of the tail current against the amplitude of the p e a k current, both normalized to their respective initial values, is p r e s e n t e d in Fig. l b . The d a t a showed no deviation from linearity ( P = 0.24). In another experi-
a 80 mV
-120 " ~ - -
TTX
TTX 7 rain
control
1hA I
ment similar results were o b t a i n e d (regression lines identical; P = 0.22). T h e slope of the regression line through the results from both cells a m o u n t e d to 0.99 + 0.04 ( + 9 5 % confidence limit). T h e linear relationship demonstrates that the tail current, which is carried entirely by d e l t a m e t h r i n - m o d i f i e d sodium channels, and the p e a k current were equally affected by T T X as the concentration gradually increased to its final value of 0.5 /~M. Effects of lidocaine and its derivatives Q X 314 and Q X 222 on the sodium current in the presence of 10 p M deltamethrin are d e p i c t e d in Fig. 2. T h e steady effect of 0.6 m M lidocaine (Fig. 2a) consisted of a reduction of the p e a k amplitude to 66% of its initial value, while at the same time the tail a m p l i t u d e was r e d u c e d to 52%. The time course of the sodium current was not significantly altered by lidocaine. A f t e r raising the lidocaine concentration to 1.4 m M the p e a k and the tail a m p l i t u d e further r e d u c e d to 37% and 23% (Fig. 2a) of the initial values, respectively. External application of the lidocaine derivatives Q X
50 ms
/
/
// i/
8 0 _120 ~
"~
.80 mV
/
/// // •
t.4
mM
1hA[ 50 m s
0//~//
0
I 15 min
QX 222 30 min
2 ,^{ / / / /
0.0 0.0
~
50 ms
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/ /
'
~
'
'
'
0.5
'
'
'
'
1.0
relative peak current
Fig. 1. Effects of "I"TX on the sodium current after exposure to 10 /~M deltamethrin. Deltamethrin-modified sodium currents were evoked by the step depolarization indicated, a: deltamethrinmodified sodium current (left), and sodium currents 5 min (middle) and 7 min (right) after subsequent addition of a small amount of I T X to a final concentration of 0.5 /~M. b: two-dimensional representation of the onset of the blocking effect of 0.5/~M TTX on the peak and the tail current. The amplitude of the peak current (abscissa) and that of the deitamethrin-induced tail current have both been normalized to the respective values before addition of I"TX. Data marked with asterisks were obtained from the records in (a). Linear regression analysis showed no deviation of the tail-to-peak relationship from linsarity (P = 0.24). The slope was 1.03 + 0.07 (_+95% confidence limits).
3 nAL~ 5O ms
Fig. 2. Effects of the local anesthetics lidocaine, QX 222 and QX 314 on the sodium current after exposure to 10 /~M deltamethrin. Deltamethrin-modified sodium currents were evoked by the step depolarization indicated, a: deltamethrin-modified sodium current (left), and subsequent steady effects of 0.6 mM (middle) and 1.4 mM lidocaine (right). b: deltamethrin-modified sodium current (left), and sodium currents 15 min (middle) and 30 min (right) after internal perfusion of the cell with 1 mM QX 222. c: deltamethrinmodified sodium current (left), and sodium currents 14 min (middle) and 25 min (right) after internal perfusion of the cell with 1 mM QX 314.
14 314 and Q X 222 did not affect the sodium current in the presence of deltamethrin. H o w e v e r , internal perfusion with 1 m M of Q X 222 and of O X 314 blocked both the p e a k and the tail current. With O X 222 the peak and the tail current were r e d u c e d in parallel to 66% of their initial values after 15 min and to 5% 30 min after switching the pipette solution to one containing 1 m M of the local anesthetic (Fig. 2b). Similar to lidocaine, O X 314 blocked the tail current to a greater extent than the peak current. A f t e r 14 min of internal perfusion with a pipette solution containing 1 m M Q X 314 the amplitude of the peak current was reduced to 74% of its initial value, while at the same time the amplitude of the tail current was reduced to 50%. A f t e r 25 min of internal perfusion with 1 m M O X 314 the peak and the tail current were r e d u c e d to 16% and 12%, respectively (Fig. 2c). In contrast to the other local anesthetics O X 314 caused an acceleration of the decay of the slow c o m p o n e n t of the sodium current during m e m b r a n e depolarization. The gradual block of the peak and the tail current after adding 1.6 m M lidocaine to the external solution was investigated in two experiments. In contrast to TTX, lidocaine initially blocked the tail current more than the p e a k current resulting in the non-linear plot of the tail against the p e a k amplitude presented in Fig. 3. The slope of the curve at the onset of block, i.e., in the concen-
tration range in which lidocaine r e d u c e d the p e a k as well as the tail current less than 35%, a m o u n t e d to 1.5 -4-_(1.3 ( + 9 5 % confidence limit). The initial slopes of the tail-to-peak relationships o b t a i n e d during the onset of block by internal Q X 222 and Q X 314 were 1.2 + 0.4 and 1.8 + 0 .6, respectively. External application of 0.1 m M of the local anesthetic tetracaine caused nearly c o m p l e t e block of the sodium
a
1.0 mephenesin
o ~poo~ 0 0 0 0
o
c
-120 ~
-80 mV
I
= 0.5
>o o
1 nA J__._ 100 m~
I
0.0 q 0,0
i
i
i
i
i 0.5
l
i
i
i 1.0
1.0
o phenyloin
1,0 lidocaine
{ o
O
a) o 0.5
O
E
0 0 0
0
oo
•®
- 120~-J
I
-80 mV
0
0.5
8 0.0 0,0
o.5 relative
0.0
,
0.0
,
i
,
i
,
~
0.5
,
~
,
1.0
relative peak current
Fig. 3. The tail-to-peak relationship during the onset of the blocking effect of 1.6 mM lidocaine in a 10 /~M deltamethrin-treated neuroblastoma cell. The amplitude of the peak current {abscissa) and that of the deltamethrin-induced tail current have both been normalized to the respective values before addition of lidocaine. The initial slope of the tail-to-peak relationship was 1.5 + 0.3 (+95% confidence limits) and was obtained by least-squares linear regression of datapoints with values >0.65.
peak
1.0
current
Fig. 4. The tail-to-peak relationship during the onset of the blocking effect of 1 mM mephenesin and 1 mM phenytoin in 10 pM deltamethrin-treated neuroblastoma cells. The amplitude of the peak current (abscissa) and that of the deltamethrin-induced tail current have both been normalized to the respective values before addition of mephenesin or phenytoin. The initial slopes of the tail-to-peak relationship have been fitted by least-squares linear regression of datapoints with values >0.65. a: the inset shows the deltamethrin-modified sodium current and the steady effect of 1 mM mephenesin. The initial slope of the tail-to-peak relationship during the onset of block by mephenesin was 0.5 + 0.1 (+95% confidence limits), b: the inset shows the deltamethrin-modified sodium current and the steady effect of 0.5 mM phenytoin. The initial slope of the tail-to-peak relationship during the onset of block by phenytoin was 1.3 _+ 0.4.
15
current in the presence o f deltamethrin. During the onset
o b t a i n e d in the presence of external lidocaine and
of this effect the tail current was blocked to a greater extent than the p e a k current. T h e initial slope of the tail-to-peak relationship at the onset of block by tetracaine a m o u n t e d to 1.4 + 0.3. T h e effects of various e x p e r i m e n t a l pyrethroid antidotes and some c o m m o n anticonvulsants have also been investigated. M e p h e n e s i n caused only partial block of the v o l t a g e - d e p e n d e n t sodium current in the presence of deltamethrin. T h e inset of Fig. 4a shows two superimp o s e d sodium current records o b t a i n e d before and at equilibrium after adding 1 m M mephenesin to the external solution. T h e p e a k current was r e d u c e d to 57% of its initial value and the tail current to 68%. The slope of the tail-to-peak relationship during the onset of block by m e p h e n e s i n in two cells a m o u n t e d to 0.5 + 0.2 (Fig. 4a). P h e n y t o i n at 0.5 m M r e d u c e d the p e a k current to 60% and the tail current to 54% of the initial values in 13 min (Fig. 4b, inset). A t 1 m M phenytoin both current c o m p o n e n t s were almost completely blocked. The slope of the tail-to-peak relationship during the onset of block by 1 m M p h e n y t o i n in two cells a m o u n t e d to 1.3 + 0.4 (Fig. 4b). T h e effects of 12 c o m p o u n d s on the sodium current in the presence of 10 # M deltamethrin are summarized in Table I. The slopes of the tail-to-peak relationships
tetracaine and internal Q X 314 were significantly greater than 1.0 (two-tailed Student's t-test; P < 0.05), indicating that low concentrations o f these local anesthetics blocked the tail current to a greater extent than the p e a k current. In contrast, mephenesin, d i a z e p a m and urethane m o r e effectively blocked the p e a k than the tail current as the slopes of the tail-to-peak relationships were significantly smaller than 1.0 ( P < 0.05). Internal Q X 222, phenobarbital, phenytoin and v a l p r o a t e a p p e a r e d to block the p e a k and the tail current to the same extent. Vitamin E, which has been r e p o r t e d to suppress cutaneous paresthesia caused by d e r m a l exposure to a - c y a n o pyrethroids in man 29, did not affect the d e l t a m e t h r i n - m o d i f i e d sodium current when present in the external solution at a concentration of 1 m M for a p e r i o d of up to 2 h.
1.0
o--o control H lidocaine g ,~ 0.s
TABLE I
Effects o f various sodium channel blockers and experimental antidotes on the voltage-dependent sodium current o f cultured mouse neuroblastoma cells in the presence o[ lO ttM deltamethrin
Compound
Final concentration (M)
TTX Lidocalne QX 222 (int) QX314 (int) Tetracalne
1.6.10-3 10-3 10-3 10-~
Urethane Mephenesin
10 -3 10-3
5'10 -7
Diazepam
5.104
Phenobarbital
5.10 -3
Phenytoin Valproate Vitamin E
10-3 10-3 10-3
0.0
Initial bloc~ Steady effec~ slope tail vs peak peak tail 0.9 + 0.2 1.5 + 0.3* 1.2 + 0.4 1.8 + 0.6* 1.4 _+0.3* 0.6 + 0.3* 0.5 + 0.1" 0.4 + 0.3* 1.2 + 0.3 1.3 + 0.4 1.0 + 0.3 -
0.00 0.19 0.04 0.14 0.19 0.75 0.54 0.21 0.32 0.09 0.59 1.0
0.00 0.13 0.03 0.11 0.14 0.79 0.77 0.24 0.18 0.04 0.53 1.0
Slope of the two-dimensional plot of tail vs. peak amplitude (+95% confidence limit) determined by linear regression of datapoints in the concentration range in which the compounds reduced the amplitudes of the peak and tail current to 65-100% of the initial values. Slope values < 1 indicates a selective block of peak current at low concentrations and slope values > 1 indicate a selective block of tail current. z Fraction of the initial amplitude remaining at steady effect, i.e., at final concentration. * Slope significantly different from 1.0 (two tailed Student's t-test, P < 0.05).
'
,
'
0
b
,
,
500
1.0 ~
,
,
1000 ms
1
o--o control H
phenytoin
g
o 0.5 >=
:
°
o
m
0.0
,
0
,
,
,
,
,
500
1000 ms t
Fig. 5. Relation between the period of depolarization and the
deltamethrin-induced tail current before and after exposure to lidocalne (a) and phenytoin (b). Tail currents were evoked by depolarizations to 0 mV for 10-1000 ms. The tail current amplitudes have been normalized to the values obtained with 10 ms depolarizations, a: at equilibrium 0.8 mM lidocaine reduced the tail current evoked by a 20 ms depolarization to 41% of its initial value. With increasing the duration of depolarization a time-dependent increase of block is observed, which declined again after depolarizations >200 ms. b: the steady block of the tail current by 0.6 mM phenytoin remained virtually constant for the range of depolarizations investigated.
16 Vm -80 mV
-4O 0.0
0.8 mM lidocaine and 0.6 external solution, except This result indicates that lidocaine and phenytoin potential.
mM phenytoin (Fig. 6) to the for a constant scaling factor. block of the tail current by is independent of membrane
DISCUSSION
o
control
•
phonyloin 1.0
Fig. 6. I- V relationship of the 10 pM deltamethrin-induced tail current before and after exposure to 0.6 mM phenytoin. Tail currents were measured at different repolarization potentials (Vm) after conditioning membrane depolarizations to 0 mV for 20 ms. The tail current amplitudes have been normalized to the values obtained at -80 mV. The block of the tail current by phenytoin remained virtually constant at membrane potentials between 0 and -80 inV. As pyrethroids induce long-lasting repetitive activity and nerve membrane depolarization, the time dependence and the potential dependence of the effects of lidocaine and phenytoin on the deltamethrin-induced tail current have been investigated. In the presence of 10 ktM deltamethrin maximum tail current amplitudes were obtained with depolarizations of 10-20 ms. The tail current amplitude decreased when the depolarization period was further increased to 1 s. The peak current amplitude was reduced by 0.8 mM lidocaine to 41% and by 0.6 mM phenytoin to 69% of the initial value, whereas the amplitudes of tail currents after 20 ms depolarizations were reduced to 27% and 56%, respectively. Fig. 5, in which normalized values of the deltamethrin-induced tail currents are presented, shows the dependence of block of the tail current by lidocaine and phenytoin on the duration of the membrane depolarization. Although the control curves obtained in the presence of deltamethrin varied between experiments, possibly because of several degrees temperature differences, similar effects were obtained in 3 experiments with each blocker. The effect of lidocaine increased with depolarizations lasting up to 200 ms, but after longer depolarizations this increase gradually vanished (Fig. 5a). The effect of phenytoin remained virtually the same for depolarizations ranging from 10 ms to 1 s (Fig. 5b). In the same cells the dependence of the effects of |idocaine and phenytoin on membrane potential was examined. The membrane was repolarized to potentials between -80 and 0 mV after depolarization to 0 mV for 20 ms. I - V curves obtained for the deltamethrin-induced tail current were identical to those obtained after adding
Deltamethrin greatly affects the voltage-dependent sodium current in voltage-clamped mouse neuroblastoma cells. The peak sodium current during depolarization is enhanced and a slowly decaying sodium tail current appears after repolarization of the membrane. Patch clamp experiments have shown that normal as well as pyrethroid-modified sodium channels contribute to the peak current during membrane depolarization TM. However, it is likely that at the time of peak, the sodium current is mainly carried by normal sodium channels, as after deltamethrin only a small increase of the peak amplitude is observed (R.G.D.M. van Kleef and H.P.M. Vijverberg, unpublished result) and the opening of pyrethroid-modified sodium channels is delayed TM. On the other hand, the deltamethrin-induced slow tail current is entirely carried by modified sodium channels. The experiments, in which TTX gradually caused a complete block of the sodium inward current in the presence of deltamethrin without changing the tailto-peak ratio, indicate that pyrethroid-modified and normal sodium channels are equally TTX-sensitive. In addition, the results are consistent with the notion that the effect of TTX is restricted to a reduction of the maximum sodium conductance. At the start of the experiments the tail-to-peak ratio varied. When the membrane was depolarized for the first time after voltage clamping neuroblastoma cells exposed to 10 p M deltamethrin for 40 min to 6 h, the tail-to-peak ratio showed significant variation irrespective of the period of exposure. This indicates that the fraction of deltamethrin-modified sodium channels varied between cells. Further, the increase of the tail current amplitude during the first membrane depolarizations appeared independent of the initial tail-to-peak ratio and the period of exposure. Without excluding the previous hypothesis that pyrethroids affect voltage-dependent sodium channels in the resting configuration 3~, this result indicates that the effect of deltamethrin is enhanced by channel opening. Voltage-dependent sodium channels are regarded as the main target site of pyrethroids. Receptor-operated ion channels, which have been postulated as potential targets of pyrethroids as well, were recently demonstrated not to be directly responsible for the toxic symptoms induced by pyrethroids 6'23'24. Therefore, se-
17 lective antidotes should block pyrethroid-modified sodium channels completely without affecting normal sodium channels. Although a highly selective block of the deltamethrin-induced tail current was not observed in the present experiments, lidocaine, QX 314 and tetracaine more effectively blocked the tail current. The selectivity of QX 222 could neither be distinguished from the other local anesthetics, nor from that of TTX (see Table I). The greater sensitivity of the tail current to local anesthetics was anticipated, since these compounds block open sodium channels 4 and the pyrethroid-modified channels remain open for prolonged periods 34'38. It has also been reported that octylguanidine, which has a similar mechanism of action as local anesthetics, selectively blocks open sodium channels in pronase-treated and in pyrethroid-treated squid giant axons 16"35. Open channel block by local anesthetics is most clearly observed as a time-dependent block of the prolonged sodium current in pronase-treated squid axons 5. In the present experiments block of the deltamethrin-induced tail current by lidocaine was time-dependent, but did not depend on the membrane potential during the tail current. Differences in use-dependent block by lidocaine, QX 222 and QX 314 have been found in voltage-clamped squid giant axons 37, but the presently observed blocking effects of these local anesthetics at low concentrations (see Table I) are not statistically different. This result and the transient character of the time-dependence of the tail current block by lidocaine (see Fig. 5a) suggest that deltamethrin affects the interaction of lidocaine with the sodium channel. Additional experiments are needed for a more detailed description of the interaction of local anesthetics with pyrethroid-modified sodium channels. The anticonvulsants phenytoin, phenobarbital and valproate blocked the sodium current without affecting the tail-to-peak ratio. Similar to local anesthetics phenytoin causes resting as well as use-dependent block of the sodium current in neuroblastoma cells21. However, unlike the present results with lidocaine, phenytoin block of the tail current is time-independent (see Fig. 5b), which REFERENCES 1 Amano, T., Richelson, E. and Nirenberg, P.G., Neurotransmitter synthesis by neuroblastoma clones, Proc. Natl. Acad. Sci. U.S.A., 6 (1972) 258-263. 2 Bradbury, J.E., Forshaw, P.J., Gray, A.J. and Ray, D.E., The action of mephenesin and other agents on the effects produced by two neurotoxic pyrethroids in the intact and spinal rat, Neuropharmacology, 22 (1983) 907-914. 3 Bradbury, J.E., Gray, A.J. and Forshaw, P., Protection against pyrethroid toxicity in rats with mephenesin, Toxicol. Appl. Pharmacol., 60 (1981) 382-384. 4 Cahalan, M., Molecular properties of sodium channels in excitable membranes. In C.W. Cotman, G. Poste and G.L. Nicolson (Eds.), The Cell Surface and Neuronal Function,
agrees with the previous finding that octylguanidine and pyrethroids independently modify voltage-dependent sodium channels in squid axons 35. Mephenesin, urethane and diazepam, which have been reported to block voltage-dependent sodium channels in different preparations 17'25'36, initially blocked the peak current to a greater extent than the tail current (see Table I). The apparent selectivity for blocking normal sodium channels with respect to deltamethrin-modified sodium channels shows that these compounds do not simply reduce the maximum sodium conductance, as has been suggested for urethane before 25. The poor block of the deltamethrin-induced tail current by most of the anticonvulsants at relatively high concentration indicates that their prophylactic and therapeutic effects in the treatment of the neuroexcitatory symptoms of pyrethroids are not mediated by a selective interaction with deltamethrin-modified sodium channels. The multiple actions of these compounds suggest that indirect effects, which generally cause depression of nervous system excitability, predominate. This conclusion is supported by the finding that the batrachotoxinand the veratridine-induced sodium influx in N18 neuroblastoma cells are inhibited by various anticonvulsants at concentrations in excess of therapeutic levels 36. The suppression of pyrethroid-induced skin sensations by vitamin E 28 also seems to be mediated by indirect effects, because the sodium current is not affected at all. In contrast, local anesthetics, which appear to block pyrethroid-modified sodium channels more effectively, are potentially useful antidotes in the treatment of accidental pyrethroid poisoning. However, the therapeutic value of local anesthetics as antidotes against pyrethroid poisoning symptoms remains to be evaluated.
Acknowledgements. The authors wish to thank Ms. P. Martens for maintaining the cell culture, Ing. A. de Groot for expert technical assistance. Prof. J. van den Bercken is thanked for his support during the experiments. This work was financially supported by Shell Internationale Research Maatschappij B.V.
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