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Effects of pyrethroids on neurotransmitter-operated ion channels in cultured mouse neuroblastoma cells
PESYKXDE
BIOCHEMISTRY
AND
PHYSIOLOGY
34, 164173 (1989)
Effects of Pyrethroids on Neurotransmitter-Operated Ion Channels in Cultured Mouse Neuroblastoma Cells MARGA OORTGIESEN,REGINA G.D.M.
VAN KLEEF, AND HENK P. M. VUVERBERG
Department of Veterinary Pharmacology, Pharmacy, and Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands Received February 15, 1989; accepted April 20, 1989 The effects of the pyrethroids allethrin, lR-cis-cyphenothrin, lR-cis-fentluthrin, and of the noninsecticidal isomer lb-cis-fenfluthrin on nemotransmitter receptor-mediated membrane responses have been investigated in the mouse neuroblastoma cell line NlE-115 using electrophysiological methods. At a concentration of 10 PM, allethrin and lR-cis-cyphenothrin caused a reduction of the peak amplitude of the nicotinic membrane depolarization to half of the control value. In voltageclamped cells the peak amplitude of the nicotinic ACh receptor-mediated inward current was reduced to 50-75% of the control value by the pyrethroids at a concentration of 1 @4. The effects of the insecticidal and the noninsecticidal isomer of fentluthrin were identical. The amplitudes of the ACh and the serotonin S-HT, receptor-mediated inward currents were equally reduced by 10 ~.uV IR-cis-cyphenothrin. The effects were partially reversed by washing. Steady-state desensitization, induced with a low concentration of ACh, was increased by 1 ~uV allethrin and by 1 l&f lb-cis-fenfluthrin to a similar extent. The rate of desensitization was not affected. Ion channel block, additionally observed at high concentrations of ACh, was facilitated by all pyrethroids investigated. It is concluded that the pyrethroids exert nonspecific, inhibitory effects on neurotransmitter receptor-operated ion channels. These effects are unlikely to contribute to the excitatory symptoms of these insecticides. 8 1989 Academic press, Inc.
INTRODUCTION
Pyrethroids are potent neuroactive insecticides with a strong excitatory action. These compounds induce repetitive activity in various parts of the peripheral and central nervous system. Electrophysiological investigation has demonstrated that this repetitive fuing originates from a prolongation of the increase in nerve sodium channel permeability associated with excitation (1, a Although it is generally accepted that voltage-dependent sodium channels are the main target site of pyrethroids, effects of these insecticides on neurotransmission have been reported as well. In rat brain synaptic membranes the binding of cage convulsants to the GABA receptor complex was demonstrated to be partially inhibited by cyanopyrethroids (3). However, in crayfish stretch receptor neurones and in rat dorsal root ganglion cells the cyanopyrethroid deltamethrin had no effect on the GABA-mediated electrophysiological re-
sponse (4, 5). Studies on the nicotinic neurotransmission in the frog motor end-plate revealed that pyrethroids induce repetitive activity in the presynaptic motor nerve terminal, whereas the postsynaptic nicotinic acetylcholine (ACh)’ receptors appeared not affected (6-8). On the other hand, a reduction of receptor desensitization has been suggested from inhibitory effects of pyrethroids on radioligand binding to the nicotinic receptor-ion channel complex and on nicotinic receptor-mediated calcium influx in Torpedo electric organ preparations (9).
In cultured neuroblastoma cells, properties of different types of ion channels, that are expressed in the plasma membrane, can be investigated independently. In cells of the clone NlE-115, effects of a range of pyrethroids on voltage-dependent sodium channels have already been described in * ACh, acetylcholine; DMSO, dimethyl sulfoxide; Garb, carbachol; HTX, perhydrohistrionicotoxin; 5HT, serotonin.
164 0048-3575189 $3.00 Copyright 0 1989 by Academic Press, Inc. AU rights of reproduction in any form reserved.
PYRETHROIDS
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detail (IO, I I). In addition, tetramethrin has been reported to block transient and, to a lesser degree, slow voltage-dependent calcium channels (12). Recently, serotonin 5-HT, receptors and neuronal type nicotinic ACh receptors have been characterized pharmacologically in NlE-115 neuroblastoma cells (13, 14). In voltage-clamped cells, serotonin (5HT) and ACh cause transient inward currents, due to the opening of distinct types of ion channels, that rapidly desensitize in the continued presence of the agonists. Despite differences in the sensitivity to snake toxins, functional properties, like desensitization, and channel block of neuronal nicotinic receptor-operated ion channels in NlE-115 cells and of nicotinic receptors in muscle end-plates are very similar (14). Neuronal type nicotinic receptors and 5HT, receptors have also been described in sympathetic and parasympathetic neurones and in the central nervous system (15-17). In addition, 5-HT, receptors present in the heart and in skin sensory nerve endings are involved in reflex bradycardia, i.e., the von Bezold-Jarisch effect, and in the transmission of pain, respectively (16). This is of particular interest with respect to cardiovascular symptoms (18) and peripheral sensory phenomena (19) potentially induced by pyrethroids in intact animals. We have investigated direct postsynaptic effects of pyrethroids on the electrophysiological responses mediated by nicotinic ACh receptors and by serotonin 5-HT, receptors in cultured mouse neuroblastoma cells. MATERIALS
AND
METHODS
Cell culture. Mouse neuroblastoma cells of the clone NlE-115 (20) were grown in Dulbecco’s modified Eagle medium supplemented with 7.5% fetal calf serum and the following amino acids (in mM): L-cysteine * HCl, 0.3; L-alanine, 0.4; L-asparagine, 0.45; L-aspartic acid, 0.4; L-proline, 0.4; and L-glutamic acid, 0.4. The cultures
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were maintained at 37°C in a humidified atmosphere containing 5% C02. Cells of passages 30-45 were subcultured in 35mm plastic tissue culture dishes. Cell differentiation was initiated 2 to 3 days after plating by adding 1 mM N6,2’-U-dibutyryladenosine 3’,5’-cyclic monophosphate and 1 n-&f 3-isobutyl-l-methylxanthine to the culture medium. This medium was refreshed every 2-3 days. The cells were used in the experiments 8-12 days after induction of differentiation. Experimental procedure. Experiments were carried out using the standard wholecell patch clamp technique (21). The resistance of fire-polished glass pipettes for intracellular recording (internal tip diameter l-l.5 pm) was 3-5 Ma. The liquid junction potential at the tip of the electrode was compensated before each experiment and remained constant within 1 mV. In voltage clamp experiments the membrane potential was held at - 80 mV and the holding current was recorded continuously. The series conductance of approximately 0.15 ~.LSwas compensated for 60-70%. All responses were low-pass filtered (- 3 dB at 1000-500 Hz; 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): NaCl, 125; KCI, 5.5; CaCl,, 1.8; MgCl,, 0.8; Hepes, 20; glucose, 25; and sucrose, 36.5. The pH was adjusted to 7.3 with approximately 10 mill NaOH. Tetrodotoxin (0.5 l&f) was added to the external solution before each experiment to eliminate interference from effects of pyrethroids on voltage-dependent sodium channels. The glass pipettes were filled with an internal solution containing (in n-&Q: KCl, 150; NaCl, 10; Hepes, 10; and MgCl,, 1. The pH was adjusted to 7.2 with approximately 3 mM KOH. Agonists were applied either by ionophoresis or by whole-cell super-fusion. The micropipettes for ionophoresis (50-60 Ma) were filled with 1 M ACh chloride. To min-
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imize agonist leakage from the tip of the pipette, a retention current (1-5 nA) was applied. To allow recovery from desensitization, the frequency of ionophoretic stimulation was low (0.005 Hz) and the external solution was exchanged in between ionophoretically induced responses. In wholecell superfusion experiments a continuous flow (1.5 ml/min) of external solution was applied to the cell from a capillary with a diameter of 1 mm positioned within a distance of 1mm from the cell. The cells were super-fused with known concentrations of agonist and/or pyrethroid for adjustable periods (2 1set) using a servo-motor operated valve (13). Data were obtained from cells that responded to superfusion with ACh with a nicotinic receptor-mediated inward current only. To allow recovery from desensitization, the cell was washed between subsequent responses with control external solution for 3-4 min. Experiments were carried out at room temperature (20-24°C). In desensitization experiments the external solution was maintained at a constant temperature of 21°C with the use of a recirculating water jacket around the superfusion capillary. Pyrethroids were dissolved in dimethyl sulfoxide (DMSO). Shortly before each experiment a suspension was made from the stock solution with a final DMSO concentration of 0.1%. At this concentration, DMSO did not affect the ACh-induced inward current in control experiments. Exponential curve fitting was performed using a Levenberg-Marquardt nonlinear least squares algorithm (22). Results are presented as mean -’ SD and are compared using Student’s t test (23). Chemicals. Acetylcholine chloride, carbamylcholine chloride, dimethyl sulfoxide, and tetrodotoxin were obtained from Sigma; allethrin[(RIS)-2-allyl-4-hydroxy-3methyl-2-cyclopenten-I-one-(lR/S,cisltrans)3-(2,2-dimethylvinyl)-2,2-dimethylcyclopropanecarboxylate; purity >96%1 was obtained from K&K Laboratories Inc.; IR-cis-cyphenothrin[(Y(S)-cyano-3-phenoxy-
AND
VUVERRERG
benzyl-(lR,cis)-3-(2,2-dimethylvinyl)-2,2dimethylcyclopropanecarboxylate; RU 29209; purity >98%] was obtained from Roussel Uclaf; and lR-cis-fenfluthrin [pentafluorobenzyl-(lR,cis)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; NAK 1901; purity >99%] and IScis-fenfluthrin [pentafluorobenzyl-( IS, cis)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; NAK 1902; purity >99%] were obtained from Bayer. RESULTS
ACh-Induced
Membrane
Depolarization
Ionophoretic application of ACh to cells with a resting membrane potential adjusted to - 65 mV by injecting a small hyperpolarizing current (0.04-0.25 nA) resulted in a depolarization which is mediated by nicotinic ACh receptors. The resting potential of the cells used in the experiments ranged from -40 to - 68 mV, the mean resting potential amounted to 52 -+ 9 mV (mean ? SD; n = IS). During membrane potential measurements, atropine (0.5 p&Q was present in the external solution to block occasionally observed muscarinic receptormediated responses. The pyrethroids caused a gradual decrease of the peak amplitude of the ACh-induced depolarization. Superfusion of the cell with 10 PM allethrin for 15 min reduced the amplitude of the ACh response to a steady level, which was 51 + 20% (n = 4) of the control value (Fig. la). After 15 min of superfusion with 10 pJ4 lR-cis-cyphenothrin the peak amplitude was reduced to 54 -+ 33% (n = 5) of the control value (Fig. lb). The blocking effects of the noncyano and the cyan0 pyrethroid on the ACh-induced membrane depolarization cannot be distinguished statistically (P = 0.91). Neurotransmitter-Znduced Inward Currents
Under voltage clamp conditions, at a holding potential of - 80 mV, stimulation of the nicotinic ACh receptors by direct su-
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same concentration of lR-cis-cyphenothrin on the Carb-induced and the 5HT-induced inward currents cannot be distinguished statistically (P = 0.65). The time course of the agonist-induced inward currents was not significantly altered by the pyrethroids. During the exposure to the pyrethroids the fluctuations of the holding current increased up to 2.5-fold. The control holding current level of 0.25-l nA was only slightly increased by 35 ‘I 51 pA (n = 9) in the presence of pyrethroid.
’:c mvliiT t
allethrin
1 R,c;s-
cyphenothrm
FIG. 1. Effect of allethrin and IR-cis-cyphenothrin on the membrane depolarization evoked by ionophoretic stimulation with ACh (indicated by arrows). The resting membrane potential was -65 mV. Left traces show depolarizations in the control situation, right traces show depolarization after 15 min of superfusion with 10 JLM allethrin (a) and 10 FM IRcis-cyphenothrin (b). During superfusion with allethrin and lR-cis-cyphenothrin, the peak amplitude of the ACh-induced depolarization was reduced to 55 and 67% of the control value, respectively. Ionophoretic pulses were 100 nC (a) and 300 nC (b).
perfusion of the cell with ACh or carbachol (Garb) resulted in a transient inward current. Exposure to IR-cis-cyphenothrin caused a gradual decrease of the amplitude of the inward current induced by 1 mM Carb. This effect was partially reversed after IO-15 min of washing with control external solution. Superfusion with 10 FM lR-cis-cyphenothrin for 10min reduced the peak amplitude of the 1 mM Carb-induced inward current to a steady level, which was 67 +. 4% (n = 4) of the control value (Fig. 2a). Superfusion with 5-HT also caused a transient inward current by the activation of S-HT, receptors. After 10 min of superfusion with 10 @U la-cis-cyphenothrin, the peak amplitude of the 5-HTinduced inward current was reduced to 70 + 12% (n = 5) of the control value (Fig. 2b). Little recovery was obtained during subsequent washing with control external solution for 10-15 min. The effects of the
Zon Channel Block by ACh
The ACh-induced inward current can be blocked by high concentrations of ACh (24). The reopening of ion channels that were blocked in the presence of ACh is measured as a transient increase of the tail current during the removal of high concentrations of ACh by washing, as shown in Fig. 3a (14). In addition to a reduction of the peak amplitude, 1 JLM allethrin and 1 $M lR-cis-cyphenothrin caused a change in the time course of the inward current induced by 3 mM ACh. Within a few minutes after superfusion with the pyrethroids, the peak amplitude of the ACh-induced inward current and the peak amplitude of the tail current were decreased. In addition, the decay of the ACh-induced inward current was accelerated, whereas the decay of the tail current was delayed (Fig. 3a and 3b). After l&15 min of super-fusion with the pyrethroids, the transient increase of the tail current was no longer visible. Partial recovery was obtained after la-15 min of washing (Fig. 3~). The peak amplitude of the 3 rnA4 AChinduced inward current was reduced after 10 min of superfusion with 1 ~JM allethrin and with 1 l&f la-cis-cyphenothrin to 75 + 13% (n = 3) and 71 it 11% (n = 4) of the control value, respectively. These values do not differ significantly (P = 0.68). Allethrin (1 u&f) also caused a reduction of the peak amplitude of the 1 mM ACh-induced inward current to 67 ? 8% (n = 5) of the
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Carb a
//“”
control
1 R CIS- cyphenothrm
I5HT
lR.cis-
cyphenothrln
recovery
FIG. 2. Effect of IR-cis-cyphenothrin on nicotinic and S-HT, receptor-mediated inward currents under voltage clamp conditions. (a) Recordings of inward currents induced by 1 mM Carb in control external solution, after 10 min of supetjkion with 10 PM lR-cis-cyphenothrin and after 10 min of recovery by washing with control external solution. In the presence of 10 fl IR-cis-cyphenothrin the amplitude of the Carb-induced inward current was reduced to 63% of the control value. (b) Recordings of inward currents induced by 3 PM 5-HT in control external solution, after 10 mitt of superfusion with IO )IM IR-cis-cyphenothrin and after 10 min of recovery by washing with control external solution. In the presence of 10 FM IR-cis-cyphenothrin the amplitude of the SHT-induced inward current was reduced to 62% of the control value. Superfusion periods with ACh and 5-HT are indicated by bars.
control value, which cannot be distinguished from its effect on the 3 mM AChinduced peak inward current (P = 0.35).
ACh-induced the transient (Fig. 4~).
inward current nor blocked increase of the tail current
Stereospecijkity
Desensitization
Superfusion with 1 tMI4 IR-cis-fenfluthrin and with 1 @I4 19cis-fenfluthrin for 10 min reduced the peak amplitude of the 3 mM ACh-induced inward current to 52 t 13% (n = 4) and 52 ? 12% (n = 3) of the control value, respectively. The effects of the insecticidal and the noninsecticidal isomers are identical (P = 0.98). The transient tail current during the removal of ACh was also suppressed by both fenfluthrin isomers (Fig. 4a and 4b). The possibility that the effects of Weis-fenfluthrin were due to contamination (~1%) with the II?-cisisomer was excluded. Superfusion with 0.01 t.&f lR-cis-fenfluthrin, i.e., 1% of the concentration used in the previous experiments, for 10 min neither caused a reduction of the peak amplitude of the 3 rnM
The time constants fitted to the decay of the inward current induced with the relatively low concentration of 9 p&I ACh, which causes little or no ion channel block, reflect the onset of desensitization (14). In a single cell an inward current was induced by 50 set of superfusion with 9 t&f ACh before and during exposure to pyrethroid (Fig. 5). The time constants fitted to the double exponential decay of the control inward current was 5.2 and 28.9 sec. After 10 min of superfusion with 1 @f allethrin, the two time constants were 4.6 and 33.2 set, respectively. At the same time, the peak amplitude of the inward current was reduced to 64% of the control value, which is similar to the effect of allethrin on the peak inward current induced by 1 and 3 mM
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b
r
1 R.crs3 ml”
cyphenothrln
1 R,os-
CyDhenolhlln
-
recovery 4 n-l,* Y
FIG. 3. Effect of allethrin and lR-cis-cyphenothrin on ion channel block by ACh under voltage clamp conditions. The reversal of ion channel block is visible as the transient increase of the tail current immediately following removal of 3 mM ACh (a,b). (a) Recordings of inward current induced by 3 mM ACh in control external solution, after 2 and 9 min of superfusion with 1 FM allethrin. (b) Recordings of 3 mM ACh-induced inward current in control external solution, after 3 and 10 min of superfusion with 1 PM lR-cis-cyphenothrin. (c) Recordings of 3 mM ACh-induced inward current after I hr of superfusion with 1 FM allethrin, and partial recovery after 4 and 9 min of washing with control external solution. Superfusion periods with ACh are indicated by bars.
ACh. In another cell the two time constants obtained in the control situation were 4.7 and 52.4 sec. After 9 min of exposure to 1 PM lR-cis-cyphenothrin, the time constants were 4.5 and 47.6 set, respectively, whereas the peak inward current decreased to 77% of the control value. In another series of experiments, cells were super-fused with 9 FM ACh for variable periods before evoking an inward current by 1 mM ACh. Increasing the superfusion period with 9 pM ACh caused a gradual reduction of the peak amplitude of the 1 mM ACh-induced inward current due to desensitization (Fig. 6). The rate of the onset of desensitization was fitted by a double exponential function and a steady-state level. The time constants obtained from the curve
fitted to the results of three control experiments were 5.3 and 55.6 sec. At steady state, which was reached after 5 min of superfusion with 9 pM ACh, the peak amplitude of the 1 mM ACh-induced inward current was reduced to 27 -+ 4% (n = 3) of the control value. The relative contributions of the fast and the slow time constants to the rate of the onset of desensitization were 66 and 34%, respectively. In the presence of 1 pM allethrin the two time constants of the onset of desensitization were 4.5 and 53 sec. At steady-state desensitization the peak amplitude of the 1 mM ACh-induced inward current was reduced to 16 + 3% (n = 4). The relative contributions of the fast and slow components to the onset of desensitization were 64 and 36%, respectively. A
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AND
ACh -
VIJVERRERG
ACh a,
b-,
1 n* I.10 a ‘i-
1 S.cis- fenfluthrin (1 AIM)
b
r “?G
-
1 f?.&fentluthrin (1 AIM)
control Y
c-
control
1 I?.&-
fenfluthrin
I” FIG. 4. Effect of cis-fenfluthrin isomers on AChinduced inward current under voltage clamp conditions. Left traces are inward currents induced with 3 mM ACh in control external solution. Right traces are recorded after WO min of superfusion with 1 WM IScis-fenfluthrin (a), with 1 PM IR-cis-fenfluthrin (b), and with 0.01 fl IR-cis-fenfluthrin (c). Superfiision periods with ACh are indicated by bars.
comparison of the degree of desensitization at different times in the absence and in the presence of 1 fl allethrin revealed that only the values obtained at steady-state can be distinguished statistically (paired t test; P < 0.01). In the presence of I t& LS-&s-fen& thrin, the amplitude of the 1 mM AChinduced inward current measured after 5 min of superfusion with 9 pJ4 ACh was reduced to 8 r: 6% (n = 3) of the nondesensitized value. The degree of steady-state desensitization in the presence of lScis-fenfhrthrin is also significantly different from that in the absence of pyrethroid (P < 0.01). DISCUSSION
The present results show inhibitory ef-
FIG. 5. Absence of effect of allethrin on the kinetics of the decay of inward current induced by 9 PM ACh. Inward currents were induced by 50 see of superfusion with 9 ).&i ACh in control external solution (a) and after 12 min of superfusion with I JLM allethrin (b). A double exponential function fitted to the decay of the inward current yielded time constants of5.2 and 28.9 set in the control situation and of4.6 and 33.2 set in the presence of allethrin. Allethrin caused a decrease of the peak amplitude of the inward current induced by 9 )sM ACh to 64% of the control value. Superfusion periods with ACh are indicated by bars.
fects of pyrethroids on electrophysiological membrane responses mediated by neuronal nicotinic ACh receptors in cultured mouse neuroblastoma cells. The amplitude of the agonist-induced response is reduced and steady-state desensitization is increased. In addition, pyrethroids indirectly inhibit the
FIG. 6. The effect of allethrin on the onset of desensitization. Ordinate represents the peak amplitude of inward current induced by 1 mM ACh after various periods of superfusion with 9 +I4 ACh normalized to the control value in normal external solution (*) and in the presence of I fl allethrin (0). The data obtained from three control and three allethrin-treated cells were fitted by a double exponential function and a steady level. The two time constants with their relative contributions and the steady-state level are 5.3 set (4&F%),55.6 set (25%). and 27 2 4% in control external solution and4.5 set (54%), 53 set (30%), and I6 + 3 % in the presence of a1Iethn.n.
PYRETHROIDS
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NEUROTRANSMITTER
ACh-induced inward current by enhancing ion channel block at high concentrations of ACh. These effects are produced to the same extent by a cyano and a noncyano pyrethroid as well as by an active and an insecticidally inactive pyrethroid isomer. In central neurones of the cockroach the amplitude of membrane depolarizations, induced by ionophoresis of ACh, was reduced by 1 p,M deltamethrin and tralomethrin to 30-50% of the control value (25). This is very similar to the blocking effects of pyrethroids presently observed in neuroblastoma cells. Under voltage clamp conditions, all pyrethroids block the ACh-induced inward current to a similar degree, despite large differences in insecticidal activity, mammalian toxicity, and poisoning symptoms. Further, la-cis-cyphenothrin equally blocks the SHT- and the ACh-induced inward currents, which are mediated by distinct receptor-operated ion channels (14). The results show that the blocking effect of pyrethroids is neither selective with respect to the two types of receptor-operated ion channels nor related to pyrethroid structure and activity. The absence of stereoselectivity suggests that pyrethroids do not interact with a specific binding site to produce their blocking effect. From the increase in holding current fluctuations consistently observed, it could be speculated that the pyrethroids cause a general membrane disturbance. However, pyrethroids were previously shown not to affect the fluidity of lipid bilayers measured by fluorescense polarization (26). The time course of the decay of the inward current induced by a low concentration of ACh as well as the rate of onset of desensitization, which are independent measures of the kinetics of desensitization (14), remain unaffected in the presence of pyrethroids. However, the degree of steadystate desensitization is significantly increased by allethrin as well as by the noninsecticidal 1S-cis-fenthithrin. This finding contrasts with the results of a previous
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study in which the effects of pyrethroids on radioligand binding and on nicotinic receptor-mediated ion flux in Torpedo electric organ membrane preparations were interpreted as a reduction of desensitization (9). The decay of inward currents evoked with a high concentration of ACh is accelerated, the amplitude of the transient tail current, which reflects the unblocking of channels, is reduced, and the decay of the tail current is delayed. These results indicate that the rate at which ACh blocks the nicotinic receptor-operated ion channels is increased and that the unblocking rate is decreased by pyrethroids. Pyrethroids have been reported to decrease perhydrohistrionicotoxin (HTX) binding to membrane preparations of the Torpedo electric organ (27). HTX has been described to block nicotinic receptor-operated ion channels in a voltage-dependent way (28). The apparent discrepancy between the effects of pyrethroids on HTX binding and on ion channel block by ACh might be due to this voltagedependent interaction of HTX with the nicotinic receptor-operated ion channels. The binding experiments were performed in the absence of TTX and it is unclear to what extent the microsacs were depolarized by an effect of the pyrethroids on voltagedependent sodium channels. At the concentrations used, pyrethroids cause an increase of the amplitude of the voltage-dependent sodium current and a pronounced delay in sodium current kinetics in NIE-115 cells (ll), which has been related to poisoning symptoms observed in vivo (29). The inhibitory effects of pyrethroids on the neuronal nicotinic ACh receptor- and the 5-HT, receptor-operated ion channels are not likely to contribute to the generally observed excitatory action of these insecticides on the nervous system. The functional consequences of a partial block of neuronal nicotinic and 5-HT, receptor-mediated inward currents remain to be established. Previous studies reported the absence of effects of allethrin on nerve evoked end-plate nicotinic receptor-med-
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iated depolarizations in frog muscle (7). A decrease of the amplitude of the end-plate potential could have been concealed by an increase of neurotransmitter release due to the prolongation of the presynaptic action potential in the presence of pyrethroids. Block of the SHT, response, which mediates electrical activity from peripheral nerve endings as well as reflex bradycardia, would result in symptoms opposite to the peripheral sensory phenomena (18) and the cardiovascular changes (19) described for pyrethroids in vivo. It can be concluded that the effects of pyrethroids on postsynaptic neurotransmitter function are of a nonspecific inhibitory nature and, unlike effects on sodium channels, are not related to the excitatory symptoms caused by these insecticides. ACKNOWLEDGMENTS
The authors thank Ing. A. de Groot and Ms. P. Martens for their assistance during the experiments and Bayer AG and Roussel Uclaf for their kind supply of pyrethroids. Professor J. van den Bercken is thanked for his comments on the manuscript and Ms. I. Duran for help in preparing the manuscript. This work was supported by Shell Internationale Research Maatschappij, B.V. REFERENCES
1. H. P. M. Vijverberg, G. S. F. Ruigt, and J. van den Bercken, Structure-related effects of pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis, Pestic. Biochem. Physiol. 18, 315 (1982). 2. H. P. M. Vijverberg, J. M. van der Zalm, and J. van den Bercken, Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves, Nature (London) 295,601 (1982). 3. L. J. Lawrence and J. E. Casida, Stereospecitic action of pyrethroid insecticides on the gammaaminobutyric acid receptor-ionophore complex, Science 221, 1399 (1983). 4. N. Ogata, S. M. Vogel, and T. Narahashi, Lindane but not deltamethrin blocks a component of GABA-activated chloride channels, FASEB J. 2, 2895 (1988). 5. A. E. Chalmers, T. A. Miller, and R. W. Olsen, Deltamethrin: A neurophysiological study of the sites of action, Pestic. Biochem. Physiol. 27, 36 (1987).
AND
VIJVERFIERG
6. M. H. Evans, End-plate potentials in frog muscle exposed to a synthetic pyrethroid, Pestic. Biothem. Physiol. 6, 547 (1976). 7. W. Wouters, J. van den Bercken, and A. van Ginneken, Presynaptic action of the pyrethroid insecticide allethrin in the frog motor end-plate, Eur. J. Pharmacol. 43, 163 (1977). 8. G. S. F. Ruigt and J. van den Bercken, Action of pyrethroids on a nerve-muscle preparation of the clawed frog, Xenopus laevis, Pestic. Biothem. Pharmacol. 25, 176 (1986). 9. S. M. Sherby, A. T. Eldefrawi, S. S. Deshpande, E. X. Albuquerque, and M. E. Eldefrawi, Effects of pyrethroids on nicotinic acetylcholine receptor binding and function, Pestic. Biochem. Physiol. 26, 107 (1986). 10. G. S. F. Ruigt, H. C. Neijt, J. M. van der Zalm, and J. van den Bercken, Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells, Brain Res. 437,309 (1987). 11. K. Chinn and T. Narahashi, Stabilization of sodium channel states by deltamethrin in mouse neuroblastoma cells. J. Physiol. 380, 191 (1986). 12. T. Narahashi, Mechanisms of actions of pyrethroids on sodium and calcium channel gating, in “Neuropharmacology and Pesticide Action” (M. G. Ford, G. G. Lunt, R. C. Reay, and P. N. R. Usherwood, Eds.), pp. 36-60, Ellis Horwood, Chichester, 1986. 13. H. C. Neijt, I. J. te Duits, and H. P. M. Vijverberg, Pharmacological characterization of serotonin 5-HT, receptor-mediated electrical response in cultured mouse neuroblastoma cells, Neuropharmacology 27, 301 (1988). 14. M. Oortgiesen and H. P. M. Vijverberg, Properties of nicotinic acetylcholine receptor in voltage clamped neuroblastoma cells, Neuroscience, in press. 15. P. B. S. Clarck, Recent progress in identifying nicotinic cholinoceptors in mammalian brain, TIPS 8, 32 (1987). 16. B. P. Richardson, G. Engel, P. Donatsch, and P. A. Stadler, Identification of serotonin Mreceptor subtypes and their specific blockade by a new class of drugs, Nature (London) 316, 126 (1985). 17. K. J. Watling, Radioligand binding studies identify S-HT, recognition sites in neuroblastoma cell lines and mammalian CNS, TIPS 6, 227 (1988). 18. P. H. Chanh, C. Navarro-Delmasure, A. P. H. Chanh, Ph. Clavel, and P. Gayrel, Toxicity and cardiovascular effects of decamethrin on anaesthetized dogs. IRCS Med. Sci. 8, 388 (1980). 19. S. B. Tucker and S. A. Flannigan, Cutaneous effects from occupational exposure to fenvalerate, Arch. Toxicol. 54, 195 (1983). 20. T. Amano, E. Richelson, and P. G. Nirenberg,
PYRETHROIDS
AND
NEUROTRANSMITTER
Neurotransmitter synthesis by neuroblastoma clones, Proc. Natl. Acad. Sci. USA 6, 258 (1972). 21. 0. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pftigers Arch. 391, 85 (1981). 22. D. W. Marquardt, An algorithm for least-squares estimation of nonlinear parameters, .I. Sot. Indust. Appl.
Math.
11, 341 (1963).
23. K. Diem and C. Lentner, “Wissenschaftliche Tabellen,” Ciba-Geigy AG, Basle, 1968. 24. D. Colquhoun and D. C. Ogden, Activation of ion channels in the frog end-plate by high concentrations of acetylcholine, J. Physiol. 395, 131 (1983). 25. B. Hue and L. Mony, Actions of deltamethrin and tralomethrin on cholinergic synaptic transmission in the central nervous system of the cock-
RECEPTOR
roach (Periplaneta them.
Physiol.
173
FUNCTION americana), C 86, 349 (1987).
Comp.
Bio-
26. 0. T. Jones and A. G. Lee, Effects of pyrethroids on the activity of a purified (Ca2+-Mg’+)ATPase, Pestic. Biochem. Physiol. 25, 420 (1986). 27. M. A. Abbassy, M. E. Eldefrawi, and A. T. Eldefrawi, Pyrethroid action on the nicotinic acetylcholine receptor/channel, Pestic. Biochem. Physiol.
19, 299 (1983).
28. C. E. Spivak, M. A. Maleque, A. C. Oliviera, L. M. Masukawa, T. Tokuyama, J. W. Daly, and E. X. Albuquerque, Actions of the histrionicotoxins at the ion channel of the nicotinic acetylcholine receptor and at the voltagesensitive ion channels of muscle membranes, Mol.
Pharmacol
21, 351 (1982).
29. H. P. M. Vijverberg and J. R. de Weille, The interaction of pyrethroids with voltage-dependent Na channels, Neurotoxicology 6, 23 (1985).
BIOCHEMISTRY
AND
PHYSIOLOGY
34, 164173 (1989)
Effects of Pyrethroids on Neurotransmitter-Operated Ion Channels in Cultured Mouse Neuroblastoma Cells MARGA OORTGIESEN,REGINA G.D.M.
VAN KLEEF, AND HENK P. M. VUVERBERG
Department of Veterinary Pharmacology, Pharmacy, and Toxicology, University of Utrecht, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands Received February 15, 1989; accepted April 20, 1989 The effects of the pyrethroids allethrin, lR-cis-cyphenothrin, lR-cis-fentluthrin, and of the noninsecticidal isomer lb-cis-fenfluthrin on nemotransmitter receptor-mediated membrane responses have been investigated in the mouse neuroblastoma cell line NlE-115 using electrophysiological methods. At a concentration of 10 PM, allethrin and lR-cis-cyphenothrin caused a reduction of the peak amplitude of the nicotinic membrane depolarization to half of the control value. In voltageclamped cells the peak amplitude of the nicotinic ACh receptor-mediated inward current was reduced to 50-75% of the control value by the pyrethroids at a concentration of 1 @4. The effects of the insecticidal and the noninsecticidal isomer of fentluthrin were identical. The amplitudes of the ACh and the serotonin S-HT, receptor-mediated inward currents were equally reduced by 10 ~.uV IR-cis-cyphenothrin. The effects were partially reversed by washing. Steady-state desensitization, induced with a low concentration of ACh, was increased by 1 ~uV allethrin and by 1 l&f lb-cis-fenfluthrin to a similar extent. The rate of desensitization was not affected. Ion channel block, additionally observed at high concentrations of ACh, was facilitated by all pyrethroids investigated. It is concluded that the pyrethroids exert nonspecific, inhibitory effects on neurotransmitter receptor-operated ion channels. These effects are unlikely to contribute to the excitatory symptoms of these insecticides. 8 1989 Academic press, Inc.
INTRODUCTION
Pyrethroids are potent neuroactive insecticides with a strong excitatory action. These compounds induce repetitive activity in various parts of the peripheral and central nervous system. Electrophysiological investigation has demonstrated that this repetitive fuing originates from a prolongation of the increase in nerve sodium channel permeability associated with excitation (1, a Although it is generally accepted that voltage-dependent sodium channels are the main target site of pyrethroids, effects of these insecticides on neurotransmission have been reported as well. In rat brain synaptic membranes the binding of cage convulsants to the GABA receptor complex was demonstrated to be partially inhibited by cyanopyrethroids (3). However, in crayfish stretch receptor neurones and in rat dorsal root ganglion cells the cyanopyrethroid deltamethrin had no effect on the GABA-mediated electrophysiological re-
sponse (4, 5). Studies on the nicotinic neurotransmission in the frog motor end-plate revealed that pyrethroids induce repetitive activity in the presynaptic motor nerve terminal, whereas the postsynaptic nicotinic acetylcholine (ACh)’ receptors appeared not affected (6-8). On the other hand, a reduction of receptor desensitization has been suggested from inhibitory effects of pyrethroids on radioligand binding to the nicotinic receptor-ion channel complex and on nicotinic receptor-mediated calcium influx in Torpedo electric organ preparations (9).
In cultured neuroblastoma cells, properties of different types of ion channels, that are expressed in the plasma membrane, can be investigated independently. In cells of the clone NlE-115, effects of a range of pyrethroids on voltage-dependent sodium channels have already been described in * ACh, acetylcholine; DMSO, dimethyl sulfoxide; Garb, carbachol; HTX, perhydrohistrionicotoxin; 5HT, serotonin.
164 0048-3575189 $3.00 Copyright 0 1989 by Academic Press, Inc. AU rights of reproduction in any form reserved.
PYRETHROIDS
AND
NEUROTRANSMITTER
detail (IO, I I). In addition, tetramethrin has been reported to block transient and, to a lesser degree, slow voltage-dependent calcium channels (12). Recently, serotonin 5-HT, receptors and neuronal type nicotinic ACh receptors have been characterized pharmacologically in NlE-115 neuroblastoma cells (13, 14). In voltage-clamped cells, serotonin (5HT) and ACh cause transient inward currents, due to the opening of distinct types of ion channels, that rapidly desensitize in the continued presence of the agonists. Despite differences in the sensitivity to snake toxins, functional properties, like desensitization, and channel block of neuronal nicotinic receptor-operated ion channels in NlE-115 cells and of nicotinic receptors in muscle end-plates are very similar (14). Neuronal type nicotinic receptors and 5HT, receptors have also been described in sympathetic and parasympathetic neurones and in the central nervous system (15-17). In addition, 5-HT, receptors present in the heart and in skin sensory nerve endings are involved in reflex bradycardia, i.e., the von Bezold-Jarisch effect, and in the transmission of pain, respectively (16). This is of particular interest with respect to cardiovascular symptoms (18) and peripheral sensory phenomena (19) potentially induced by pyrethroids in intact animals. We have investigated direct postsynaptic effects of pyrethroids on the electrophysiological responses mediated by nicotinic ACh receptors and by serotonin 5-HT, receptors in cultured mouse neuroblastoma cells. MATERIALS
AND
METHODS
Cell culture. Mouse neuroblastoma cells of the clone NlE-115 (20) were grown in Dulbecco’s modified Eagle medium supplemented with 7.5% fetal calf serum and the following amino acids (in mM): L-cysteine * HCl, 0.3; L-alanine, 0.4; L-asparagine, 0.45; L-aspartic acid, 0.4; L-proline, 0.4; and L-glutamic acid, 0.4. The cultures
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165
were maintained at 37°C in a humidified atmosphere containing 5% C02. Cells of passages 30-45 were subcultured in 35mm plastic tissue culture dishes. Cell differentiation was initiated 2 to 3 days after plating by adding 1 mM N6,2’-U-dibutyryladenosine 3’,5’-cyclic monophosphate and 1 n-&f 3-isobutyl-l-methylxanthine to the culture medium. This medium was refreshed every 2-3 days. The cells were used in the experiments 8-12 days after induction of differentiation. Experimental procedure. Experiments were carried out using the standard wholecell patch clamp technique (21). The resistance of fire-polished glass pipettes for intracellular recording (internal tip diameter l-l.5 pm) was 3-5 Ma. The liquid junction potential at the tip of the electrode was compensated before each experiment and remained constant within 1 mV. In voltage clamp experiments the membrane potential was held at - 80 mV and the holding current was recorded continuously. The series conductance of approximately 0.15 ~.LSwas compensated for 60-70%. All responses were low-pass filtered (- 3 dB at 1000-500 Hz; 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): NaCl, 125; KCI, 5.5; CaCl,, 1.8; MgCl,, 0.8; Hepes, 20; glucose, 25; and sucrose, 36.5. The pH was adjusted to 7.3 with approximately 10 mill NaOH. Tetrodotoxin (0.5 l&f) was added to the external solution before each experiment to eliminate interference from effects of pyrethroids on voltage-dependent sodium channels. The glass pipettes were filled with an internal solution containing (in n-&Q: KCl, 150; NaCl, 10; Hepes, 10; and MgCl,, 1. The pH was adjusted to 7.2 with approximately 3 mM KOH. Agonists were applied either by ionophoresis or by whole-cell super-fusion. The micropipettes for ionophoresis (50-60 Ma) were filled with 1 M ACh chloride. To min-
166
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imize agonist leakage from the tip of the pipette, a retention current (1-5 nA) was applied. To allow recovery from desensitization, the frequency of ionophoretic stimulation was low (0.005 Hz) and the external solution was exchanged in between ionophoretically induced responses. In wholecell superfusion experiments a continuous flow (1.5 ml/min) of external solution was applied to the cell from a capillary with a diameter of 1 mm positioned within a distance of 1mm from the cell. The cells were super-fused with known concentrations of agonist and/or pyrethroid for adjustable periods (2 1set) using a servo-motor operated valve (13). Data were obtained from cells that responded to superfusion with ACh with a nicotinic receptor-mediated inward current only. To allow recovery from desensitization, the cell was washed between subsequent responses with control external solution for 3-4 min. Experiments were carried out at room temperature (20-24°C). In desensitization experiments the external solution was maintained at a constant temperature of 21°C with the use of a recirculating water jacket around the superfusion capillary. Pyrethroids were dissolved in dimethyl sulfoxide (DMSO). Shortly before each experiment a suspension was made from the stock solution with a final DMSO concentration of 0.1%. At this concentration, DMSO did not affect the ACh-induced inward current in control experiments. Exponential curve fitting was performed using a Levenberg-Marquardt nonlinear least squares algorithm (22). Results are presented as mean -’ SD and are compared using Student’s t test (23). Chemicals. Acetylcholine chloride, carbamylcholine chloride, dimethyl sulfoxide, and tetrodotoxin were obtained from Sigma; allethrin[(RIS)-2-allyl-4-hydroxy-3methyl-2-cyclopenten-I-one-(lR/S,cisltrans)3-(2,2-dimethylvinyl)-2,2-dimethylcyclopropanecarboxylate; purity >96%1 was obtained from K&K Laboratories Inc.; IR-cis-cyphenothrin[(Y(S)-cyano-3-phenoxy-
AND
VUVERRERG
benzyl-(lR,cis)-3-(2,2-dimethylvinyl)-2,2dimethylcyclopropanecarboxylate; RU 29209; purity >98%] was obtained from Roussel Uclaf; and lR-cis-fenfluthrin [pentafluorobenzyl-(lR,cis)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; NAK 1901; purity >99%] and IScis-fenfluthrin [pentafluorobenzyl-( IS, cis)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; NAK 1902; purity >99%] were obtained from Bayer. RESULTS
ACh-Induced
Membrane
Depolarization
Ionophoretic application of ACh to cells with a resting membrane potential adjusted to - 65 mV by injecting a small hyperpolarizing current (0.04-0.25 nA) resulted in a depolarization which is mediated by nicotinic ACh receptors. The resting potential of the cells used in the experiments ranged from -40 to - 68 mV, the mean resting potential amounted to 52 -+ 9 mV (mean ? SD; n = IS). During membrane potential measurements, atropine (0.5 p&Q was present in the external solution to block occasionally observed muscarinic receptormediated responses. The pyrethroids caused a gradual decrease of the peak amplitude of the ACh-induced depolarization. Superfusion of the cell with 10 PM allethrin for 15 min reduced the amplitude of the ACh response to a steady level, which was 51 + 20% (n = 4) of the control value (Fig. la). After 15 min of superfusion with 10 pJ4 lR-cis-cyphenothrin the peak amplitude was reduced to 54 -+ 33% (n = 5) of the control value (Fig. lb). The blocking effects of the noncyano and the cyan0 pyrethroid on the ACh-induced membrane depolarization cannot be distinguished statistically (P = 0.91). Neurotransmitter-Znduced Inward Currents
Under voltage clamp conditions, at a holding potential of - 80 mV, stimulation of the nicotinic ACh receptors by direct su-
PYRETHROIDS
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NEUROTRANSMITTER
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FUNCTION
167
same concentration of lR-cis-cyphenothrin on the Carb-induced and the 5HT-induced inward currents cannot be distinguished statistically (P = 0.65). The time course of the agonist-induced inward currents was not significantly altered by the pyrethroids. During the exposure to the pyrethroids the fluctuations of the holding current increased up to 2.5-fold. The control holding current level of 0.25-l nA was only slightly increased by 35 ‘I 51 pA (n = 9) in the presence of pyrethroid.
’:c mvliiT t
allethrin
1 R,c;s-
cyphenothrm
FIG. 1. Effect of allethrin and IR-cis-cyphenothrin on the membrane depolarization evoked by ionophoretic stimulation with ACh (indicated by arrows). The resting membrane potential was -65 mV. Left traces show depolarizations in the control situation, right traces show depolarization after 15 min of superfusion with 10 JLM allethrin (a) and 10 FM IRcis-cyphenothrin (b). During superfusion with allethrin and lR-cis-cyphenothrin, the peak amplitude of the ACh-induced depolarization was reduced to 55 and 67% of the control value, respectively. Ionophoretic pulses were 100 nC (a) and 300 nC (b).
perfusion of the cell with ACh or carbachol (Garb) resulted in a transient inward current. Exposure to IR-cis-cyphenothrin caused a gradual decrease of the amplitude of the inward current induced by 1 mM Carb. This effect was partially reversed after IO-15 min of washing with control external solution. Superfusion with 10 FM lR-cis-cyphenothrin for 10min reduced the peak amplitude of the 1 mM Carb-induced inward current to a steady level, which was 67 +. 4% (n = 4) of the control value (Fig. 2a). Superfusion with 5-HT also caused a transient inward current by the activation of S-HT, receptors. After 10 min of superfusion with 10 @U la-cis-cyphenothrin, the peak amplitude of the 5-HTinduced inward current was reduced to 70 + 12% (n = 5) of the control value (Fig. 2b). Little recovery was obtained during subsequent washing with control external solution for 10-15 min. The effects of the
Zon Channel Block by ACh
The ACh-induced inward current can be blocked by high concentrations of ACh (24). The reopening of ion channels that were blocked in the presence of ACh is measured as a transient increase of the tail current during the removal of high concentrations of ACh by washing, as shown in Fig. 3a (14). In addition to a reduction of the peak amplitude, 1 JLM allethrin and 1 $M lR-cis-cyphenothrin caused a change in the time course of the inward current induced by 3 mM ACh. Within a few minutes after superfusion with the pyrethroids, the peak amplitude of the ACh-induced inward current and the peak amplitude of the tail current were decreased. In addition, the decay of the ACh-induced inward current was accelerated, whereas the decay of the tail current was delayed (Fig. 3a and 3b). After l&15 min of super-fusion with the pyrethroids, the transient increase of the tail current was no longer visible. Partial recovery was obtained after la-15 min of washing (Fig. 3~). The peak amplitude of the 3 rnA4 AChinduced inward current was reduced after 10 min of superfusion with 1 ~JM allethrin and with 1 l&f la-cis-cyphenothrin to 75 + 13% (n = 3) and 71 it 11% (n = 4) of the control value, respectively. These values do not differ significantly (P = 0.68). Allethrin (1 u&f) also caused a reduction of the peak amplitude of the 1 mM ACh-induced inward current to 67 ? 8% (n = 5) of the
168
OORTGIESEN,
VAN
KLEEF,
AND
VIJVERBERG
Carb a
//“”
control
1 R CIS- cyphenothrm
I5HT
lR.cis-
cyphenothrln
recovery
FIG. 2. Effect of IR-cis-cyphenothrin on nicotinic and S-HT, receptor-mediated inward currents under voltage clamp conditions. (a) Recordings of inward currents induced by 1 mM Carb in control external solution, after 10 min of supetjkion with 10 PM lR-cis-cyphenothrin and after 10 min of recovery by washing with control external solution. In the presence of 10 fl IR-cis-cyphenothrin the amplitude of the Carb-induced inward current was reduced to 63% of the control value. (b) Recordings of inward currents induced by 3 PM 5-HT in control external solution, after 10 mitt of superfusion with IO )IM IR-cis-cyphenothrin and after 10 min of recovery by washing with control external solution. In the presence of 10 FM IR-cis-cyphenothrin the amplitude of the SHT-induced inward current was reduced to 62% of the control value. Superfusion periods with ACh and 5-HT are indicated by bars.
control value, which cannot be distinguished from its effect on the 3 mM AChinduced peak inward current (P = 0.35).
ACh-induced the transient (Fig. 4~).
inward current nor blocked increase of the tail current
Stereospecijkity
Desensitization
Superfusion with 1 tMI4 IR-cis-fenfluthrin and with 1 @I4 19cis-fenfluthrin for 10 min reduced the peak amplitude of the 3 mM ACh-induced inward current to 52 t 13% (n = 4) and 52 ? 12% (n = 3) of the control value, respectively. The effects of the insecticidal and the noninsecticidal isomers are identical (P = 0.98). The transient tail current during the removal of ACh was also suppressed by both fenfluthrin isomers (Fig. 4a and 4b). The possibility that the effects of Weis-fenfluthrin were due to contamination (~1%) with the II?-cisisomer was excluded. Superfusion with 0.01 t.&f lR-cis-fenfluthrin, i.e., 1% of the concentration used in the previous experiments, for 10 min neither caused a reduction of the peak amplitude of the 3 rnM
The time constants fitted to the decay of the inward current induced with the relatively low concentration of 9 p&I ACh, which causes little or no ion channel block, reflect the onset of desensitization (14). In a single cell an inward current was induced by 50 set of superfusion with 9 t&f ACh before and during exposure to pyrethroid (Fig. 5). The time constants fitted to the double exponential decay of the control inward current was 5.2 and 28.9 sec. After 10 min of superfusion with 1 @f allethrin, the two time constants were 4.6 and 33.2 set, respectively. At the same time, the peak amplitude of the inward current was reduced to 64% of the control value, which is similar to the effect of allethrin on the peak inward current induced by 1 and 3 mM
PYRETHROIDS
AND
NEUROTRANSMITTER
RECEPTOR
FUNCTION
169
b
r
1 R.crs3 ml”
cyphenothrln
1 R,os-
CyDhenolhlln
-
recovery 4 n-l,* Y
FIG. 3. Effect of allethrin and lR-cis-cyphenothrin on ion channel block by ACh under voltage clamp conditions. The reversal of ion channel block is visible as the transient increase of the tail current immediately following removal of 3 mM ACh (a,b). (a) Recordings of inward current induced by 3 mM ACh in control external solution, after 2 and 9 min of superfusion with 1 FM allethrin. (b) Recordings of 3 mM ACh-induced inward current in control external solution, after 3 and 10 min of superfusion with 1 PM lR-cis-cyphenothrin. (c) Recordings of 3 mM ACh-induced inward current after I hr of superfusion with 1 FM allethrin, and partial recovery after 4 and 9 min of washing with control external solution. Superfusion periods with ACh are indicated by bars.
ACh. In another cell the two time constants obtained in the control situation were 4.7 and 52.4 sec. After 9 min of exposure to 1 PM lR-cis-cyphenothrin, the time constants were 4.5 and 47.6 set, respectively, whereas the peak inward current decreased to 77% of the control value. In another series of experiments, cells were super-fused with 9 FM ACh for variable periods before evoking an inward current by 1 mM ACh. Increasing the superfusion period with 9 pM ACh caused a gradual reduction of the peak amplitude of the 1 mM ACh-induced inward current due to desensitization (Fig. 6). The rate of the onset of desensitization was fitted by a double exponential function and a steady-state level. The time constants obtained from the curve
fitted to the results of three control experiments were 5.3 and 55.6 sec. At steady state, which was reached after 5 min of superfusion with 9 pM ACh, the peak amplitude of the 1 mM ACh-induced inward current was reduced to 27 -+ 4% (n = 3) of the control value. The relative contributions of the fast and the slow time constants to the rate of the onset of desensitization were 66 and 34%, respectively. In the presence of 1 pM allethrin the two time constants of the onset of desensitization were 4.5 and 53 sec. At steady-state desensitization the peak amplitude of the 1 mM ACh-induced inward current was reduced to 16 + 3% (n = 4). The relative contributions of the fast and slow components to the onset of desensitization were 64 and 36%, respectively. A
170
OORTGIESEN,
VAN
KLEEF.
AND
ACh -
VIJVERRERG
ACh a,
b-,
1 n* I.10 a ‘i-
1 S.cis- fenfluthrin (1 AIM)
b
r “?G
-
1 f?.&fentluthrin (1 AIM)
control Y
c-
control
1 I?.&-
fenfluthrin
I” FIG. 4. Effect of cis-fenfluthrin isomers on AChinduced inward current under voltage clamp conditions. Left traces are inward currents induced with 3 mM ACh in control external solution. Right traces are recorded after WO min of superfusion with 1 WM IScis-fenfluthrin (a), with 1 PM IR-cis-fenfluthrin (b), and with 0.01 fl IR-cis-fenfluthrin (c). Superfiision periods with ACh are indicated by bars.
comparison of the degree of desensitization at different times in the absence and in the presence of 1 fl allethrin revealed that only the values obtained at steady-state can be distinguished statistically (paired t test; P < 0.01). In the presence of I t& LS-&s-fen& thrin, the amplitude of the 1 mM AChinduced inward current measured after 5 min of superfusion with 9 pJ4 ACh was reduced to 8 r: 6% (n = 3) of the nondesensitized value. The degree of steady-state desensitization in the presence of lScis-fenfhrthrin is also significantly different from that in the absence of pyrethroid (P < 0.01). DISCUSSION
The present results show inhibitory ef-
FIG. 5. Absence of effect of allethrin on the kinetics of the decay of inward current induced by 9 PM ACh. Inward currents were induced by 50 see of superfusion with 9 ).&i ACh in control external solution (a) and after 12 min of superfusion with I JLM allethrin (b). A double exponential function fitted to the decay of the inward current yielded time constants of5.2 and 28.9 set in the control situation and of4.6 and 33.2 set in the presence of allethrin. Allethrin caused a decrease of the peak amplitude of the inward current induced by 9 )sM ACh to 64% of the control value. Superfusion periods with ACh are indicated by bars.
fects of pyrethroids on electrophysiological membrane responses mediated by neuronal nicotinic ACh receptors in cultured mouse neuroblastoma cells. The amplitude of the agonist-induced response is reduced and steady-state desensitization is increased. In addition, pyrethroids indirectly inhibit the
FIG. 6. The effect of allethrin on the onset of desensitization. Ordinate represents the peak amplitude of inward current induced by 1 mM ACh after various periods of superfusion with 9 +I4 ACh normalized to the control value in normal external solution (*) and in the presence of I fl allethrin (0). The data obtained from three control and three allethrin-treated cells were fitted by a double exponential function and a steady level. The two time constants with their relative contributions and the steady-state level are 5.3 set (4&F%),55.6 set (25%). and 27 2 4% in control external solution and4.5 set (54%), 53 set (30%), and I6 + 3 % in the presence of a1Iethn.n.
PYRETHROIDS
AND
NEUROTRANSMITTER
ACh-induced inward current by enhancing ion channel block at high concentrations of ACh. These effects are produced to the same extent by a cyano and a noncyano pyrethroid as well as by an active and an insecticidally inactive pyrethroid isomer. In central neurones of the cockroach the amplitude of membrane depolarizations, induced by ionophoresis of ACh, was reduced by 1 p,M deltamethrin and tralomethrin to 30-50% of the control value (25). This is very similar to the blocking effects of pyrethroids presently observed in neuroblastoma cells. Under voltage clamp conditions, all pyrethroids block the ACh-induced inward current to a similar degree, despite large differences in insecticidal activity, mammalian toxicity, and poisoning symptoms. Further, la-cis-cyphenothrin equally blocks the SHT- and the ACh-induced inward currents, which are mediated by distinct receptor-operated ion channels (14). The results show that the blocking effect of pyrethroids is neither selective with respect to the two types of receptor-operated ion channels nor related to pyrethroid structure and activity. The absence of stereoselectivity suggests that pyrethroids do not interact with a specific binding site to produce their blocking effect. From the increase in holding current fluctuations consistently observed, it could be speculated that the pyrethroids cause a general membrane disturbance. However, pyrethroids were previously shown not to affect the fluidity of lipid bilayers measured by fluorescense polarization (26). The time course of the decay of the inward current induced by a low concentration of ACh as well as the rate of onset of desensitization, which are independent measures of the kinetics of desensitization (14), remain unaffected in the presence of pyrethroids. However, the degree of steadystate desensitization is significantly increased by allethrin as well as by the noninsecticidal 1S-cis-fenthithrin. This finding contrasts with the results of a previous
RECEPTOR
FUNCTION
171
study in which the effects of pyrethroids on radioligand binding and on nicotinic receptor-mediated ion flux in Torpedo electric organ membrane preparations were interpreted as a reduction of desensitization (9). The decay of inward currents evoked with a high concentration of ACh is accelerated, the amplitude of the transient tail current, which reflects the unblocking of channels, is reduced, and the decay of the tail current is delayed. These results indicate that the rate at which ACh blocks the nicotinic receptor-operated ion channels is increased and that the unblocking rate is decreased by pyrethroids. Pyrethroids have been reported to decrease perhydrohistrionicotoxin (HTX) binding to membrane preparations of the Torpedo electric organ (27). HTX has been described to block nicotinic receptor-operated ion channels in a voltage-dependent way (28). The apparent discrepancy between the effects of pyrethroids on HTX binding and on ion channel block by ACh might be due to this voltagedependent interaction of HTX with the nicotinic receptor-operated ion channels. The binding experiments were performed in the absence of TTX and it is unclear to what extent the microsacs were depolarized by an effect of the pyrethroids on voltagedependent sodium channels. At the concentrations used, pyrethroids cause an increase of the amplitude of the voltage-dependent sodium current and a pronounced delay in sodium current kinetics in NIE-115 cells (ll), which has been related to poisoning symptoms observed in vivo (29). The inhibitory effects of pyrethroids on the neuronal nicotinic ACh receptor- and the 5-HT, receptor-operated ion channels are not likely to contribute to the generally observed excitatory action of these insecticides on the nervous system. The functional consequences of a partial block of neuronal nicotinic and 5-HT, receptor-mediated inward currents remain to be established. Previous studies reported the absence of effects of allethrin on nerve evoked end-plate nicotinic receptor-med-
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iated depolarizations in frog muscle (7). A decrease of the amplitude of the end-plate potential could have been concealed by an increase of neurotransmitter release due to the prolongation of the presynaptic action potential in the presence of pyrethroids. Block of the SHT, response, which mediates electrical activity from peripheral nerve endings as well as reflex bradycardia, would result in symptoms opposite to the peripheral sensory phenomena (18) and the cardiovascular changes (19) described for pyrethroids in vivo. It can be concluded that the effects of pyrethroids on postsynaptic neurotransmitter function are of a nonspecific inhibitory nature and, unlike effects on sodium channels, are not related to the excitatory symptoms caused by these insecticides. ACKNOWLEDGMENTS
The authors thank Ing. A. de Groot and Ms. P. Martens for their assistance during the experiments and Bayer AG and Roussel Uclaf for their kind supply of pyrethroids. Professor J. van den Bercken is thanked for his comments on the manuscript and Ms. I. Duran for help in preparing the manuscript. This work was supported by Shell Internationale Research Maatschappij, B.V. REFERENCES
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