![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois (X.Z., J.Z.Y., T.N.); and Bayer AG, Bayer CropScience, Global Biology Insecticides, Monheim, Germany (V.L.S.)
Received March 19, 2003; accepted May 15, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Matsumura,
1985; Brooks, 1986;
Mahajna et al., 1997;
Hainzl et al., 1998), in many
other cases it is related to higher insecticide sensitivities of target
neuroreceptors and ion channels of insects than their mammalian counterparts
as exemplified by pyrethroids (Song and
Narahashi, 1996; Warmke et
al., 1997).
GABA is a major inhibitory neurotransmitter in the nervous system of
vertebrates as well as invertebrates
(Osborne, 1996), and the GABA
receptor is an important target of cyclodiene and hexachlorocyclohexane
insecticides (Ghiasuddin and Matsumura,
1982; Narahashi,
2001). Dieldrin (Fig.
1), a cyclodiene, has been shown to exert a potent blocking action
on both insect GABA receptors (Bermudez et
al., 1991) and vertebrate GABAA receptors
(Abalis et al., 1986;
Nagata and Narahashi, 1994;
Pomés et al., 1994).
Fipronil (Fig. 1), a
phenylpyrazole compound, was developed in the mid-1990s and became an
excellent insecticide, mainly due to its high effectiveness against some of
the dieldrin-resistant strains of insects and its low toxicity to mammals
(Tingle et al., 2003).
Although both fipronil and dieldrin are known to block GABA receptors
(Millar et al., 1994;
Hosie et al., 1995;
Ikeda et al., 2001), the
mechanisms of their high and selective toxicity against insects are not fully
understood. Our hypothesis is that the differential sensitivities of the GABA
receptors in insect and mammalian neurons are the basis of the selective
toxicity of fipronil and dieldrin. Insect GABA receptors are distinctly
different from mammalian GABAA receptors
(Lees et al., 1987;
Sattelle et al., 1991).
Although both GABA receptors are blocked by picrotoxinin, bicuculline blocks
the mammalian GABAA receptor but not the insect GABA receptor
(Buckingham et al., 1994).
|
To explore the differential sensitivities of the GABA receptors between
insects and mammals to insecticides, the actions of fipronil and dieldrin on
GABA-induced currents were examined in cockroach thoracic ganglion neurons
using the whole-cell patch-clamp technique. Both dieldrin and fipronil
potently blocked the insect GABA-gated chloride channels at nanomolar
concentrations. Compared with our previous studies with rat dorsal root
ganglion (DRG) neurons (Nagata and
Narahashi, 1994; Ikeda et al.,
2001), the blocking action of fipronil on cockroach GABA receptors
was much more potent than its action on mammalian GABAA receptors.
In contrast, the action of dieldrin on insect GABA receptors was similar to
its actions on mammalian GABAA receptors, exhibiting a dual action
on GABA-induced chloride currents that manifested as an initial enhancement
followed by a dramatic inhibition. It was concluded that the high sensitivity
of insect GABA receptors to fipronil is one of the crucial mechanisms that
underlie the selective toxicity between mammals and insects.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Because
the synaptic transmission across the thoracic ganglion of the cockroach was
more susceptible to the action of dieldrin than that in the last abdominal
ganglion (Wang et al., 1971),
the thoracic ganglia were selected in the present study. Briefly, cockroaches
were immobilized with pins dorsal side up on a dissection dish. The dorsal
cuticle, gut, and some muscles were removed to allow access to the ventral
nerve cord. Three thoracic ganglia were carefully dissected and placed in
normal cockroach saline solution containing 200 mM NaCl, 3.1 mM KCl, 4 mM
MgCl2, 20 mM D-glucose, and 10 mM HEPES-acid, with pH adjusted to
7.4 with 1 mM NaOH. The ganglia were incubated for 30 min at room temperature
(22-24°C) in the cockroach saline solution containing collagenase (type
IA, 0.5 mg/ml; Sigma-Aldrich, St. Louis, MO) and hyaluronidase (type I-S, 1
mg/ml; Sigma-Aldrich). The ganglia were then rinsed twice in normal saline
solution supplemented with 5 mM CaCl2, fetal calf serum (5% by
volume), and mechanically dissociated by gentle repeated trituration through a
fire-polished Pasteur pipette. The dissociated neurons, suspended in the
supplemented normal saline solution, were allowed to settle on coverslips
coated with poly-L-lysine hydrobromide (mol. wt. >30,000; Sigma-Aldrich) in
55-mm tissue culture petri dishes. The neurons were incubated at 24°C for
12 to 24 h before recording GABA-induced currents.
Whole-Cell Current Recordings. Neurons were continuously perfused
with the cockroach external solution containing 167 mM NaCl, 3.1 mM KCl, 33 mM
D-gluconic acid, 5 mM CaCl2, 4 mM MgCl2, and 10 mM
HEPES-acid (Alix et al., 2002).
The pH was adjusted to 7.4 with 1 mM NaOH, and the osmolarity was 420 mOsM.
Ionic currents were recorded using the whole-cell patch-clamp technique at
room temperature (23°C). Pipette electrodes were made from 1.5-mm (o.d.)
borosilicate glass capillary tubes and had a resistance of 2 to 3 M
when filled with the standard internal solution containing: 15 mM NaCl, 170 mM
KCl, 0.5 mM CaCl2,1mM MgCl2, 10 mM EGTA, 10 mM
phosphocreatine diTris, 20 mM HEPES-acid, and 3 mM
ATP-Mg2+ (Alix et
al., 2002). The pH was adjusted to 7.4 with KOH, and the
osmolarity was 420 mOsM. The membrane potential was clamped at -60 mV unless
otherwise stated. The recording of whole-cell currents began 10 min after
membrane rupture so that the cell interior milieu was adequately equilibrated
with the pipette solution. Currents through the electrode were recorded with
an Axopatch 200A amplifier (Axon Instruments, Inc., Union City, CA), filtered
at 2 kHz, and stored by a PC-based data acquisition system that also provided
preliminary data analysis. Data, when quantified, were expressed as the mean
±S.E.M.
Drug Application. Two methods of drug application were used. The fast application of test solution to the cell chamber through a U-tube was controlled by the computer-operated magnetic valve, which, when opened, allowed the test solution to bypass the chamber. When it was closed, the test solution was ejected through the hole of the U-tube that was located close to the cell. At the same time, another valve controlling the suction tube was opened, allowing the test solution to be sucked away quickly. The external solution surrounding the cell could be completely changed with a test solution within 30 ms. Test compound was coapplied with GABA. Alternatively, a test drug was added to the external solution that was continuously perfused through the recording chamber.
Chemicals. GABA (Sigma-Aldrich) was first dissolved in deionized water as the stock solution and then diluted with the cockroach external solution immediately before use. Dieldrin and fipronil (provided by Rhone-Poulenc Yuka Agro K.K., Akeno, Japan) were first dissolved in dimethyl sulfoxide to make stock solutions and then diluted with the external solution shortly before experiments. The final concentrations of dimethyl sulfoxide in test solutions were 0.1% (v/v) or less, which had no effect on the GABA-induced currents.
Analysis. Whole-cell currents were initially analyzed with the pClamp version 6.0.4 software to measure the current amplitudes and decay kinetics. The statistical analysis and the nonlinear regression analysis were carried out using the Sigmaplot 2001 software (SPSS Science, Chicago, IL). The dose-response relationship for GABA to activate the GABA receptor was evaluated by fitting the data to Hill equation: I = ImaxC50n /(Cn + C50n), where I is the current amplitude relative to the control maximum current, Imax; C is the chemical concentration; and n is the Hill coefficient. The dose-response relationship for insecticides to modulate the GABA response was evaluated with a similar Hill equation to estimate EC50 or IC50 values.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The concentration-response relationship for GABA to activate GABA receptors was determined by applying various concentrations of GABA (1-3,000 µM) via a U-tube at an interval of 60 s (Fig. 2A). Little or no currents were discernible at GABA concentrations equal to or less than 3 µM. The current amplitude increased steeply as the GABA concentration was increased from 30 to 100 µM, reaching a maximum at 1000 µM. At low GABA concentrations, the current rose slowly and was maintained during application of GABA. For instance, the time constant of the rising phase of the current activated by 30 µM GABA was 265.8 ± 15.8 ms (n = 64). As the GABA concentration was increased, the initial rising phase of the GABA-induced current was accelerated, and the current amplitude reached a peak and was followed by a prominent decay phase.
|
The concentration-response relationship was constructed by plotting the peak amplitudes, normalized to the one induced by 1000 µM GABA, as a function of the GABA concentrations as shown in Fig. 2B. The concentration-response relationship was fitted to a sigmoid curve with an EC50 value of 52.9 ± 5.6 µM (n = 4) and a Hill coefficient of 2.11 ± 0.1 (n = 4), indicating that the activation of GABA receptor in cockroach neurons requires binding of two agonist molecules.
To test whether GABA-induced currents are carried by chloride ions, currents were evoked by GABA at a holding potential ranging from -60 mV to +40 mV using internal and external solutions with symmetrical chloride concentrations. As shown in Fig. 3A, a large GABA-induced inward current was generated at -40 mV, the current decreased to near zero at 0 mV, and it became outward in direction at +40 mV. The current-voltage (I-V) relationship is shown in Fig. 3B. The inward currents were larger than the outward currents. The current reversed in polarity at a membrane potential of -2.8 mV (n = 10), which was very close to the calculated chloride equilibrium potential of -0.1 mV after correction of 3.1-mV junction potential. This result indicated that the GABA-induced current was carried by chloride ions as seen with mammalian GABAA receptors.
|
The GABA-induced chloride current in cockroach neurons, however, was not blocked by 10 µM bicuculline, an antagonist of the mammalian GABAA receptor (Fig. 4). In contrast, the GABA response was almost completely blocked by 10 µM picrotoxinin, a GABAA receptor channel blocker (Fig. 4). The GABA current suppressed by picrotoxinin was partially restored after washout with drug-free external solution.
|
Fipronil Blocks GABA Receptors in Both Resting and Activated States.
Fipronil is known to block the GABAA receptor in rat DRG neurons
with an IC50 value of 1.6 µM
(Ikeda et al., 2001). It
blocks both the resting receptors and activated receptors to the same extent.
To examine whether fipronil exerted these two types of block in cockroach GABA
receptors, protocols similar to those used by Ikeda et al.
(2001) were used here. To
examine the resting receptor block by fipronil, two protocols were used. In
the first protocol, the effect of bath-applied fipronil on the GABA current
was monitored during 2-s applications of 30 µM GABA pulses at an interval
of 30 s at a holding potential of -60 mV
(Fig. 5A). After several stable
control current recordings had been established, 1 µM fipronil was
continuously perfused into the bath and GABA and fipronil were coapplied every
30 s for 2 s via a U-tube. The current amplitude was gradually decreased
during a 5-min treatment of fipronil (Fig.
5A). To examine whether the activation of the receptor by brief
GABA test pulses was required for block caused by 1 µM fipronil, a second
protocol was used (Fig. 5B). Fipronil was continuously perfused to the bath for 5 min during which time no
GABA pulse was given and then GABA and fipronil were coapplied to examine the
degree of block. This second protocol revealed that the GABA current was
reduced to the same degree as that in the first protocol with repeated GABA
stimulations during fipronil bath application. In both cases, no recovery of
the GABA current was seen after 20-min washout (n = 3). The lack of
use-dependent block indicated that fipronil was capable of blocking the
resting GABA receptor without activation and that the first protocol using
short GABA test pulses can be used for monitoring the time course of fipronil
block of the resting GABA receptor.
|
To observe the rate of fipronil block of the activated GABA receptor, fipronil was coapplied with 100 µM GABA for 30 s at a holding potential of -60 mV via the U-tube perfusion system. As shown in Fig. 5C, in the absence of insecticide, the current evoked by 30-s application of 100 µM GABA decayed slowly, which is mostly likely due to desensitization of the receptors. After coapplication of 100 nM fipronil and 100 µM GABA, the GABA current showed the same rising phase and reached nearly the same peak as the control, but decayed with a faster time course to a very small steady-state current. These observations indicated that fipronil blocked the GABA receptor because it was activated by GABA. Receptor desensitization seems to play a small role in the acceleration of current decay in the presence of fipronil because the peak GABA current remained small during the second and third applications at a 2-min interval (Fig. 5C) and because desensitization in the absence of fipronil recovered within 2 min. The peak current during the second coapplication of fipronil and GABA was greater than the steady-state level of the first coapplication. This suggested that the receptor desensitization prevented fipronil block of the activated receptor. The use-dependent inhibitory action of fipronil on the peak current also indicated that fipronil molecules could not dissociate from the receptor with 2-min interpulse intervals. This was consistent with the result of washout experiment, in which recovery was very small even after a 10-min washout with fipronil-free solution. Coapplications of fipronil at a higher concentration of 1 µM induced a similar but more rapid decay and more extensive inhibitory action.
Comparison of the Kinetics of Fipronil Block of Resting and Activated GABA Receptors. The time course of the inhibitory actions of fipronil on the resting GABA receptors is illustrated in Fig. 6A. Inward chloride currents were induced by a 2-s application of 30 µM GABA at an interval of 30 s when the membrane potential was hold at -60 mV. After bath and U-tube application of fipronil, the time course of fipronil block was accelerated as the fipronil concentration was increased from 10 to 1000 nM. The time constants for the block were 249 ± 18 s (n = 5) in the presence of 30 nM fipronil, 267 ± 47 s at 100 nM (n = 3), 172 ± 13 s at 300 nM (n = 4), and 63 ± 7 s at 1,000 nM (n = 4).
|
To examine the blocking kinetics of fipronil on the activated GABA receptors, the GABA current induced by the first coapplication of fipronil with 100 µM GABA was normalized to the control current because the control current exhibited some decay (Fig. 5Ca). Figure 6B illustrates the time course of normalized currents evoked by 30-s 100 µM GABA pulses. The time constants estimated from the fit were 17.5 ± 2.9 s in 100 nM fipronil (n = 4) and 3.7 ± 0.8 s in 1000 nM fipronil (n = 4).
To compare the fipronil blocking kinetics of the resting receptor with
those of the activated receptors, the reciprocals of their time constants of
block are plotted as a function of fipronil concentrations as shown in
Fig. 6C. Binding
(k+1) and unbinding
(k-1) rate constants were calculated from the
relationship 1/
= [F]k+1 +
k-1, where is the time constant of
current decay and [F] is the fipronil concentration. The linear fit
to the data of the resting receptor block gave a binding rate constant of 1.3
x 104 M-1 s-1 and
an intercept gave an unbinding rate constant of 2.3 x
10-3 s-1. This resulted in a
Kd value of 179 nM. The linear fit to the data of the
activated receptor gave a slope of 5.4 x 105
M-1 s-1 and an intercept of 5.2
x 10-2 s-1. Assuming this
additional decay in current caused by fipronil is due to an open channel
blocking action, the value of 5.4 x 105
M-1 s-1 gives the binding rate
constant, the value of 5.2 x 10-2
s-1 gives the unbinding rate constant, and the
Kd value is 98 nM.
Figure 6D compares the dose-response relationships for block of the resting receptors to that of the activated receptors. For the resting receptor block, the current amplitudes recorded 7 min after fipronil application as shown in Fig. 6A were normalized to the control and are plotted against the fipronil concentration. The data were fitted to a sigmoid Hill equation with an IC50 value of 28 nM and a Hill coefficient of 2.1 (n = 4). Because the equilibrium was not reached by 7 min at concentrations lower than 100 nM, the true IC50 value is somewhat lower than 28 nM.
For the block of the activated receptor, the steady-state block was calculated by comparing the current amplitude at the end of the 30-s pulse of GABA-fipronil coapplication (Fig. 5Cb) to the control current (Fig. 5Ca). The block amounted to 13.5 ± 3.7% (n = 5), 43.1 ± 4.2% (n = 5), 80.8 ± 3.1% (n = 4), 88.8 ± 3.2% (n = 5), and 98.9 ± 1.5% (n = 4), respectively, at 10, 30, 100, 300, and 1000 nM fipronil. When plotted on the same graph as that for the resting receptor block (Fig. 6D), these values are fitted to a sigmoid Hill equation with an IC50 value of 35 nM and a Hill coefficient of 1.4. Thus, fipronil has the same affinity for the resting and activated states of the GABA receptor, despite the different rates of blocking action on these two states.
Dual Action of Dieldrin on GABA-Induced Currents. Several protocols used to examine the fipronil action were also used to study the effects of dieldrin on cockroach GABA receptors. Bath and U-tube applications of 1 µM dieldrin exhibited a biphasic effect on the GABA current: the current was first potentiated and then inhibited (Fig. 7A). The current did not recover 20 min after washing with dieldrin-free external solutions. The time course of the dual action of dieldrin on GABA currents is illustrated in Fig. 7B. The potentiating action and inhibitory action seemed to have different dose dependencies as shown in Fig. 7C, in which the increase and the subsequent decrease of the peak current are plotted as a function of dieldrin concentration. The fit to the dose-potentiation data gave an EC50 value of 4.4 nM and a maximum potentiation to 40% of the control, whereas the fit to the dose-inhibition relationship gave an IC50 value of 15.5 nM and almost 100% maximum inhibition. Thus, it seems that dieldrin exerts the transient potentiation more potently than the delayed inhibition.
|
Experiments were conducted to test the use dependence of the blocking action of dieldrin using the same protocol as that for fipronil. GABA at 30 µM was applied for 2 s at an interval of 30 s at a holding potential of -60 mV. The current amplitude initially increased and was followed by a gradual decrease during 5-min treatment of dieldrin applied in U-tube and bath solutions (Fig. 8A). In another neuron, no GABA pulse via a U-tube was given during the 5-min bath application of dieldrin, and GABA-dieldrin coapplication via a U-tube was resumed after 5 min of dieldrin perfusion (Fig. 8B). The GABA current was reduced to the same degree in both protocols (n = 3). The lack of use-dependent block indicated that dieldrin blocked the GABA receptor in the resting state.
|
The kinetics of dieldrin block of the resting GABA receptor was estimated from the time course of the inhibitory action on the GABA-induced current. The reciprocals of the time constants are plotted as a function of dieldrin concentration in Fig. 9. The linear fit to the data of the resting receptor block gave a binding rate constant of 5.0 x 104 M-1 s-1 and an intercept gave an unbinding rate constant of 2.0 x 10-3s-1. The block of the activated GABA receptors by dieldrin could not be estimated from the current decay during coapplication of dieldrin with GABA, because the blocking action was complicated by the potentiating action. In a few experiments in which the potentiating action was not seen, the rate constants for blocking the activated receptor were estimated to be 187.9 ± 17.2 s (n = 10) and 161.3 ± 25.3 s (n = 3) in the presence of 100 and 1000 nM dieldrin, respectively. The reciprocals of these rate constants are plotted in Fig. 9 for comparison with those for the resting receptor block. The fit to the data of the activated receptor block gave a slope of 5.4 x 104 M-1 s-1 which represented the binding rate constant, and an intercept of 3.5 x 10-2 s-1 which represented the unbinding rate constant.
|
The potentiating action of dieldrin was dependent on GABA concentrations whereas the inhibitory action was not (Fig. 10A). With a near maximum inward chloride currents induced by high concentration of GABA (300 µM), no initial potentiating phase was observed after application of 100 nM dieldrin. The time course of the dieldrin inhibition of the current induced by 300 µM GABA was nearly identical to that of the inhibitory phase of the current induced by 30 µM GABA. Dieldrin at 100 nM inhibited the current induced by 300 µM GABA by 95.2 ± 1.5% (n = 5), which is not significantly different from its inhibition on the current induced by 30 µM GABA.
|
In contrast to the inhibitory action, the potentiating action of dieldrin varied among the neurons tested. The potentiating action was observed in 64% of the neurons tested, whereas the inhibitory action was observed in all neurons tested. However, when the time course of current changes obtained from the cells exhibiting both enhancing and inhibitory responses was compared to that from the cells exhibiting inhibition alone, the two inhibitory curves overlapped with a similar time course (Fig. 10B).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). In the present study, the GABA-activated currents were
recorded from neurons isolated from the thoracic ganglia of American
cockroaches. GABA activated the receptors to generate an inward chloride
current at -60 mV with an EC50 value of 53 µM and a Hill
coefficient of 2.1, which suggested that at least two GABA molecules are
required for activation of the receptor. The shape of dose-response
relationship resembled that of a previous study
(Alix et al., 2002), but our
EC50 value cannot directly be compared with their value because
pressure injection used to apply GABA to activate the receptor in their study
makes it difficult to estimate the exact concentration of GABA. Although there
are reports describing bicuculline antagonism against the insect GABAergic
neuronal activity (Roberts et al.,
1981; Waldrop et al.,
1987), the present study demonstrated that the GABA receptors of
isolated cockroach neurons are insensitive to bicuculline, a potent mammalian
GABAA receptor antagonist, but are sensitive to the blocking action
of picrotoxinin, a channel blocker of mammalian GABAA receptors.
The results are in agreement with the reports from other laboratories
(Lees et al., 1987;
Sattelle et al., 1988). As the
critical target of insecticides such as cyclodienes, avermectins, fipronil,
and spinosyns (Lummis, 1990;
Gant et al., 1998;
Watson, 2001), insect GABA
receptors with pharmacological properties distinct from mammalian
GABAA receptors (Sattelle et
al., 1991) seem to be a basis for the selective toxicity of these
insecticides in mammals and insects.
Fipronil Inhibition of GABA-Induced Current. The present study
demonstrated that fipronil exerted a potent inhibitory action on GABA-induced
chloride channel currents in cockroach thoracic ganglion neurons with an
IC50 value of 28 nM. The rate at which fipronil interacted with the
resting receptor was estimated from the onset of inhibition of the current
activated by GABA applications. The binding rate constant of fipronil was
estimated to be 1.3 x 104 M-1
s-1 and its unbinding rate constant 2.33 x
10-3 s-1, both of which differ
from the kinetics of interactions of fipronil with GABAA receptors
in rat DRG neurons (Ikeda et al.,
2001). The binding constant of fipronil in the cockroach GABA
receptor is about 20 times greater than that in rat DRG GABAA
receptors, whereas the unbinding rate constant is about 10 times smaller,
resulting in a higher affinity of fipronil for the cockroach GABA receptor.
These results corroborate those of the previous study
(Gant et al., 1998), showing
that fipronil binds more tightly to the insect GABA receptor than to the
GABAA receptor in mammals.
Fipronil inhibits the GABA receptor without requiring channel opening
(Fig. 5). The activation of the
receptor, however, greatly facilitates fipronil block of the receptor as
illustrated in Fig. 5C. For
example, in the presence of 1000 nM fipronil, the time constant for the
resting receptor block was 73 s, and the time constant for the activated
receptor block was 1.7 s. The concentration dependencies illustrated in
Fig. 6C indicate that fipronil
binds to the activated receptor 41 times faster than to the resting receptor,
whereas it unbinds from the activated receptor 23 times faster than from the
resting receptor. Thus, the activation of the receptor facilitates fipronil to
interact with its binding site. Consistent with its blocking action of the
activated receptor is the observation that, at the single-channel level,
fipronil reduced the duration of channel opening in S2 cell line expressing
the wild-type Rdl(ac) Drosophila melanogaster
homomer-forming ionotropic GABA receptor subunits
(Grolleau and Sattelle,
2000).
The studies using recombinant dieldrin-sensitive and dieldrin-resistant
Rdl receptors of D. melanogaster expressed
inXenopus oocytes (Buckingham et
al., 1994; Hosie et al.,
1995) revealed that fipronil at 1 to 100 µM blocked
dieldrin-sensitive GABA-gated chloride channels by 30 to 95% and
dieldrin-resistant GABA receptor channels by -25%. In these studies, the
blocking action of fipronil on both types of GABA receptors is similar to that
of picrotoxinin, indicating that fipronil and picrotoxinin may share a common
mechanism in blocking GABA responses. In rat DRG neurons, however, the
previous study shows that fipronil and picrotoxinin have their own binding
sites without sharing a common binding site on GABAA receptors
(Ikeda et al., 2001). They
conclude that fipronil and picrotoxinin may act as allosteric modulators at
different sites to block the GABAA receptors. This point remains to
be examined in the cockroach GABA receptors.
Dual Actions of Dieldrin on GABA Receptors. The present study showed that dieldrin exerted a dual action on cockroach GABA receptor channels: an initial enhancement of the GABA-induced current was followed by an inhibition. The potentiating action of dieldrin was observed in 64% of the neurons tested, whereas the inhibitory action was seen in all of the neurons tested. In addition, the potentiating action was not observed at high GABA concentrations, whereas the inhibitory action was seen regardless of GABA concentrations. Furthermore, dieldrin exerted these two actions with different potencies and efficacies: it potentiated the current with an EC50 value of 4.4 nM and a maximum enhancement of 40% of the control, and it inhibited the current with an IC50 value of 15.5 nM, amounting to nearly 100% inhibition. In the other 40% of the neurons tested, dieldrin did not exert its potentiating action on the GABA-induced current, and its inhibitory action was similar to the inhibitory action observed in neurons in which the dual action was seen.
The dual action may be mediated at two different sites on the same GABA
receptor or on two subtypes of GABA receptors. To further examine these two
hypotheses, dieldrin action was studied in the GABAA receptors with
known subunits expressed in human embryonic kidney cells
(Nagata et al., 1994). They
found that the dual action of dieldrin was observed in the GABA receptor with
1
2
2s or 622s combinations. However,
only suppression was observed in the 12 combination of
GABAA receptors. These results indicate that subunit is
necessary for the enhancing effect and that there is no subunit selectivity
for the suppressive effect (Nagata et al.,
1994). It is tempting to speculate that the dual action of
dieldrin on cockroach GABA receptors may be related to the differences in
subtypes of GABA receptors.
In dissociated cockroach neurons, multiple conductance states of 11, 17,
and 19 pS were detected in GABA receptor single-channel currents
(Shimahara et al., 1987;
Malecot and Sattelle, 1990).
The open-time distributions were fitted to two exponential functions and the
close-time distributions were fitted to three exponential functions. Noise
analysis demonstrated that lindane and dieldrin decreased the frequency of
GABA chloride channel openings without changing the mean open time
(Bermudez et al., 1991).
Therefore, the dual action of dieldrin may also be related to different gating
properties of the cockroach GABA-receptor chloride channels. Despite the
uncertainty of the toxicological significance of the enhancement of
GABA-induced currents by dieldrin, the dual action of dieldrin is worthy of
further study to elucidate the mechanism of interactions of dieldrin with the
GABA system.
Comparison of Blocking Kinetics between Fipronil and Dieldrin. Fipronil and dieldrin inhibited the resting GABA receptor in cockroach neurons at a comparable rate both in terms of binding and unbinding rate constants (Figs. 6C and 9). The slightly faster onset of fipronil block is due to its faster binding rate constants: 1.4 x 104 M-1 s-1 for fipronil and 5.0 x 103 M-1 s-1 for dieldrin. In contrast, fipronil interacted with the activated receptor at a rate at least 10 times faster than that for dieldrin. The binding rate constants were 5.4 x 105 M-1 s-1 for fipronil and 5.4 x 104 M-1 s-1 for dieldrin, and the unbinding rate constants were 5.2 x 10-3 s-1 for fipronil and 3.5 x 10-2 s-1 for dieldrin. It remains to be seen how these differences in blocking kinetics are reflected in their insecticidal activities.
Selective Toxicity of Insecticides. Both dieldrin and fipronil have
selective toxicity between insects and mammals. For dieldrin, the oral
LD50 in mammals was estimated to be 38 to 87 mg/kg b.wt. in rats
(Allen et al., 1979). In
comparison, the topical LD50 in American cockroaches was 1.3 to 1.5
mg/kg (Giannotti et al., 1959;
O'Brien, 1967). For fipronil,
the oral LD50 in rats was 91 mg/kg, and the LD50 values
in insects were 0.07 mg/kg in corn rootworm
(Scharf and Siegfried, 1999)
and 0.13 mg/kg in house fly (Hainzl and
Casida, 1996). With the topical application of dieldrin to
cockroaches, convulsions developed followed by paralysis. The synaptic
transmission across the metathoracic ganglion but not the last abdominal
ganglion was markedly facilitated, suggesting that the synapse in the
metathoracic ganglion was one of the important loci of the dieldrin action
(Wang et al., 1971).
There are many biological and physiological differences between vertebrates and invertebrates, especially the enzymatic metabolic degradation of insecticides. Any of these differences might be a factor for the selective toxicity of compounds in insects and mammals. The primary goal of the present study was to compare the sensitivity of insect GABA receptors with that of mammalian GABAA receptors to dieldrin and fipronil.
Fipronil inhibited the cockroach GABA receptors with an IC50
value of 28 nM (present study) and the rat GABAA receptors with an
IC50 value of 1660 nM (Ikeda et
al., 2001). Thus, the cockroach GABA receptors are 59 times more
sensitive to fipronil than the rat GABAA receptors. Therefore, the
fipronil's higher blocking potency in the insect GABA receptors compared with
that in the mammalian GABAA receptors accounts at least in part for
the selective toxicity between insects and mammals. Because different
combinations of GABA receptor subunits and/or different amino acid
compositions confer differential sensitivity to fipronil, these differences
may be partly responsible for the selectivity of fipronil block of GABA
receptors between cockroach and rat neurons
(Ratra and Casida, 2001).
In contrast, the mechanism of the selective toxicity of dieldrin must lie
in factors other than the GABA receptor sensitivity, because dieldrin exerts
both potentiating and inhibitory actions on cockroach and rat GABA receptors
with comparable potencies. The EC50 values for potentiation are 4.4
and 5 nM in cockroach and rat receptors, respectively, and the IC50
values for inhibition are 15.5 and 92 nM in cockroach and rat receptors,
respectively (present study; Nagata and
Narahashi, 1994). Therefore, the inhibitory potency in cockroach
is only 6 times higher than that in rat. The difference in metabolic
degradation of dieldrin in insects and mammals is among the most likely causes
of the selective toxicity. In addition, the blocking action of dieldrin on the
glycine receptor (Vale et al.,
2003) and invertebrate-specific glutamate-activated
slow-desensitizing chloride channels (our unpublished data) may also account
for the selective toxicity of dieldrin in insects.
Recently, our studies and other reports demonstrate that fipronil also
modulates the glutamate-activated chloride channel, a unique ligand-gated
anion channel present in insects but not in vertebrates
(Raymond et al., 2000;
Horoszok et al., 2001).
Fipronil is much more potent than dieldrin in blocking the glutamate-gated
chloride channels (X. Zhao, V. L. Salgado, J. Z. Yeh, and T. Narahashi,
unpublished data). The modulation of the insect-specific ion channels may also
play an important role in the selective toxicity between mammals and insects,
and offers a unique approach to the development of newer insecticides.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DRG, dorsal root ganglion; I-V, current-voltage.
Address correspondence to: Dr. Toshio Narahashi, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611-3008. E-mail: narahashi{at}northwestern.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abalis IM, Eldefrawi ME, and Eldefrawi AT (1986) Effects of insecticides on GABA-induced chloride influx into rat brain microsacs. J Toxicol Environ Health 18: 13-23.[Medline]
Alix P, Grolleau F, and Hue B (2002)
Ca2+/calmodulin-dependent protein kinase regulates
GABA-activated Cl- current in cockroach dorsal unpaired median
neurons. J Neurophysiol
87:
2972-2982.
Allen JR, Hargraves WA, Hsia MTS, and Lin FSD (1979) Comparative toxicology of chlorinated compounds on mammalian species. Pharmacol Ther 7: 513-547.[CrossRef][Medline]
Bermudez I, Hawkins CA, Taylor AM, and Beadle DJ (1991) Actions of insecticides on the insect GABA receptor complex. J Receptor Res 11: 221-232.[Medline]
Buckingham SD, Hoise AM, Roush RL, and Sattelle DB (1994) Action of agonists and convulsants antagonists on a Drosophila melanogaster GABA receptor (Rdl) homo-oligomer expressed in Xenopus oocytes. Neurosci Lett 181: 137-140.[CrossRef][Medline]
Brooks GT (1986) Insecticide metabolism and selective toxicity. Xenobiotica 16: 989-1002.[Medline]
Gant DB, Chalmers AE, Wolff MA, Hoffman HB, and Bushey DF (1998) Fipronil: action at the GABA receptor. Rev Toxicol 2: 147-156.
Ghiasuddin SM and Matsumura F (1982) Inhibition of gamma-aminobutyric acid (GABA)-induced chloride uptake by gamma-BHC and heptachlor epoxide. Comp Biochem Physiol 73C: 141-144.[CrossRef]
Giannotti O, Metcalf RL, and March RB (1959) The mode of action of aldrin and dieldrin in Periplaneta americana (L.). Ann Entomol Soc Am 119: 588-592.
Grolleau F and Sattelle DB (2000) Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line. Br J Pharmacol 130: 1833-1842.[CrossRef][Medline]
Hainzl D and Casida JE (1996) Fipronil insecticide:
novel photochemical desulfinylation with retention of neurotoxicity.
Proc Natl Acad Sci USA
93:
12764-12767.
Hainzl D, Cole LM, and Casida JE (1998) Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem Res Toxicol 11: 1529-1535.[CrossRef][Medline]
Horoszok L, Raymond V, Sattelle DB, and Wolstenholme AJ (2001) GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans. Br J Pharmacol 132: 1247-1254.[CrossRef][Medline]
Hosie AM, Baylis HA, Buckingham SD, and Sattelle DB (1995) Actions of the insecticide fipronil, on dieldrin-sensitive and -resistant GABA receptors of Drosophila melanogaster. Br J Pharmacol 115: 909-912.[Medline]
Ikeda T, Zhao X, Nagata K, Kono Y, Shono T, Yeh JZ, and Narahashi T
(2001) Fipronil modulation of -aminobutyric
acidA receptors in rat dorsal root ganglion neurons. J
Pharmacol Exp Ther 296:
914-921.
Lees G, Beadle DJ, Neumann R, and Benson JA (1987) Responses to GABA by isolated insect neuronal somata: pharmacology and modulation by a benzodiazepines and a barbiturate. Brain Res 401: 267-278.[CrossRef][Medline]
Lummis SCR (1990) GABA receptors in insects. Comp Biochem Physiol 95C: 1-8.[CrossRef]
Mahajna M, Quistad GB, and Casida JE (1997) Acephate insecticide toxicity: safety conferred by inhibition of the bioactivating carboxyamidase by the metabolite methamidophos. Chem Res Toxicol 10: 64-69.[CrossRef][Medline]
Malecot C and Sattelle DB (1990) Single channel
recordings from insect neuronal GABA-activated chloride channels. J
Exp Biol 151:
495-501.
Matsumura F (1985) Toxicology of Insecticides, 2nd ed, Plenum Press, New York.
Millar NS, Buckingham SD, and Sattelle DB (1994) Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line. Proc R Soc Lond B Biol Sci 258: 307-314.[Medline]
Nagata K, Hamilton BJ, Carter DB, and Narahashi T (1994) Selective effects dieldrin on the GABAA receptor-channel subunits expressed in human embryonic kidney cells. Brain Res 645: 19-26.[CrossRef][Medline]
Nagata K and Narahashi T (1994) Dual action of the
cyclodiene insecticide dieldrin on the -aminobutyric acid
receptor-channel complex of rat dorsal root ganglion neurons. J
Pharmacol Exp Ther 269:
164-171.
Narahashi T (2001) Neurophysiological effects of insecticides, in Handbook of Pesticide Toxicology, 2nd ed (Krieger RI ed) vol 1, pp 335-351, Academic Press, Inc., San Diego, CA.
O'Brien RD (1967) Insecticides: Action and Metabolism, Academic Press, Inc., New York.
Osborne RH (1996) Insect neurotransmission: neurotransmitters and their receptors. Pharmacol Ther 69: 117-142.[CrossRef][Medline]
Pomés A, Rodriguez-Farré E, and Suñol C
(1994) Disruption of GABA-dependent chloride flux by cyclodienes
and hexachlorocyclohexanes in primary cultures of cortical neurons.
J Pharmacol Exp Ther
271:
1616-1623.
Ratra GS and Casida JE (2001) GABA receptor subunit composition relative to insecticide potency and selectivity. Toxicol Lett 122: 215-222.[CrossRef][Medline]
Raymond V, Sattelle DB, and Lapied B (2000) Co-existence in DUM neurons of two GluCl channels that differ in their picrotoxin sensitivity. Neuroreport 11: 2695-2701.[Medline]
Roberts CJ, Krogsgaard-Larsen P, and Walker RJ (1981) Studies of the action of GABA, muscimol and related compounds on Periplaneta and Limulus neurons. Comp Biochem Physiol 69C: 7-11.[CrossRef]
Sattelle DB (1992) Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana). Comp Biochem Physiol 103C: 429-438.[CrossRef]
Sattelle DB, Lummis SCR, Wong JFH, and Rauh JJ (1991) Pharmacology of insect GABA receptors. Neurochem Res 16: 363-374.[CrossRef][Medline]
Sattelle DB, Pinnock RD, Wafford KA, and David JA (1988) GABA receptors on the cell-body membrane of an identified insect motor neuron. Proc R Soc Lond B Biol Sci 232: 443-456.[Medline]
Scharf M and Siegfried B (1999) Toxicity and neurophysiological effects of fipronil and fipronil sulfone on the western corn rootworm (Coleoptera: Chrysomelidae). Arch Insect Biochem Physiol 40: 150-156.[CrossRef]
Shimahara T, Pichon Y, Lees G, Beadle VA, and Beadle DJ
(1987) -Aminobutyric acid receptors on cultured cockroach
brain neurons. J Exp Biol
131:
231-244.
Song J-H and Narahashi T (1996) Modulation of sodium
channels of rat cerebellar Prukinje neurons by the pyrethroid tetramethrin.
J Pharmacol Exp Ther
277:
445-453.
Tingle CC, Rother JA, Dewhurst CF, Lauer S, and King WJ (2003) Fipronil: environmental fate, ecotoxicology and human health concerns. Rev Environ Contam Toxicol 176: 1-66.[Medline]
Vale C, Fonfria E, Bujons J, Messeguer A, Rodriguez-Farre E, and
Sunol C (2003) The organochlorine pesticides
-hexachlorocyclohexane (lindane), -endosulfan and dieldrin
differentially interact with GABAA and glycine-gated chloride
channels in primary cultures of cerebellar granule cells.
Neuroscience 117:
397-403.[CrossRef][Medline]
Waldrop B, Christensen TA, and Hildebrand JG (1987) GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth Manduca sexta. J Comp Physiol 161: 23-32.
Wang CM, Narahashi T, and Yamada M (1971) The neurotoxic action of dieldrin and its derivatives in the cockroach. Pestic Biochem Physiol 1: 84-91.
Warmke JW, Reenan RA, Wang P, Qian S, Arena JP, Wang J, Wunderler
D, Liu K, Kaczorowski GJ, Van der Ploeg LH, et al. (1997)
Functional expression of Drosophila para sodium channels. Modulation
by the membrane protein TipE and toxin pharmacology. J Gen
Physiol 110:
119-133.
Watson GB (2001) Actions of insecticidal spinosyns on
-aminobutyric acid responses from small-diameter cockroach neurons.
Pestic Biochem Physiol
71: 20-28.[CrossRef]
This article has been cited by other articles:
|
|
 |
|
  T. Narahashi, X. Zhao, T. Ikeda, K. Nagata, and J. Yeh Differential actions of insecticides on target sites: basis for selective toxicity Human and Experimental Toxicology, April 1, 2007; 26(4): 361 - 366. [Abstract] [PDF]
|
|
|
|
 |
|
 X. Zhao, J. Z. Yeh, V. L. Salgado, and T. Narahashi Sulfone Metabolite of Fipronil Blocks {gamma}-Aminobutyric Acid- and Glutamate-Activated Chloride Channels in Mammalian and Insect Neurons J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 363 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhao, J. Z. Yeh, V. L. Salgado, and T. Narahashi Fipronil Is a Potent Open Channel Blocker of Glutamate-Activated Chloride Channels in Cockroach Neurons J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 192 - 201. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), k+1 = 1.30 x
104 M-1 s-1,
k-1 = 2.33 x 10-3
s-1, and Kd = 179 nM; for the
activated receptor block (
), k+1 = 5.4
x 105 M-1 s-1,
k-1 = 5.2 x 10-2
s-1, and Kd = 98 nM. D, comparison
of the dose-response relationships for fipronil block of the resting and
activated GABA receptors. The fit to the dose-response data gave an
IC50 value of 27.6 nM and a Hill coefficient of 2.09 for the
resting receptor block (
