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, 132 (2), 587-95

Indoxacarb, an Oxadiazine Insecticide, Blocks Insect Neuronal Sodium Channels

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Indoxacarb, an Oxadiazine Insecticide, Blocks Insect Neuronal Sodium Channels

B Lapied et al. Br J Pharmacol.

Abstract

1. Decarbomethoxyllated JW062 (DCJW), the active component of a new oxadiazine insecticide DPX-JW062 (Indoxacarb), was tested on action potentials and the inward sodium current recorded from short-term cultured dorsal unpaired median neurones of the cockroach Periplaneta americana. 2. Under whole-cell current-clamp conditions, 100 nM DCJW reduced the amplitude of action potentials and induced a large hyperpolarization of the resting membrane potential associated with a 41% increase in input resistance. 3. In voltage-clamp, DCJW resulted in a dose-dependent inhibition (IC(50) 28 nM) of the peak sodium current. Based on IC(50) values, the effect of DCJW was about 10 fold less potent than tetrodotoxin (TTX) but 1000 fold more potent than the local anaesthetic lidocaine. DCJW (100 nM) was without effect on activation properties of the sodium current, reversal potential, voltage dependence of sodium conductance and on both fast and slow steady-state inactivations. 4. TTX (2 nM) resulted in 48% inhibition of the peak inward sodium current. Co-application of TTX (2 nM) with various concentrations of DCJW produced an additional inhibition of the peak inward current, indicating that the blocking actions of DCJW and TTX were distinct. Co-application of lidocaine (IC(50) 30 microM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. 5. DCJW (100 nM) did not affect inward calcium or outward potassium currents. 6. This study describes, for the first time, the action on insect neuronal voltage-dependent sodium channels of Indoxacarb, a new class of insecticides.

Figures

Figure 1
Figure 1
The chemical structure of the oxadiazine insecticide DCJW is shown together with structures of the local anaesthetic lidocaine, the dihydropyrazole RH 3421 and the well-known sodium channel active insecticide the pyrethroïd deltamethrin. *Denotes chiral center.
Figure 2
Figure 2
Using the whole cell current-clamp recording configuration, the effect of DCJW on membrane potential and triggered action potentials recorded from an isolated DUM neurone cell body are demonstrated. Action potentials elicited by a 40-ms depolarizing current pulse (0.8 nA) were recorded from an isolated cell body held at −52 mV prior to (A, control) and following the application of 100 nM DCJW (B,C). DCJW reduced the action potential amplitude and produced a large hyperpolarization (B, arrow). Artificial depolarization of the neurone to bring its resting potential back to the control value was ineffective in reversing the DCJW blocking effect (C). (D) The amplitude of the DCJW-induced hyperpolarization is plotted as a function of time of application (n=5). Inset: shows the membrane potential recorded in response to a hyperpolarizing current pulse (150 ms in duration) in saline (control 1) and 15 min (2) after the application of 100 nM DCJW.
Figure 3
Figure 3
Effects of DCJW on the DUM neurone voltage-dependent inward sodium current. (A) Sodium inward current traces obtained by a 30-ms depolarizing pulse to −10 mV from a holding potential of −90 mV, in the absence (control) and presence of 100 nM DCJW. Currents are leak- and capacity-corrected. (B) Effect of DCJW on the current-voltage relationship of the inward sodium current. The maximum peak current amplitude was plotted versus membrane potential before (control) and after application of 100 nM DCJW. (C) Voltage dependence of the normalized sodium conductance of the inward current was calculated according to equation 2, in normal saline (control) and after the application of 100 nM DCJW. (D) Superimposed voltage dependence of the fast and slow steady-state inactivation curves of the inward sodium current in normal saline (control) and in the presence of 100 nM DCJW. The smooth lines are fitted through the mean data points using the single Boltzmann distribution (equation 3). Data are means±s.e.mean.
Figure 4
Figure 4
Effects of TTX, DCJW and lidocaine on DUM neurone voltage-dependent inward sodium current. (A) Inward sodium currents evoked by 30-ms depolarizing steps from −90 to −10 mV before (control) and following application of 100 nM and 1 μM DCJW. (B) Semi-logarithmic dose-response curves for the blockade of inward sodium current by TTX, DCJW and lidocaine. The percentage inhibition of the peak inward sodium current was plotted as a function of log [TTX], log [DCJW] and log [lidocaine]. The smooth line represents the best fit to the mean data according to the Hill equation. (C) Comparative histograms of the percentage of residual inward current measured after application of 2 nM TTX and a solution containing 2 nM TTX+28 nM DCJW. These concentrations correspond to the IC50 values calculated from the semi-logarithmic dose-response curves shown in (B). (D) Effects of co-application of 2 nM TTX (corresponding to the IC50) with various concentrations of DCJW. Data plotted are mean values±s.e.mean. (n=6). (E) Comparative histograms of the percentage of residual response measured after application of 30 μM lidocaine and a solution containing 30 μM lidocaine and 28 nM DCJW. These concentrations are the IC50 values obtained from the semi-logarithmic dose response curves shown in (B). (F) Effects of co-application of 30 μM lidocaine (IC50) with various concentrations of DCJW. Data are mean values±s.e.mean (n=7).
Figure 5
Figure 5
Absence of blocking effect of DCJW on inward calcium and outward potassium currents. Whole-cell high-voltage activated (HVA) calcium current (Aa) elicited by 100-ms depolarizing voltage pulse to −10 mV from a holding potential of −100 mV, before (control) and after application of 100 nM DCJW. (b) Current-voltage relationships constructed from values of peak current plotted as a function of test potentials, in control and after application of 100 nM DCJW. (c) Histogram comparing the peak HVA current recorded before (control) and after bath application of DCJW (n=4). (Ba) Superimposed global outward potassium current traces recorded in saline containing 100 nM TTX (control) and following application of 100 nM DCJW. Outward currents were evoked by depolarization to +10 mV (100 ms in duration) from a holding potential of −80 mV. (b) Current-voltage relationships of both peak and late outward potassium current before and after application of 100 nM DCJW. (c) Histogram comparing the effect of 100 nM DCJW on the peak outward current measured at +10 mV. In both cases, the Student's t-test (•) was used to indicate that the difference was not significant (P>0.05, n=4).

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