Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin, a Pyrethroid Insecticide

Abstract

Deltamethrin (DLT) is a type-II pyrethroid ester insecticide used in agricultural and domestic applications as well as in public health. However, transmembrane ionic channels perturbed by this compound remain largely unclear, although the agent is thought to alter the gating characteristics of voltage-gated Na⁺ (Na_V) channel current. In this study, we reappraised whether and how it and other related compounds can make any further modifications on voltage-gated Na⁺ current (I_Na) in pituitary tumor (GH₃) cells. Cell exposure to DLT produced a differential and dose-dependent stimulation of peak (transient, I_Na(T)) or sustained (late, I_Na(L)) I_Na; consequently, the EC₅₀ value required for DLT-stimulated I_Na(T) or I_Na(L) was determined to be 11.2 or 2.5 μM, respectively. However, neither the fast nor slow component in the inactivation time constant of I_Na(T) activated by short depolarizing pulse was changed with the DLT presence; conversely, tefluthrin (Tef), a type-I pyrethroid insecticide, can accentuate I_Na with a slowing in inactivation time course of the current. The I_Na(L) augmented by DLT was attenuated by further application of either dapagliflozin (Dapa) or amiloride, but not by chlorotoxin. During pulse train (PT) stimulation, with the Tef or DLT presence, the cumulative inhibition of I_Na(T) became slowed; moreover, following PT stimuli, a large tail current with a slowly recovering process was observed. Alternatively, during rapid depolarizing pulse, the amplitude of I_Na(L) and tail I_Na (I_Na(Tail)) for each depolarizing pulse became progressively increased by adding DLT, not by Tef. The recovery time constant following PT stimulation with continued presence of Tef or DLT was shortened by further addition of Dapa. The voltage-dependent hysteresis (Hys_(V)) of persistent I_Na was differentially augmented by Tef or DLT. Taken together, the magnitude, gating, frequency dependence, as well as Hys_(V) behavior of I_Na exerted by the presence of DLT or Tef might exert a synergistic impact on varying functional activities of excitable cells in culture or in vivo.

Keywords: late Na+ current; persistent Na+ current; pyrethroids; transient Na+ current; voltage-gated Na+ current.

Figure 1

Effect of deltamethrin (DLT) or tefluthrin (Tef) on voltage-gated Na⁺ current (I_Na) measured from pituitary GH₃ lactotrophs. This set of experiments was made in cells placed in Ca²⁺-free, Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), and the measuring electrode was filled with an internal solution enriched with Cs⁺. (A) Exemplar current traces obtained in (a, blue color) the control conditions (i.e., neither DLT nor Tef was present) and during cell exposure to either 10 μM DLT (b, upper, red color) or 10 μM Tef (b, lower, red color). The voltage-clamp protocol is illustrated atop recorded current traces. The graph shown in the right side of (A) denotes the expanded record from the observed current trace (red color) in the presence of 10 μM DLT and the definition of transient Na⁺ current (I_Na(T)), late Na⁺ current (I_Na(L)), total Na⁺ current (I_Na(Tot)), or tail Na⁺ current (I_Na(Tail)) is marked (indicated with blue double arrows). (B) Time course of effects of 10 μM DLT on the amplitude of I_Na(Tot) (upper), I_Na(L) (middle), and I_Na(T) (lower). Each point was taken at a rate of 0.1 Hz. The horizontal bar shown above indicated the application of DLT. (C) Concentration-dependent relationship of DLT on I_Na(T) (purple open circles) or I_Na(L) (blue solid circles) activated by short depolarizing step. Each data point in this graph represents mean ± SEM of 9 cells. According to the averaged data, the smooth line represents the best fit to the Hill equation as described in Materials and Methods.

Figure 1

Effect of deltamethrin (DLT) or tefluthrin (Tef) on voltage-gated Na⁺ current (I_Na) measured from pituitary GH₃ lactotrophs. This set of experiments was made in cells placed in Ca²⁺-free, Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), and the measuring electrode was filled with an internal solution enriched with Cs⁺. (A) Exemplar current traces obtained in (a, blue color) the control conditions (i.e., neither DLT nor Tef was present) and during cell exposure to either 10 μM DLT (b, upper, red color) or 10 μM Tef (b, lower, red color). The voltage-clamp protocol is illustrated atop recorded current traces. The graph shown in the right side of (A) denotes the expanded record from the observed current trace (red color) in the presence of 10 μM DLT and the definition of transient Na⁺ current (I_Na(T)), late Na⁺ current (I_Na(L)), total Na⁺ current (I_Na(Tot)), or tail Na⁺ current (I_Na(Tail)) is marked (indicated with blue double arrows). (B) Time course of effects of 10 μM DLT on the amplitude of I_Na(Tot) (upper), I_Na(L) (middle), and I_Na(T) (lower). Each point was taken at a rate of 0.1 Hz. The horizontal bar shown above indicated the application of DLT. (C) Concentration-dependent relationship of DLT on I_Na(T) (purple open circles) or I_Na(L) (blue solid circles) activated by short depolarizing step. Each data point in this graph represents mean ± SEM of 9 cells. According to the averaged data, the smooth line represents the best fit to the Hill equation as described in Materials and Methods.

Figure 2

Comparison among effects of Tef, DLT, Tef plus chlorotoxin (ChloroTx), DLT plus ChloroTx, Tef plus dapagliflozin (Dapa), DLT plus Dapa, and DLT plus amiloride on the amplitude of I_Na(L) measured from GH₃ cells. The I_Na was elicited by 20 ms depolarizing voltage command from −80 to −10 mV for a duration of 20 ms at a rate of 0.2 Hz. The I_Na(L) amplitudes during exposure to different tested compounds were measured at the end of each depolarizing step. Each bar represents the mean ± SEM (n = 8). ^* Significantly different from control (p < 0.05), ** significantly different from Tef (10 μM) alone group (p < 0.05), and ⁺ significant different from DLT (10 μM) alone group (p < 0.05).

Figure 3

Effect of DLT on the steady-state current versus voltage (I-V) relationship of I_Na(T) and I_Na(L) identified from GH₃ cells. In this set of experiments, we held each cell at −80 mV, and varying depolarizing command voltages from −80 to +10 mV in 10-mV steps were delivered to evoke I_Na(T) and I_Na(L). (A) Exemplar current traces obtained either in the control condition (upper) or with the presence of 10 μM DLT (lower). The uppermost part is the voltage-clamp protocol given. (B) The mean I-V relationship of I_Na(T) (black symbols) or I_Na(L) (red symbols) in control (upper, solid symbols) and during exposure to 10 μM DLT (lower, open symbols) (mean ± SEM; n = 8 for each point). Either I_Na(T) or I_Na(L) was measured at the beginning or end of each depolarizing pulse. (C) Conductance versus voltage relationship of I_Na(T) (black symbols) or I_Na(L) (red symbols) in the control period (left side) and during cell exposure to 10 μM DLT (right side) (mean ± SEM; n = 8 for each point).

Figure 4

Effects of Tef (A) or DLT (B,C) on I_Na evoked by a train of depolarizing pulses (i.e., pulse train [PT] stimulation) in GH₃ cells. The train given consists of 40–20 ms pulses (stepped to −10 mV) separated 5 ms intervals at −80 mV for a total duration of 1 sec. In (A) or (B), exemplar current traces acquired in the control period (i.e., neither Tef nor DLT was present, upper part, blue color) and during cell exposure to 10 μM Tef (lower part, red color) or 10 μM DLT (lower part, red color) are illustrated, respectively. The voltage-clamp protocol (black color) atop current traces in (A–C) is illustrated. The black dashed arrows in (A) or (B), respectively, indicate the direction of current changes (i.e., either decay or rise) over time in an exponential fashion, while the asterisk shows a large inward deflection following PT stimulation with cell exposure to 10 μM Tef (upper) or 10 μM DLT (lower). (C) Expanded records (i.e., potential or current traces) from the broken box in (B).

Figure 4

Effects of Tef (A) or DLT (B,C) on I_Na evoked by a train of depolarizing pulses (i.e., pulse train [PT] stimulation) in GH₃ cells. The train given consists of 40–20 ms pulses (stepped to −10 mV) separated 5 ms intervals at −80 mV for a total duration of 1 sec. In (A) or (B), exemplar current traces acquired in the control period (i.e., neither Tef nor DLT was present, upper part, blue color) and during cell exposure to 10 μM Tef (lower part, red color) or 10 μM DLT (lower part, red color) are illustrated, respectively. The voltage-clamp protocol (black color) atop current traces in (A–C) is illustrated. The black dashed arrows in (A) or (B), respectively, indicate the direction of current changes (i.e., either decay or rise) over time in an exponential fashion, while the asterisk shows a large inward deflection following PT stimulation with cell exposure to 10 μM Tef (upper) or 10 μM DLT (lower). (C) Expanded records (i.e., potential or current traces) from the broken box in (B).

Figure 5

Relationship of I_Na(T) or I_Na(Tail) versus the pulse train (PT) duration in the absence (blue filled circles) and presence (orange open circles or blue open triangles) of 10 μM DLT (mean ± SEM, n = 8 for each point). The observed I_Na(T) or I_Na(L) was measured as indicated in the right side of Figure 1. The continuous smooth lines, over which the experimental data points are overlaid, were optimally fitted by a single exponential (i.e., exponential decrease or increase). Notably, during PT stimulation, cell exposure to DLT can increase the decaying time constant of I_Na(T) inactivation; however, it led to a progressive increase (i.e., staircase increase) in the amplitude of I_Na(Tail).

Figure 6

Effect of DLT or DLT plus Dapa on I_Na evoked by PT stimulation identified from GH₃ cells. The PT stimulation was applied in exactly the same way as utilized in Figure 4. (A) Exemplar current traces obtained in the presence of DLT (10 μM) alone (black color) or DLT (10 μM) plus Dapa (10 μM) (red color). The upper part shows the voltage-clamp protocol (blue color) given, whereas asterisk denotes the emergence of the current recovery immediately following PT stimulation. (B) Summary bar graph demonstrating effects of Tef, DLT, Tef plus Dapa, and DLT plus Dapa on the recovery time constant of I_Na following PT stimulation (mean ± SEM; n = 8 for each bar). * Significantly different from control (p < 0.01), ** significantly different from Tef (10 μM) alone group (p < 0.05), ⁺ significantly different from Tef (10 μM) alone group (p < 0.05), and ⁺⁺ significantly different from DLT (10 μM) alone group (p < 0.05).

Figure 7

Modifications by Tef or DLT on the strength of voltage-dependent hysteresis (Hys_(V)) in persistent I_Na (I_Na(P)) present in GH₃ cells. In this set of whole-cell current recordings, the examined cell was voltage-clamped at −80 mV and we then delivered the isosceles-triangular ramp voltage (V_ramp) for a duration of 1 s (i.e., a ramp speed of ±0.26 mV/ms) to activate I_Na(P). (A) Exemplar current traces obtained in the control period (upper) and in the presence of 10 μM Tef (middle) or 10 μM DLT (lower). The ascending (upsloping) limb is indicated in black color, where the descending (downsloping) one is in the red color. Inset in the upper part of (A) shows the voltage-clamp protocol applied, whereas the dashed arrow indicates the direction of potential or current trajectory by which time goes. In (B) or (C), summary bar graph, respectively, demonstrates the effect of Tef (10 μM), DLT (10 μM), Tef (10 μM) plus Dapa (10 μM), and DLT (10 μM) plus Dapa (10 μM) on I_Na(P) amplitude activated by upsloping (at −10 mV) or downsloping limb (at the level of −70 mV) of double V_ramp (mean ± SEM; n = 8 for each bar). * Significantly different from control (p < 0.05), ** significantly different from Tef (10 μM) alone group (p < 0.05), and ⁺ significantly different from DLT (10 μM) alone group (p < 0.05).