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.

1. Introduction

Deltamethrin (DLT, decamethrin) is a cyclopropanecarboxylate ester obtained by formal condensation between 3-(2,2-dibromovinyl)-2,2-dimethylcyclopanecarboxylic acid and cyano(3-phenoxyphenyl)methanol [1,2]. It is viewed to be the active insecticide of the proinsecticide tralomethrin [3,4,5]. Pyrethroids like DLT or tefluthrin (Tef) have been demonstrated to modify the gating characteristics of voltage-gated Na⁺ (Na_V) channels [6,7,8,9,10]. Deltamethrin (DLT), a neurotoxic type-II pyrethroid ester insecticide [3,4,5], has been demonstrated previously to cause a reversible sequence of motor symptoms in rats involving hind limb rigidity and choreoathetosis [2,11,12,13]. Alternatively, DLT was also reported to decrease Cl⁻ currents through voltage-dependent Cl⁻ channels and this action probably contributes the most to the features of poisoning with DLT or other type II pyrethroids [14,15,16]. At relatively high concentrations, pyrethroids can also act on GABA-gated Cl⁻ channels, which may be responsible for the seizures seen with severe type-II poisoning [2,17].

It has been established that nine isoforms (i.e., Na_V1.1–1.9 [or SCN1A–SCN5A and SCN8A–SCN11A]) of the voltage-gated Na⁺ (Na_V) channels are widely distributed in mammalian excitable tissues, which include the central or peripheral nervous system, and the endocrine or neuroendocrine system [18,19,20]. The eukaryotic versions of these Na_V-channel proteins are comprised of a single subunit which contains four six-transmembrane pseudodomains [20,21]. Upon rapid depolarization, the Na_V channels, by which macroscopic voltage-gated Na⁺ currents (I_Na) are encoded, are characterized by going through rapid transitions from the closed (resting) state to the open state, and then by swiftly changing to the inactivated state [20,21]. The inactivation of I_Na has been also demonstrated to accumulate before being stimulated during repetitive short depolarizing pulses [22,23,24,25]. Consequently, once being evoked, the increased magnitude of I_Na can quickly depolarize the cell membrane through positive feedback cycle and, in turn, elicit the upstroke of the action potentials, thereby intrinsically governing the amplitude, frequency, and/or pattern of firing action potentials, as well as hormonal secretion, in an array of excitable cells [20,21,26,27]. On the other hand, the aberrant changes in Na_V (i.e., Na_V1.2) channel activity occurring in corticostriatal circuits of adult mice were also reported to elevate neuronal excitability [28].

Like the action of tefluthrin (Tef) [7,8,16,26,29], deltamethrin (DLT) was used to kill a wide range of insects [2,6,30]. There is a growing concern over human or animal poisoning as aberrant use in these esters. However, whether and how deltamethrin (DLT) or other structurally similar pyrethroids (e.g., Tef) is able to modify the magnitude, gating kinetics, frequency dependence, and/or voltage-dependent hysteresis (Hys_(V)) of I_Na remains mostly obscure, although they are recognized to augment the I_Na magnitude [6,7,8,9].

In light of the aforementioned considerations, we wanted to extensively explore the electrophysiological effects of DLT and other related compounds (e.g., Tef) in pituitary GH₃ sommatolactotrophs with either a single voltage-clamp pulse or pulse train (PT) stimulation. The tetrodotoxin (TTX)-sensitive I_Na, which is responsible for the generation of action potentials, has been identified in pituitary tumor (GH₃) cells [18]. The GH₃ cell line has been demonstrated previously to express the α-subunits of Na_V1.1, Na_V1.2, Na_V1.3, and Na_V1.6, as well as the β1 and β3-subunits of Na_V channel [18,31]. In the current investigations, we intended to (1) evaluate if DLT has any perturbations on the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na intrinsically in these cells; (2) examine if this compound affects either magnitude or time course of I_Na during as well as following 1-s pulse train (PT) stimulation; (3) explore whether or not the Hys_(V)’s behavior of persistent I_Na (I_Na(P)) could be seriously disturbed by the presence of DLT; and (4) the molecular docking between the DLT molecule and the hNa_V1.5 channel was also predicted. The present results disclosed that the differential and dose-dependent stimulation of I_Na(T) and I_Na(L) by DLT as well as its perturbations either on I_Na occurring during or following PT stimulation, or on Hys_(V) properties of I_Na(P) may potentially converge to engage in a great impact on electrical behaviors of mammalian excitable cells (e.g., GH₃ cells).

2. Results

2.1. Modification by Deltamethrin (DLT) or Tefluthrin (Tef) on Voltage-Gated Na⁺ Current (I_Na) Measured from Pituitary GH₃ Cells

In the first stage of whole-cell current recordings, we measured the effects of DLT or Tef on the magnitude and inactivation time course of I_Na activated in response to abrupt depolarizing pulse. We placed cells in Ca²⁺-free, Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA), and the pipette used was filled up with a Cs⁺-containing solution. As demonstrated in Figure 1A, two minutes after cells were continually exposed to DLT or Tef at a concentration of 10 μM, the amplitude in the transient (I_Na(T)) or late (I_Na(L)) component of I_Na activated by 20-ms depolarizing pulse from −80 to −10 mV was progressive raised. For example, as the rectangular voltage step from −80 to −10 mV with a duration of 20 ms was given (indicated in the uppermost part of Figure 1A) to activate I_Na, the addition of 10 μM DLT was found to result in a striking increase in either I_Na(T) or I_Na(L) amplitude to 512 ± 17 pA (n = 9, p < 0.05) or 128 ± 9 pA (n = 9, p < 0.05) from control values of 401 ± 15 or 22 ± 5 pA (n = 9), respectively. After washout of DLT, I_Na(T) or I_Na(L) was returned to 409 ± 17 or 28 ± 7 pA (n = 9). Likewise, the presence of 10 μM Tef also measurably increased I_Na(T) or I_Na(L) amplitude from 409 ± 14 pA (n = 9) or 32 ± 6 pA (n = 9) to 499 ± 16 pA (n = 9, p < 0.05) or 162 ± 11 pA (n = 9, p < 0.05), respectively. However, with cell exposure to 10 μM DLT, neither fast nor slow time constant of I_Na(T) inactivation in response to rapid membrane depolarization was evidently changed. Alternatively, with the presence of 10 μM Tef, the slow component in the time constant of I_Na(T) inactivation was strikingly raised to 19 ± 2 msec (n = 9, p < 0.05) from a control value of 0.9 ± 0.2 msec (n = 9). The time course of effects of DLT (10 μM) on the amplitude of I_Na(Tot), I_Na(L) or I_Na(T) is illustrated in Figure 1B. Of note, during exposure to 10 mM DLT, the amplitude of I_Na(Tot), I_Na(L), or I_Na(T) was increased to 177 ± 21 % (n = 8), 1752 ± 105 % (i.e., around 1.7-fold) (n = 8), or 24 ± 6% (n = 8), respectively.

Figure 1C demonstrates that the addition of DLT to the bath can concentration-dependently increase the amplitude of I_Na(T) or I_Na(L) activated by short depolarizing step. According to the Hill equation stated under Materials and Methods, the EC₅₀ value needed for DLT-stimulated I_Na(T) or I_Na(L) observed in GH₃ cells was calculated as 11.2 or 2.5 µM, respectively. Consistent with previous studies [6,8], the experimental observations, therefore, enable us to reflect that the DLT presence exerts a stimulatory action on the magnitude of I_Na(T) and I_Na(L) natively expressed in GH₃ cells, and that this compound tends to be selective for I_Na(L) over I_Na(T) during rectangular depolarizing pulse.

2.2. Comparison among Effects of Tef, DLT, Tef plus Chlorotoxin (ChloroTx), DLT plus ChloroTx, Tef plus Dapaglifozin (Dapa), DLT plus Dapa, and DLT plus Amiloride on I_Na(L) Amplitude Measured from GH₃ Cells

Exposure to pyrethroids (e.g., DLT) has been previously demonstrated to activate Cl⁻ currents [14,15,16]. We further compared the effects of Tef, DLT or their combinations with ChloroTx, Dapa, or amiloride on I_Na(L) amplitude. ChloroTx was reported to suppress Cl⁻ current, Dapa was an inhibitor of I_Na(L) [32,33], and amiloride can attenuate the pyrethroids-stimulated sodium transport [16]. As summarized in Figure 2, with continued presence of Tef or DLT, the further exposure to ChloroTx (1 μM) failed to modify their stimulation of I_Na(L). Dapa (10 mM) or amiloride (10 mM) alone decrease the I_Na(L) amptitude to 31 ± 2 pA (n = 8, p < 0.05) or 28 ± 2 pA (n = 8, p < 0.05) from control value of 50.3 ± 3 pA (n = 8). Moreover, the subsequent presence of either Dapa (10 μM) or amiloride (10 μM) was able to attenuate Tef- or DLT-mediated increase of I_Na(L) effectively. The results prompted us to suggest that either Dapa or amiloride could directly cause an inhibitory effect on the amplitude of I_Na(L) observed in GH₃ cells [32,33].

2.3. Effect of DLT on Mean Current Versus Voltage (I-V) Relationship of I_Na(T) and I_Na(L)

We next explored any perturbations of this compound on the amplitude of I_Na(T) or I_Na(L) measured from the different level of membrane potentials. As demonstrated in Figure 3A,B, a steady-state I-V relationship of I_Na(T) and I_Na(L) acquired with or without the DLT (10 μM) presence was established in these cells. The appearance of 10 μM DLT resulted in a striking increase in the I_Na(T) or I_Na(L) amplitude elicited by abrupt depolarizing steps. For example, when the tested cells were rapidly depolarized from −80 to −10 mV, the addition of 10 μM DLT raised either I_Na(T) or I_Na(L) magnitude from 729 ± 54 to 952 ± 76 pA (n = 8, p < 0.05), or from 26 ± 34 to 265 ± 48 pA (n = 8, p < 0.05), respectively. However, the steady-state I-V relationship of I_Na(T) or I_Na(L) remained unaffected during exposure to 10 μM DLT, despite a marked increase in I_Na(T) or I_Na(L) magnitude. The relationship (i.e., G-V relationship) for the conductance of I_Na(T) or I_Na(L)) with or without the application of 10 μM DLT was also established and depicted in Figure 3C. The V_1/2 value for G-V relationship of I_Na(T) or I_Na(L) between the absence and presence of 10 mM DLT did not differ significantly {(−19.9 ± 1.8 mV [control] versus −20.5 ± 1.7 mV [in the presence of DLT]; n = 8, p > 0.05, for the results of I_Na(T)), or (−18.4 ± 1.6 mV [control] versus −18.5 ± 1.6 mV [in the presence of DLT]; n = 8, p > 0.05, for the results of I_Na(L))}.

2.4. Tef- or DLT-Mediated Slowing in Cumulative Inhibition of I_Na(T) during Rapid Depolarizing Stimuli

It has been demonstrated that, prior to being activated during repetitive short pulses, the inactivation of I_Na(T) is able to accumulate [22,23,25,34,35,36]. For this reason, we next explored if Tef or DLT could modify the extent of I_Na(T) activated either during or following the PT depolarizing stimuli. In this set of measurement, the stimulus protocol, consisting of repetitive depolarization of −10 mV (20 ms in each pulse with a rate of 40 Hz for 1 s), was applied to the tested cells which were voltage-clamped at −80 mV. In accordance with earlier reports [22,23,24,25,36], as demonstrated in Figure 4 and Figure 5, during the control period (i.e., neither Tef nor DLT was present), the exponential time course of I_Na(T) inactivation observed in GH₃ cells was observed during a 1-s repetitive depolarization from −80 to −10 mV, and an evolving decaying time constant of 22.1 ± 2.8 ms (n = 8) was then yielded. In other words, there appeared to be a progressive current decay (indicated with the dashed arrows in Figure 4A) with a single-exponential process. It also needs to be noted that with cell exposure to DLT (10 μM), the time constant of I_Na(T) decaying activated during the same train of depolarizing pulses was increased to 56.4 ± 3.9 ms (n = 8), apart from a progressive increase in I_Na(L) or I_Na(Tail) (i.e., appearance of tail current following 1-s PT stimulation) magnitude. Of additional notice, a significant increase in tail I_Na (I_Na(Tail)) (blue open triangles in Figure 5) with a rising time constant of 87.4 ± 4.6 ms (n = 8) was found in the presence of 10 μM DLT (Figure 4B,C and Figure 5); however, cell exposure to 10 μM Tef resulted in a gradual decay in I_Na(L) with a decaying time constant of 26.3 ± 2.7 ms (n = 8). Alternatively, with continued exposure to 10 mM DLT, further addition of Dapa (10 mM) or Ami (10 mM) significantly decreased the time constant of I_Na(L) during PT stimulation.

Table 1. Effects of DLT, DLT plus dapagliflozin (Dapa, 10 mM), and DLT plus amiloride (Ami, 10 mM) on either the decaying time constant of I_Na(T) during pulse train (PT) stimulation (i.e., cumulative inhibition of I_Na(T) during rapid depolarizing stimuli) or the rising time constant of I_Na(L) during the same PT stimulation, as well as the time constant of I_Na(Tail) recovery evoked following PT stimulation. All values are mean ± SEM.

	Control	DLT (10 mM)	DLT (10 mM) Plus Dapa (10 mM)	DLT (10 mM) Plus Ami (10 mM)	Cell Number (n)
Decaying time constant of INa(T)	22.1 ± 2.8 ms	56.4 ± 3.9 * ms	29.6 ± 6.1 * ms	30.9 ± 6.5 * ms	8
Rising time constant of INa(L)	(-)	87.4 ± 4.6 ms	19.1 ± 6.1 ** ms	21.1 ± 6.5 ** ms	8
Recovery time constant of INa(Tail)	25 ± 3 ms	1.23 ± 0.19 + s	0.56 ± 0.04 ** s	0.58 ± 0.05 ** s	8

* Significantly different from controls (p < 0.05), ** significantly different from DLT (10 mM) alone groups (p < 0.05), and ⁺ significantly different from controls (p < 0.01). (-) shown in Table 1 indicates that the time constant of I_Na(L) during PT stimulation decayed in an exponential manner.

summarizes the results showing effects of DLT, DLT plus dapagliflozin (Dapa), DLT plus amiloride (Ami) on either the decaying time constant of I_Na(T) during PT stimulation or the rising time constant of I_Na(L) during the same PT stimulation, as well as the time constant of I_Na(Tail) recovery evoked following PT stimulation.

Moreover, with the DLT presence, an exponential increase in I_Na(L) during PT stimuli occurring over time was also observed (Figure 4B,C). Following 1-s PT stimulation, as cells were continually exposed to 10 μM DLT, there appeared to be a large inward current (i.e., I_Na(Tail)) accompanied by a gradual recovery (indicated with asterisk in Figure 4B) in the second timescale with a recovery time constant of 1.23 ± 0.19 s (n = 8) (Figure 4B,C). The appearance of I_Na(Tail) could reflect changes in the magnitude of I_Na(P), and the I_Na(L) and I_Na(P) evoked during an extended period of time were thought to share the same Na_V channels [22,26]. Likewise, with the presence of 10 μM Tef, the recovery time constant of I_Na(Tail)) (or I_Na(P)) acquired following PT stimuli was estimated to be 123 ± 25 ms (n = 8), a value which is different from DLT-induced change in the recovery time constant of I_Na(Tail) following PT stimuli. In contrast, during the control period (i.e., neither Tef nor DLT was present), the recovery time constant of the current following PT stimulation was rather small (i.e., 25 ± 3 ms [n = 8]) (Figure 6B). Moreover, with continued presence of DL (10 mM), further addition of either Dapa (10 mM) or Ami (10 mM) significantly attenuated the recovery time constant of I_Na(T) evoked following PT stimulation, as summarized in Table 1.

Additionally, with continued exposure to Tef (10 μM) or DLT (10 μM), further addition of dapagliflozin (Dap) at a concentration of 10 μM resulted in an attenuation of the drastic appearance of large inward I_Na(Tail) following PT stimuli, as demonstrated by a respective reduction in the recovery time constant of the current to 54 ± 17 ms (n = 8, p < 0.05) or 564 ± 62 ms (n = 8, p < 0.05) estimated during further presence of Dapa (10 μM) (Figure 6B). Taken together, these results prompted us to reflect that the presence of DLT can act as a striking slowing the deactivating kinetics of I_Na(Tail) (or I_Na(P)) following return to the holding potential at −80 mV. Therefore, during 1-s PT stimulation, insufficient period of time was allowed for I_Na recovery. As a result, particularly during exposure to DLT, single I_Na deactivation during PT stimulation presently given (i.e., at a rate of 40 Hz) could be apparently incomplete, thereby leading to frequency-dependent ‘accumulation’ of the Na_V-channel activated state. Therefore, the response of Tef- and DLT-mediated I_Na(L) or I_Na(Tail) was overly distinguishable. In other words, one (i.e., the Tef presence) is progressive decay of I_Na(L) during a train of depolarizing pulses, while the other (i.e., the DLT presence) exhibits a staircase increase in I_Na(L). Moreover, upon continued exposure to 10 μM DLT, the subsequent addition of Dapa (10 μM) could measurably attenuate DLT-mediated increase I_Na(Tail) during PT stimuli as well as shortened the recovery time constant of I_Na(P) following repetitive depolarizing stimuli (Figure 6A,B).

2.5. Effect of Tef or DLT on the Strength of Voltage-Dependent Hysteresis (Hys_(V)) of Persistent I_Na (I_Na(P)) Elicited by an Upright Isosceles-Triangular Ramp Voltage (V_ramp)

The nonlinear Hys_(V) behavior residing in I_Na(P) has been recently disclosed with a figure-of-eight (i.e., $\infty$-shaped) configuration as current traces were robustly activated by an upright double V_ramp (i.e., ascending and descending limbs of triangular V_ramp) [33]. In this regard, efforts were made to explore if the existence of Tef or DLT could have any different adjustments on the Hys_(V)’s behavior elicited in response to such upright V_ramp. This separate set of measurements was performed in GH₃ cells which were placed in Ca²⁺-free Tyrode’s solution, and we filled up the measuring electrodes with a solution containing Cs⁺. The tested cells were maintained at −80 mV and an upright double V_ramp ranging between −80 and +50 mV for a duration of 1 s (i.e., ramp speed of ±0.26 mV/ms) was afterwards applied to them. As shown in Figure 7A, during cell exposure to Tef (10 μM) or DLT (10 μM), current traces in response to such triangular V_ramp are distinguishable, although two types of hysteretic loops (i.e., low- and high-threshold loops) became overly noticeable during the presence of Tef or DLT. In particular, the strength of low-threshold hysteretic loop during such V_ramp became considerably larger in the presence of Tef (10 μM), as compared with that during exposure to DLT. For example, as cells were continually exposed to Tef (10 μM), the amplitude of I_Na(P) at the descending limb of V_ramp (i.e., at the level of −70 mV) resulted in a striking increase by 6.6 folds from 61 ± 9 to 403 ± 24 pA (n = 8, p < 0.05); conversely, upon the presence of DLT (10 μM), I_Na(P) amplitude at the same level was increased only by 1.5 folds (Figure 7C). However, upon cell exposure to Tef (10 μM) or DLT (10 μM), the I_Na(P) amplitude at the ascending limb (i.e., at −10 mV) was increased to 3.6 or 3.7 folds, respectively, which did not differ significantly between these two compounds. From these data, it was plausible to assume that the strength of I_Na(P)’s Hys_(V) in response to long-lasting double V_ramp was susceptible to being enhanced during Tef or DLT presence; moreover, the low-threshold loop of Hys_(V) appeared to be more sensitive to augmentation by Tef than that by DLT.

3. Discussion

In the current investigations together with previous studies, we provided the evidence to unveil that the presence of DLT, known to be a type II pyrethroid, was able to exert stimulatory actions on vastly different types of I_Na, including I_Na(T), I_Na(L), I_Na(Tail), and I_Na(P), seen in pituitary tumor (GH₃) cells. It is likely, therefore, that the endocrine disrupting potential caused by the existence of DLT or other structurally similar pyrethroids, as demonstrated recently [2,16,20,27,37,38,39,40,41], could be highly linked to the excitatory actions on varying types of I_Na presented herein, presuming that similar pharmacological or toxicological actions take place in variable types of endocrine or neuroendocrine cells present in vivo [2,3,7,11,13,42], although pyrethroids are thought to be around 2250 times more toxic than mammals [12,13].

Upon cell exposure to DLT, the observed I_Na(L) activated in response to short depolarizing step was noticed to be stimulated to a greater extent than the I_Na(T). The EC₅₀ values required for DLT-stimulated I_Na(T) and I_Na(L) in GH₃ cells were estimated to be 11.2 and 2.5 μM, these values which was noted to differ significantly by 4.5 folds (Figure 1). However, the further addition of chlorotoxin (ChloroTx), still in continued presence of tefluthrin (Tef) or DLT, failed to modify Tef- or DLT-stimulated I_Na(L), although either dapagliflozin (Dapa) or amiloride could effectively reverse their increase in I_Na(L) amplitude. Tef is a Type I pyrethroid insecticide [8,10,26]. The overall steady-state I-V relationship of I_Na(T) or I_Na(L) during exposure to DLT remained unchanged; furthermore, the inactivation time course of I_Na(T) during brief step depolarizing did not differ between the absence and presence of DLT. However, the magnitude of I_Na(T) following the PT stimulation (i.e., 40-Hz repetitive depolarizing pulse) tended to be pronouncedly larger as well as its decaying time course became slowed in the presence of Tef or DLT (Figure 4).

It needs to be mentioned that a large appearance of inward current (i.e., I_Na(P)) following such PT stimulation clearly emerged during the presence of Tef or DLT, while a rather small transient current following the same PT stimuli was observed during the control conditions (i.e., neither Tef nor DLT was present). The larger magnitude of inward current immediately following PT stimuli by adding DLT was noted as compared to that by Tef. Furthermore, the exposure to Tef markedly rendered the inactivation time course of I_Na(T) during rapid membrane depolarization to become slowed, whereas DLT itself had minimal interference with the inactivation time constant of the current. However, the DLT existence, a progressive elevation of I_Na(L) and I_Na(Tail) during a train of repetitive depolarizations; moreover, it induced a larger tail I_Na following repetitive depolarizations. The experimental results can be interpreted to mean that, upon continued exposure to DLT, the I_Na deactivation elicited during PT stimuli could be apparently incomplete, thus leading to rate-dependent ‘accumulation’ of the Na_V channel activated state. The slowed inactivation caused by the exposure to Tef thus reflects that the barrier for going from the open to the inactivated state of the Na_V channel tends to be higher during its presence. The α-cyano-3-phenoxybenzyl group present in the DLT molecule tends to be a notable structure required preferentially for open/resting state of the channel.

Earlier reports have demonstrated the effectiveness of DLT either in inducing the raise in Ca²⁺ transient or exert anti-neoplastic actions in different types of neoplastic cells, including liver, oral, and prostate cancer cells, and Jurkat-J6 cell cells [43,44,45,46,47,48]. The functional expression of Na_V channels has also been reported in different neoplastic cells, including prostate cancer and glioma cells [45,49,50,51,52]. As such, whether DLT-mediated modifications on I_Na presented herein can be responsible for the actions of DLT or other structurally similar pyrethroids on intracellular Ca²⁺ or aberrant growth in neoplastic cells [44] is worth pursuing further.

Earlier reports have demonstrated the effectiveness of pyrethroids (e.g., DLT, esfenvalerate, or permethrin) in increasing long-term potentiation recorded in CA1 hippocampal region [53,54,55,56]. Indeed, a brief period of high-frequency electrical activity applied artificially to a neuronal pathway is expected to enhance the strength of synapses for various periods of time, which is called long-term potentiation. However, it needs to be stressed that with cell exposure to either Tef or DLT, following 1-s PT stimulation from −80 to −10 mV, a large inward current (i.e., I_Na(P)) with slowly decaying process was considerably observed (Figure 4A,B). Under such scenario, the observed induction of long-term potentiation (i.e., facilitation of synaptic transmitter [e.g., glutamate] release) evoked during high-frequency stimulation could have been seriously disturbed or even overestimated by the present findings showing a large recovery time course of I_Na emerging following PT stimulation in situations where cells present in tissue preparations were exposed to either Tef or DLT.

Previous studies have demonstrated that pyrethroids could affect transepithelial ion transport in the external layers of the skin and the further addition of amiloride could regulate pyrethroids-mediated change in such transepithelial ion transport [16,57]. In this study, the subsequent addition of amiloride can attenuate DLT-induced increase in I_Na(L) measured from GH₃ cells; however, further application of chlorotoxin (ChloroTx) had no effect on DLT- or Tef-stimulated I_Na. Moreover, further addition of ChloroTx failed to modify changes in DLT- or Tef-stimulated I_Na during PT timulation; however, subsequent application of either Dapa or amiloride could significantly attenuate DLT and Tef-stimulated I_Na by the same stimulation protocol. Therefore, the amiloride-mediated effect on the modifications by pyrethroids of ion transport through rabbit skin is likely associated with its direct inhibitory action on I_Na(L).

The ability of pyrethroids (e.g., DLT) to augment Cl⁻ currents has been previously demonstrated [14,16]. In our study, the subsequent addition of ChloroTx, an inhibitor of Cl⁻ currents, failed to modify DLT-stimulated I_Na(T) or I_Na(L) in GH₃ cells (Figure 2). However, the further application of dapagliflozin (Dapa) or amiloride can effectively attenuate DLT-activated I_Na(L) or I_Na(P). Dapa was recently demonstrated to ameliorate Tef-augmented Hys_(V) strength of I_Na(P) activated by double V_ramp [33]. Therefore, DLT-mediated stimulation of I_Na(T), I_Na(L), and I_Na(P) demonstrated herein is unlikely to be attributed to its activation of Cl⁻ current.

Work in our laboratory has demonstrated the non-equilibrium Hys_(V) behavior of I_Na(P) activated by the upright isosceles-triangular V_ramp [33]. The results indicated that there was a striking voltage dependence of such V_ramp-evoked I_Na(P) [29,33]. The experimental data also showed two types of Hys_(V) loops (i.e., a high-threshold counterclockwise followed by a low-threshold clockwise loop) with a figure-of-eight ($\mathrm{i}.\mathrm{e}., \infty$) configuration, which is reminiscent of the dynamics of the Lorenz-like motion [58]. Alternatively, with GH₃-cell exposure to Tef or DLT, the Hys_(V) motion of I_Na(P) activated by the upsloping (ascending) and downsloping (descending) ends of such double V_ramp as a function of time was noticed to move in both counterclockwise and clockwise directions (Figure 7A). In particular, one activated during the ascending limb of double V_ramp is called a high-threshold counterclockwise loop with a peak of around −10 mV, while the other evoked by the descending limb of V_ramp is a low-threshold clockwise loop with a peak falling at around −70 mV [33]. Moreover, as compared with the effect of DLT on Hys_(V)’s strength of I_Na(P), the exposure to Tef could augment Hys_(V)’s strength at low-threshold loop to a greater extent than that observed at high-threshold loop. However, with continued presence of either Tef or DLT, the further addition of Dapa could attenuate their stimulation of Hys_(V) strength in GH₃ cells. Thus, the presence of Tef could slow the inactivation time course of I_Na(T) activated by rapid step depolarization as well as augment magnitude of I_Na(P)’s low-threshold loop of Hys_(V) responding to double V_ramp. Conversely, as cells were exposed to DLT, the I_Na(T) inactivation time course during step depolarization was found to remain unchanged, and the increased strength of low-threshold Hys_(V) loop during double V_ramp was relatively smaller in its presence. It is therefore plausible to assume that the low-threshold loop of I_Na(P)’s Hys_(V) activated during the downsloping end of double V_ramp could be closely linked to the extent of the inactivation time course of I_Na(T).

4. Conclusions

The modifications by DLT and Tef on the magnitude, gating kinetics, frequency dependence, and Hys_(V) strength of I_Na in electrically excitable cells are noticeably different. The variable actions of pyrethroids presented here would be of clinical, pharmacological, and toxicological relevance [3].

5. Materials and Methods

5.1. Chemicals, Drugs, Reagents, and Solution Used in This Work

Deltamethrin (DLT, decamethrin, C₂₂H₁₉Br₂NO₃, IUPAC name: [(S)-cyano-(3-phenoxyphenyl)methyl](1R,3R)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane-1-carboxylate, (S)-α-cyano-3-phenoxybenzyl-cis-(1R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate) was acquired from MedChemExpress (Asia Biomed Inc., Taipei, Taiwan), dapagliflozin (Dapa, Foxiga^®) was from Cayman (Ann Arbo, MI), while amiloride, tetraethylammonium chloride (TEA), tetrodotoxin (TTX), and tefluthrin (Tef) were from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan). Chlorotoxin was a kind gift from Professor Dr. Woei-Jer Chuang (Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan). Because of a highly nonpolar nature of low water solubility (Laskowski, 2002), the stock solution of DLT (10 mM) was prepared by dissolving it in dimethylsulfoxide (DMSO), and it was wrapped in aluminum foil and then kept under −20 °C for long-term storage. Unless specified otherwise, growth media (e.g., Ham’s F-12 medium), fetal or horse bovine serum, trypsin/EDTA, and L-glutamine were mostly acquired from HyClone^TM (Thermo Fisher, Kaohsiung, Taiwan), while other chemicals or reagents were from Sigma-Aldrich or Merck (Genechain), and they were of laboratory grade and taken from standard sources.

The standard extracellular solution (i.e., normal Tyrode’s solution) used in this study had the ionic compositions containing (in mM): NaCl 136.5, CaCl₂ 1.8, KCl 5.4, MgCl₂ 0.53, glucose 5.5, HEPES 5.5, and the solution pH was titrated to 7.4 by adding NaOH. The composition of Ca²⁺-free Tyrode’s solution used for the measurement of I_Na (e.g., I_Na(T), I_Na(L), I_Na(P), and I_Na(Tail)) was the same as normal Tyrode’s solution in which CaCl₂ was removed. For the experiments on recording I_Na, the electrode used was filled up with the internal pipette solution containing (in mM): Cs-aspartate 130, CsCl 20, KH₂PO₄ 1. MgCl₂, Na₂ATP 3, Na₂GTP 0.1, and HEPES 5, and the pH was then adjusted to 7.2 with CsOH. The twice-distilled water used for the experiments was deionized with a Milli-Q ion exchange and activated carbon cartridge treatment system (Merck, Tainan, Taiwan).

5.2. Cell Preparation

Clonal pituitary (GH₃) somatolactotrophs, originally acquired from the Bioresources Collection and Research Center ([BCRC-60015], http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=60015) (access on 19 September 2022), Hsinchu, Taiwan), were revived and cultured in Ham’s F-12 growth medium, which was supplemented with 15% heat-inactivated horse serum (v/v), 2.5 % fetal calf serum (v/v), and 2 mM L-glutamine. They were commonly incubated at 37 °C in monolayer cultures in 50-mL plastic culture flasks in a humidified environment of 5% CO₂/95% air. It was confirmed that this cell line can continually secrete prolactin. We carried out electrical recordings 5 or 6 days after cells underwent subculture (60–70% confluence).

5.3. Electrophysiological Measurements (Patch-Clamp Current Recordings)

In the few hours before the experiments, GH₃ cells were detached from culture dishes with a 1% trypsin/EDTA solution, and a few drops of cell suspension (~10⁶/mL) was rapidly placed in a custom-built chamber mounted on the stage of a DM-IL inverted phase-contrast microscope (Leica; Major Instruments, Kaohsiung, Taiwan). We bathed cells at room temperature (20–25 °C) in the extracellular solution (i.e., normal Tyrode’s solution), the ionic compositions of which are described above. Before each experiment, cells were allowed to settle on the chamber’s bottom. The recording pipettes were pulled from Kimax^®-51 borosilicate glass tube (#DWK34500-99; Kimble^®, Merck, Tainan, Taiwan) and they were then polished to reach the resistances ranging between 3 and 5 MΩ. During each measurement, the electrode was mounted in an air-tight holder, which had a suction port on the side, and a silver-chloride wire was used to make good contact with the internal pipette solution. We recorded varying types of ionic currents (e.g., I_Na) with the whole-cell mode of a modified patch-clamp technique by using an RK-400 patch amplifier (Bio-Logic, Claix, France), as dealt with in our previous works [26,36,59]. All recordings were conducted inside a noise-proof Faraday cage. The junction potentials that commonly develop when the compositions of the pipette internal solution are different from those in the bath were zeroed shortly before giga-Ω formation was made, and the whole-cell data were corrected. As pulse train (PT) stimulation was applied to the tested cell, we used an Astro-Med Grass S88X dual output pulse stimulator (Grass; KYS Technology, Tainan, Taiwan).

5.4. Data Recordings and Processing

Throughout the recording period, the signal output (i.e., potential and current traces) was monitored and digitized online at 10 kH or more in an ASUS ExpertBook laptop computer (Yuan-Dai, Tainan, Taiwan). For analog-to-digital (A/D) and digital-to-analog (D/A) conversion, a Digidata^® 1550B converter equipped with the computer was controlled by pCLAMP^TM 10.6 program run under Microsoft Windows 7 (Redmond, WA, USA). Current signals were low-pass filtered at 2 kHz by using a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA). The voltage-clamp protocols with manifold rectangular or ramp waveforms were designed, and they were then given to the examined cell through D/A conversion.

5.5. Data Analyses for Whole-Cell Ionic Currents

To establish concentration-dependent stimulation of DLT on the amplitude of I_Na(T) or I_Na(L), we bathed GH₃ cells in Ca²⁺-free Tyrode’s solution which contained 10 mM tetraethylammonium chloride (TEA). During the recording period, we voltage-clamped each cell at −80 mV, and a brief step depolarization to −10 mV for a 20 ms at a rate 0.2 Hz was applied to evoke I_Na. The I_Na(T) magnitude was measured as the peak amplitude of I_Na at the beginning of depolarizing pulse was subtracted from the sustained I_Na (i.e., I_Na(L)), while the I_Na(L) magnitude was measured at the end of 20-ms depolarizing pulse in situations where different DLT concentrations were cumulatively given (as indicated in the right side of Figure 1A). The total amplitude of I_Na (I_Na(Tot)) taken from each step depolarization is equal to I_Na(T) plus I_Na(L). The amplitude of I_Na(L) obtained during the presence of DLT at a concentration of 100 µM was considered as 100% and we then compared current magnitudes (i.e., I_Na(T) and I_Na(L)) during cell exposure to varying concentrations of DLT. The concentration-dependent stimulation by DLT of I_Na(T) or I_Na(L) observed in GH₃ cells was determined by fitting experimental data set to a modified Hill function [10,25], which can be given as follows.

$$\mathrm{percentage}\mathrm{increase}(\%)=\langle {\mathrm{E}}_{\max }\times {\left[\mathrm{DLT}\right]}^{{\mathrm{n}}_{\mathrm{H}}}\rangle /\langle {\mathrm{EC}}_{50}^{{\mathrm{n}}_{\mathrm{H}}}+{\left[\mathrm{DLT}\right]}^{{\mathrm{n}}_{\mathrm{H}}}\rangle$$

In this equation, [DLT] = the deltamethrin (DLT) concentration given; n_H = the Hill coefficient (i.e., coefficient for cooperativity); EC₅₀ = the concentration required for a 50% stimulation of I_Na(T) or I_Na(L) amplitude activated in response to short depolarizing step from −80 to −10 mV; and E_max = maximal stimulation of I_Na(T) or I_Na(L) produced by the DLT presence.

5.6. Curve-Fitting Approximations and Statistical Analyses Used in This Work

To determine the model parameters, linear or nonlinear curve-fitting to the experimental data set presently obtained was optimally fitted with least-squares minimization procedure by using manifold analytical tools, such as the Microsoft “Solver” built in Excel^® 2022 (Microsoft) and OriginPro^® 2022 program (OriginLab^®; Scientific Formosa, Kaoshiung, Taiwan). The experimental results are presented as the mean ± standard error of the mean (SEM). The size of independent observations (n) is indicated in cell numbers collected during the measurements. The data distribution obtained presently was found to satisfy the tests for normality. Paired or unpaired t-tests were used for comparison between the two different groups; however, for comparison among more than two groups, we carried out analysis of variance (for one- or two-way ANOVA) with or without repeated measure followed by a post hoc Fisher’s least-significance difference test for multiple-range comparisons. A statistical significance (indicated with *, **, ⁺, or ⁺⁺ in the figures) was considered when p < 0.01 or < 0.05.