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NEUROPHARMACOLOGY
Laboratoire Récepteurs et Canaux Ioniques Membranaires Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 2647/Unité Sous Contrat Institut National de la Recherche Agronomique, Université d'Angers, Unité de Formation et de Recherche Sciences, Angers cedex, France (H.G., C.L., B.L.); and Institut de Recherche sur la Biologie de l'Insecte, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6035, Université François Rabelais, Faculté des Sciences, Parc de Grandmont, France (J.A.)
Received July 16, 2007; accepted October 16, 2007.
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Abstract |
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], we demonstrated that TRP initiated calcium influx. By contrast, 
-conotoxin GVIA (an inhibitor of N-type high-voltage-activated calcium channels), did not affect the DMDS-induced [Ca2+]i rise. Finally, the participation of the calcium-induced calcium release mechanism was investigated using thapsigargin, caffeine, and ryanodine. Our study revealed that DMDS-induced elevation in [Ca2+]i modulated IKCa in an unexpected bell-shaped manner via intracellular calcium. In conclusion, DMDS affects multiple targets, which could be an effective way to improve pest control efficacy of fumigation.
; Collins et al., 2002; Schlipalius et al., 2002). In the same context, methyl bromide has been mainly used as a quarantine treatment for plants and to control insect in buildings and commodities. However, methyl bromide is being phased out because of its role in ozone depletion. This prompts the Montreal Protocol on Substances That Deplete the Ozone Layer to eliminate this substance (Bell, 2000; UNEP 2000; Yates et al., 2003; Caddick, 2004). In addition to environmental concerns, there are serious questions regarding methyl bromide toxicology and risk assessment (Ruzo, 2006). Although the principal target site for methyl bromide seems to be the central nervous system, its precise mechanism of toxicity is unclear (Yang et al., 1995). Based on these findings, developed countries have agreed to a schedule of reductions. To date, the methyl bromide consumption in these countries is less than 12% of the baseline defined by consumption in 1991 (UNEP, 2006). Consequently, many chemicals [e.g., 1,3-D (TELONE II), chloropicrine (TELONE C-35, InLine), and metam-sodium, which is a recognized precursor to the formation of methyl isothiocyanate (VAPAM HL, BASAMID in soils)] and physical alternatives have been proposed as replacements for methyl bromide (Fields and White, 2002; Martin, 2003; Tomlin, 2003; Ruzo, 2006; UNEP, 2006).
The important efforts spent in the past 10 years to develop and make available in the short term molecules that can successfully be applied to soil fumigation have been concentrated on ubiquitous natural products detected as metabolites in numerous biological processes. Among them, sulfur volatile compounds such as dimethyl disulfide (DMDS) (Auger et al., 1989), first suggested as a grain fumigant, is undergoing extensive studied for fungus, nematode, and also insect control in soil (Dugravot et al., 2003; Church and Rosskopf, 2004). DMDS treatment, at relatively high concentration (i.e., 100 µM), results in insecticidal activity through an inhibition of cytochrome c oxidase in the insect central nervous system (Dugravot et al., 2003). This effect subsequently decreases the intracellular ATP concentration, which thereby activates insect neuronal ATP-dependent potassium channels, which produce an important hyperpolarization associated with the disappearance of action potentials. This insect neurotoxicity supported the development of DMDS as a soil fumigant used to control insects. However, the use of novel compounds as alternative to classic fumigants needs to consider an optimum insecticidal activity with an admitted paucity of definitive mammalian toxicological effect. In this context, additional studies have been planned on the insect central nervous system with much lower concentration (i.e., 1 µM) to understand in depth the pathway of neurotoxicity. This approach has allowed us, for the first time, to elucidate the neurotoxic effect of DMDS on unusual neuronal targets identified as calcium-activated potassium channels through complex regulatory pathways.
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Materials and Methods |
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; Wicher et al., 2001). Cockroaches were obtained from our laboratory stock colonies maintained under standard conditions (29°C; photoperiod of 12-h light/12-h dark). Animal care and handling procedures were in accordance with French institutional and national health guidelines. Animals were immobilized ventral side up on a dissection dish. The ventral cuticle and accessory glands were removed to allow access to the ventral nerve cord. The TAG, carefully dissected under a binocular microscope, were placed in normal cockroach saline containing (200 mM NaCl, 3.1 mM KCl, 5 mM CaCl2, 4 mM MgCl2, 50 mM sucrose, and 10 mM HEPES buffer; pH was adjusted to 7.4 with NaOH).
Cell Isolation and Electrophysiological Recordings. Isolation of adult DUM neuron cell bodies was performed under sterile conditions using enzymatic digestion and mechanical dissociation of the TAG. Briefly, ganglia were excised and incubated for 35 min at 29°C in cockroach saline supplemented with 300 IU/ml collagenase (type I; Worthington Biochemicals, Lakewood, NJ). After thoroughly washing off the enzyme, ganglia were mechanically dissociated by gentle repeated suctions through fire-polished Pasteur pipettes. According to the cobalt-filling technique together with immunohistochemical mapping and electrophysiological recordings (Sinakevitch et al., 1996; Grolleau and Lapied, 2000), it was assumed that most of TAG DUM neurons investigated formed a relatively homogeneous population of cells. The isolated DUM neuron cell bodies were maintained at 29°C for 24 h before experiments were carried out.
The whole-cell patch-clamp technique was used to record spontaneous electrical activity and voltage-gated potassium currents (voltage-clamp mode; Grolleau and Lapied, 1995). Patch pipettes were pulled from borosilicate glass capillary tubes (GC 150T-10; Clark Electromedical Instruments, Harvard Apparatus, Edenbridge, UK) using a PP83 electrode puller (Narishige, Tokyo, Japan). Pipettes had resistances ranging from 1.1 to 1.4 M
(for action potentials recordings) or from 0.8 to 1.1 M (for potassium currents recording) when filled with internal solutions (see composition below). The liquid junction potential between bath and internal solution was always corrected before the formation of a gigaohm seal (>5G).
Voltage-dependent potassium currents (Grolleau and Lapied, 1995) were recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) with the low-pass four-pole Bessel filter set to 5 kHz. Command potentials were generated by a programmable stimulator (SMP 310; Biologic Science Instruments, Claix, France) or a PC computer (Elonex, Pentium 733 MHz; sampling frequency, 33 kHz) connected to a 16-bit analog-to-digital converter (Digidata 1322A; Axon Instruments). The pClamp package, version 8.0.2 (Axon Instruments) was used for data acquisition and analysis. Although leak and capacitive currents were compensated electronically at the beginning of each experiment, subtraction of residual capacitive and leakage currents was performed with an on-line P/6 protocol provided by pClamp. Series resistance value was obtained for each experiment from the patch-clamp amplifier settings after compensation and varied between 3 and 5M. DUM neuron cell bodies were voltage-clamped at a steady-state holding potential of –80 mV, and test pulses, 100 ms in duration, were applied from the holding potential at 0.25 Hz. For current-clamp experiments, spontaneous action potentials were displayed on a digital oscilloscope (310; Thermo Electron Corporation, Waltham, MA), and they were stored on a digital audio tape (DTR-1204; Biologic Science Instruments, Claix, France) or on the hard disk of the computer for subsequent off-line analysis. Patch-clamp experiments were conducted at room temperature (20–22°C). Data, when quantified, were expressed as mean ± S.E.M. Differences between means were tested for statistical significance by Student's t test. For data analysis including fitting procedures, the software Prism 2 (GraphPad Software Inc., San Diego, CA) was used.
Calcium Imaging. Falcon 1006 Petri dishes with glass coverslips were coated with poly-D-lysine hydrobromide (mol. wt. 70,000–150,000; Sigma Chemical, l'Isle d'Abeau Chesnes, France), and isolated DUM neuron cell bodies were plated as described above. External recording solution contained 200 mM NaCl, 3.1 mM KCl, 5 mM CaCl2, 4 mM MgCl2, and 10 mM HEPES buffer; pH was adjusted to 7.4 with NaOH. The cells were incubated in the dark with 10 µM Fura-2 pentakis(acetoxy-methyl) ester (Sigma Chemical) for 60 min at 37°C. After loading, cells were washed three times in saline. The glass coverslips were then mounted in a recording chamber (Warner Instruments, Hamden, CT) connected to a gravity perfusion system allowing drug application. Imaging experiments were performed with an Olympus IX50 inverted microscope (Olympus, Rungis, France) equipped with epifluorescence. Excitation light was provided by a 75-W integral xenon lamp (Life Science Resources, Cambridge, UK). Excitation wavelengths (340 and 380 nm) were applied using a computer-driven Spectramaster (Life Science Resources). Images were collected with an Olympix digital charge-coupled device camera (AstroCam; Life Science Resources), and they were recorded in the computer with Merlin software, version 2.0 (Life Science Resources). Exposure times at 340 and 380 nm were usually 150 ms, and images were collected at various frequencies. Regions of interest were defined and analyzed off-line.
Antisense Assay. According to the protocol described by Fickbohm and Trimmer (2003), isolated DUM neurons were treated with either 10 µM antisense oligonucleotides (ASO) targeted to the pSlo coding region (GenBank accession no. AF452164) or reverse antisense oligonucleotides. For inhibition of pSlo expression, DUM neurons were incubated with a mixture of three 15-mers ASO (Europrim; Invitrogen, Cergy Pontoise, France) targeting the first 45 base pairs of the coding region of pSlo and designed with the following sequences: 5'-CTCACAGTTCGACAT-3', 5'-CACCGATGTCATCTC-3', and 5'-ATCTTCGCCTGGTGA-3'. For control experiments, the sequences of reverse antisense oligonucleotides corresponds to the three reverse sequences of ASO indicated above. We performed 24-h and 48-h incubations at 29°C. After the 24-h incubation, the cell culture medium containing oligonucleotides was half replaced.
Solutions. For voltage-clamp experiments, the extracellular solution contained 100 mM NaCl, 70 mM Trizma-HCl, 3.1 mM KCl, 5 mM CaCl2, 4 mM MgCl2, 10 mM HEPES buffer, and 100 nM tetrodotoxin; pH was adjusted to 7.4 with NaOH. Patch pipettes were filled with internal solution containing 135 mM KCl, 25 mM KF, 9 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, 3 mM ATP-Na2, and 10 mM HEPES buffer; pH was adjusted to 7.4 with KOH. Drug solutions were prepared in the external solution, and they were applied in the immediate vicinity of the cell body by a gravity perfusion system. In some cases, the tested compounds were added in the internal pipette solution immediately before use. For current-clamp recordings, cells were bathed in external solution containing 200 mM NaCl, 3.1 mM KCl, 4 mM MgCl2, 5 mM CaCl2, and 10 mM HEPES buffer; pH was adjusted to 7.4 with NaOH. The internal solution contained 160 mM K-aspartate, 10 mM K-fluoride, 10 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM EGTA, 1 mM ATP-Mg, and 10 mM HEPES buffer; pH was adjusted to 7.4 with KOH. All chemicals were purchased from Sigma Chemical, except LOE 908, an inhibitor of TRP, generously gifted by Boehringer Ingelheim (Ingelheim, Germany) and iberiotoxin (IbTx; Latoxan, Valence, France). DMDS was prepared in dimethyl sulfoxide. Final dilution never contained more than 0.1% dimethyl sulfoxide. This concentration of solvent was found to be without effect on electrophysiological properties of DUM neurons.
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12 Hz after 2 min; Fig. 1C). After 2 min, DMDS transformed the beating pacemaker activity into a burst of electrical activity containing approximately 10 spikes separated by short silent periods (500 ms; Fig. 1A). It was interesting to note that during DMDS treatment, the modified DUM neuron firing pattern was associated with a reduction of the amplitude of the action potential afterhyperpolarization (Fig. 1, B and D).
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, 2000), we examined further the mode of action of low concentration of DMDS on the voltage-dependent calcium-activated potassium currents under voltage-clamp conditions. Calcium-activated potassium currents can be isolated form the total outward current using IbTx or inorganic calcium channel blockers, such as cadmium chloride (Fig. 2E, top traces; Grolleau and Lapied, 1995). Note that the currents shown in Fig. 2E (right) were difference current obtained by subtraction of IbTx-resistant current from the corresponding total outward current (control; Fig. 2E, left).
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We first tested DMDS on the total outward potassium current recorded during a 100-ms test pulse (from –80 mV to +10 mV). After 15 min, external application of 1 µM DMDS significantly reduced both peak transient and late components at all potential tested (Fig. 2, A and B). Unexpected results were obtained when time course of inhibition by DMDS of the total outward current was measured at the beginning (peak transient component) and at the end of the depolarizing pulse (sustained component). Figure 2C compared the effect of 1 µM DMDS on transient and sustained components of the outward current. It seemed that DMDS did not affect similarly the amplitude of both components since a monotonic reduction of the amplitude of the peak transient component was mainly observed, whereas the effect on the sustained component was bell-shaped. In this last case, within the first 4 min, DMDS produced an increase of the sustained component, which was statistically significant (Fig. 2C), to reach a maximum at 4 min before decreasing for longer time exposure. By contrast, the peak transient current amplitude was relatively stable within the first 2 min, and then it progressively decreased. The first indication that DMDS affected calcium-activated potassium currents came from the results illustrated in Fig. 2, A and E (left traces). The DMDS-sensitive current (Fig. 2E, right, bottom), obtained by subtraction of the record after DMDS treatment from the control at 15 min, displayed a biphasic waveform composed of a well developed fast transient component followed by a late sustained current. This DMDS-sensitive outward current was very similar to that previously described in DUM neurons and identified as calcium-activated potassium currents (Grolleau and Lapied, 2000; Wicher et al., 2001). The second argument was obtained from experiments performed with IbTx. When DUM neuron cell bodies were pretreated for 15 min with 10 nM IbTx, known to block calcium-activated potassium currents in DUM neurons (Grolleau and Lapied, 1995; Fig. 2E, top traces), 1 µM DMDS did not affect both peak transient and sustained components of the current (Fig. 2D). Accordingly, the view that calcium-activated potassium currents were targeted by DMDS was also well illustrated in Fig. 2E (middle traces) in which the DMDS-sensitive current disappeared in the presence of 10 nM IbTx, indicating that IbTx already blocked the calcium-activated potassium current.
Finally, to substantiate that BK (pSlo; Derst et al.; 2003) channels were blocked by DMDS, additional experiments were performed using antisense oligonucleotides technology for decreasing endogenous mRNA of pSlo, with further assessment of its impact on calcium-activated potassium currents in DUM neurons (see Materials and Methods). In the initial studies, we confirmed the efficacy and specificity of antisense oligonucleotides in the down-regulation of DUM neuron BK (pSlo) channels by measuring both peak transient and sustained component amplitudes of the calcium-activated potassium currents after antisense versus reverse antisense treatments. As shown in Fig. 3, A and B, 1 mM cadmium chloride, known to block calcium-activated potassium currents in this neuronal preparation (Grolleau and Lapied, 1995), was able to inhibit both components of the current even if DUM neuron cell bodies were treated for 24 h or 48 h with reverse antisense. This indicated that treatment with reverse antisense oligonucleotides did not produce unspecific effects on calcium-activated potassium currents. Comparing DUM neurons pretreated with pSlo antisense oligonucleotides for 24 and 48 h with DUM neurons pretreated with reverse antisense oligonucleotides revealed a significant decrease of amplitudes of both the peak transient and sustained component of the calcium-activated potassium currents (Fig. 3, A–D). These effects were much more pronounced at 48 h (Fig. 3, C and D). In these last cases, bath application of 1 mM cadmium chloride, which we used to check for remaining calcium-activated currents (positive control), did not produce any additional effect on the outward current. These experiments indicated that calcium-activated potassium channel expression was suppressed in DUM neuron cell bodies pretreated by antisense oligonucleotides targeted pSlo mRNA (Fig. 3C). Next, we tested the effect of subsequent application of 1 µM DMDS on DUM neurons pretreated with pSlo antisense oligonucleotides for 24 and 48 h. This resulted in a strong reduction of DMDS-sensitive outward currents since antisense oligonucleotides reduced the conductance of the calcium-activated potassium currents (Fig. 3, E and F). Once again, the effects observed were more pronounced at 48 h rather than 24 h.
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DMDS-Induced Changes in Intracellular Calcium Concentration. The unexpected results illustrated in Fig. 2B led us to suspect an atypical DMDS-induced calcium-dependent regulation of these channels in DUM neurons. Consequently, a new set of experiments was designed to investigate the expected effect of DMDS on [Ca2+]i. The resting level of [Ca2+]i in isolated Fura-2-loaded DUM neuron cell bodies (saline containing 5 mM CaCl2) was measured as 29.6 ± 2.3 (n = 32).
Bath application of 1 µM DMDS produced a marked elevation in [Ca2+]i followed by a sustained elevated level. The maximum calcium rise did not decay to the baseline after washing (Fig. 4A). We next examined the source of the intracellular calcium rise observed in DUM neurons. When the experiments were performed in an EGTA-buffered calcium-free superfusing solution, the DMDS-induced elevation in [Ca2+]i quickly decayed toward the resting level and stabilized to a plateau phase, which was higher than the baseline [Ca2+]i level (Fig. 4B). These results indicated that the elevation in [Ca2+]i resulted, in part, from extracellular calcium through plasma membrane calcium channels. This was confirmed in Fig. 4C. If DUM neuron cell bodies were pretreated with an EGTA-buffered calcium-free superfusing solution, 1 µM DMDS was ineffective in producing any [Ca2+]i rise. By contrast, subsequent bath application of normal physiological saline restored the ability of DMDS to produce elevation in [Ca2+]i. After careful observation of the calcium response produced by 1 µM DMDS, the time course [Ca2+]i rise differed according to the cell region (Fig. 4, D and E). As indicated in Fig. 4E, a, illustrating the three-dimensional cytofluorescence intensity plot, the elevation in [Ca2+]i was detected and quantified in two defined fields, field 1 in the initial segment (basal pole) and field 2 in the apical of the soma. During the first 8 min, cytofluorescence intensity gradually increased in the apical pole of the DUM neuron, whereas there was no significant calcium response in the initial segment (Fig. 4, D and E, b and c). The [Ca2+]i rise occurred in the apical pole, with a half-maximum response reached 1 min later than in the initial segment. After [Ca2+]i reached its maximum in the apical pole, [Ca2+]i continued to rise in the initial segment (Fig. 4, D and E, e and f). In both cases, [Ca2+]i elevation slowly declined toward the same resting value and stabilized to a plateau phase after external application of EGTA-buffered calcium-free solution.
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Previous findings reported that calcium influx, in DUM neurons, might be accomplished by a functionally unusual pathway first identified as noncapacitive calcium entry (Wicher et al., 2006) and later as TRP (Wicher et al., 2006) situated at the apical region of the neurons. Based on findings presented above, it was tempting to suggest that the elevation in [Ca2+]i triggered by DMDS might involved TRP. Consequently, additional experiments were performed with LOE 908 known to specifically inhibit non-capacitative calcium entry in DUM neurons (Wicher et al., 2006). As shown in Fig. 5A, the enhancement of [Ca2+]i in response to 1 µM DMDS was strongly reduced after 40 µM LOE 908 pretreatment. DUM neurons are known to also express HVA calcium channels (Grolleau and Lapied, 2000; Wicher et al., 2001), which might be essential for triggering calcium signals. By contrast, HVA calcium channels seemed not to be involved since preincubation of DUM neuron cell bodies with 200 nM -conotoxin GVIA, one of the more potent N-type HVA calcium channel blockers, did not produce elevation of [Ca2+]i produced by 1 µM DMDS (Fig. 5B). Altogether, these results indicated that TRP might represent the first step essential for initiating calcium response.
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[Ca2+]i Rise Produced a Bell-Shaped Regulation of Calcium-Activated Potassium Currents in DUM Neurons. Time course of the effect of DMDS on both components of the outward current (i.e., fast transient and sustained components) (Fig. 2C) might suggest an unusual bell-shaped regulation, dependent on [Ca2+]i rise induced by DMDS. To substantiate this hypothesis, we examined the influence of intracellular and/or extracellular calcium changes on both amplitudes of the peak transient and sustained components of the calcium-activated potassium currents. Intracellular perfusion of DUM neurons with high [Ca2+] (i.e., without EGTA) pipette solution should allow for a first approximation of the calcium sensitivity of the calcium-activated potassium channels. As shown in Fig. 6A, immediately after establishing the whole-cell configuration, both components (i.e., peak transient and sustained components) of the currents increased in amplitude to reach a maximum at 4 min of EGTA exposure. Subsequently, when the concentration of EGTA rose on dialysis of the cell, both components were reduced, with an effect more pronounced for the peak transient component. It was interesting to note that the time for which the amplitudes of the currents were maximum was very similar to that of obtained with DMDS (Fig. 2B). We also wanted to test the influence of external calcium concentration changes on the calcium-activated potassium currents. When external calcium concentration was raised from 5 to 9 mM, an important [Ca2+]i rise was observed (Fig. 6C). In this case, a similar bell-shaped regulation of both components was also observed, with a maximum occurring at 4 min. For shorter or longer exposure than 4 min, these two components were reduced in amplitude. The difference current shown in Fig. 6D, obtained by subtraction of IbTx-resistant current from the total current, indicated that bath application of 9 mM external calcium did not affect potassium current amplitude recorded from DUM neuron cell bodies pretreated with 10 nM iberiotoxin.
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Because insect resistance to a given fumigant and/or insecticide in general (e.g., compounds based on natural products or of synthetic origin) is increasing (Raymond-Delpech et al., 2005; Dong, 2007), it seems evident that single-molecule replacements for methyl bromide, phosphine, or both, for example, may not be as effective as combination of chemicals. However, previous observations have clearly reported the incompatibility interactions between different fumigants (Ruzo, 2006). Simultaneous presence of metam-sodium with halogenated fumigants in polar organic solvents or moist soil (Guo et al., 2005) give rise to rapid degradation of the halogenated fumigants and to decomposition of metam to methyl isothiocyanate. Moreover, it has been demonstrated that simultaneous and sequential application of metam-sodium with chloropicrine and 1,3-D accelerates transformation of the two fumigants, reducing their availability in soil (Zheng et al., 2004). This results in the loss of active ingredient that occurs due to the reaction between fumigants and a poor pest control efficacy of field fumigation. Consequently, these observations have spurred research to solve such problems. A relatively recent alternative approach exists, consisting of the use of a chemical, which induces multiple metabolism alterations within the same organism via, for instance, second messenger activation (e.g., intracellular calcium), affecting distinct identified targets. In this context, DMDS may represent an exciting alternative to the phase-out of classic fumigants (e.g., methyl bromide) since, depending on the concentration used, DMDS produces significantly different alterations of the main physiological functions in insects.
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). This effect is attributed to an inhibition of cytochrome c oxidase. From these results, it is interesting to note that this mode of action observed at high concentration is very similar to that of phosphine (Singh et al., 2006). However, although this mode of action has been proposed to be similar, previous comparisons of LC50 values for 24-h treatments with each fumigant indicate that DMDS is much more toxic toward invertebrates, such as nematodes, for example, than is phosphine (Valmas and Ebert, 2006). Among other possibilities, this might suggest that DMDS could produce unexpected additional effects reflecting this higher toxicity. In this present study, we show that at 100-fold lower concentration, DMDS induces distinct complex additional effects summarized in Fig. 7. Previous studies performed on the same neuronal preparation have revealed that DMDS produces negative shift of both activation and inactivation curves of the voltage-dependent sodium current (Gautier et al., 2006). From these results, we have assumed that this hyperpolarized shift of the voltage dependence of both activation and inactivation parameters would produce channels that could function in a hyperexcitable manner, as illustrated in Figs. 1A and 7. The increase in the action potential discharge frequency associated with the slight depolarization reinforces the involvement of the low-voltage-activated currents previously identified as Na/Ca current (Defaix and Lapied, 2005) and both transient and maintained calcium currents (Grolleau and Lapied, 2000; Wicher et al., 2001). These currents are known to participate in the regulation of intracellular calcium level and to be involved in the generation of the pacemaker potential. The consecutive calcium influx modulates, in a positive manner, TRP (Grolleau et al., 2006; Wicher et al., 2006). This further enhances intracellular calcium level via the additional participation of calcium from internal stores through the calcium-induced calcium release mechanism activation. Finally, intracellular calcium changes regulate, in a bell-shaped manner, the calcium-activated potassium currents involved in the afterhyperpolarization that modulates the DUM neuron firing properties.
These last results also emphasized the involvement of unusual target sites for such insecticidal chemicals. Although the insecticidal toxic effect of fumigants is only hypothesized in insects, it is well known in the literature that the commonly used insecticides either exert their toxicity by virtue of acetylcholinesterase inhibition at the synapses and neuromuscular junctions, or they affect voltage-dependent sodium channels, ionotropic receptors such as nicotinic acetylcholine, and GABA receptors, which are considered as classic molecular targets in insects (Tomizawa and Casida, 2003; Raymond-Delpech et al., 2005). Recently, the question of the implication of another class of molecular targets that could play crucial indirect role in insecticide neurotoxicity has been raised. It has previously been indicated that ATP-dependent potassium channels, indirectly involved in toxicity, are a consequence of insecticide-induced mitochondrial dysfunction (Jenner, 2001; Dugravot et al., 2003). This downstream consequence could represent an epidemiological linkage between insecticide and neuropatholigical diseases (Jenner, 2001; Liss and Roeper, 2001).
In this study, we have provided new evidence for the participation of such an indirect target in insecticide neurotoxicity. Until now, calcium-activated potassium currents have not been identified as putative targets for such neurotoxic compounds. We have demonstrated that DMDS affects calcium balance-targeted calcium-activated potassium channels by an unexpected regulation, not reported before, for such potassium channels. Calcium-activated potassium channels identified as BK, SK (including KCa2.1, 2.2, and 2.3) channels and calcium-activated potassium channels of intermediate conductance (KCa 3.1) are known to be regulated by intracellular pathways involving calmodulin, phosphorylation process (e.g., through protein kinase A activation), and calcium-dependent and -independent protein phosphatases (calcineurin and PP1, respectively). In addition, distinct calcium-dependent regulatory mechanisms with different calcium selectivity were also known to influence BK channel-gating properties (Stocker, 2004; Latorre and Brauchi, 2006; Neylon et al., 2006), and some of calcium-activated potassium channels (e.g., SK2; Allen et al., 2007) are known to form multiprotein complexes containing associated calmodulin, both subunits of protein kinase CK2 and two different subunits of protein phosphatase PP2A. These SK2 channels seem to be regulated in a dynamic manner directly by protein kinase CK2 and PP2A and indirectly by calcium. In our case, the dual role of intracellular calcium on calcium-activated potassium currents via this bell-shaped regulation was unknown. Although the intracellular mechanisms involved in such unusual regulation still remain to be elucidated, the increase of intracellular calcium concentration that represents an important mechanism responsible for the abrogation of calcium-dependent potassium current activation may explain why exposure to sublethal levels of DMDS could potentially lead to higher toxicity. The associated increase of DUM neuron spontaneous firing frequency observed during DMDS exposure indicates a close relationship between calcium-activated potassium channels and production of changes in beating activity. Because it is well known that DUM neurons regulate vital functions in insects (e.g., heart wall muscle activity) via the release of octopamine (Sinakevitch et al., 1996), any neurotoxic mechanisms able to modulate, directly and/or indirectly, calcium-activated potassium channels could significantly alter neurosecretory properties, closely related to the firing pattern, which thereby will affect physiological functions. This reveals new important physiological features that should be considered during the design of chemical compounds.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IKCa, calcium-activated potassium current(s); DMDS, dimethyl disulfide; DUM, dorsal unpaired median; TAG, terminal abdominal ganglia; ASO, antisense oligonucleotides; LOE 908, (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxy-phenyl)ethyl]-acetamide; TRP, transient receptor potential; IbTx, iberiotoxin; BK, calcium-activated potassium channels of large conductance; HVA, high-voltage-activated; SK, calcium-activated potassium channels of small conductance; PP, protein phosphatase; 1,3-D, 1,3-dichloropropene.
Address correspondence to: Dr. Bruno Lapied, Laboratoire Récepteurs et Canaux Ioniques Membranaires, Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 2647/USC Institut National de la Recherche Agronomique, Université d'Angers, Unité de Formation et de Recherche Sciences, 2 boulevard Lavoisier, F-49045 Angers cedex, France. E-mail: bruno.lapied{at}univ-angers.fr
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ration 340/380 nm measured in the presence of 1 µM DMDS (white bar) and after subsequent application of EGTA-buffered calcium-free solution (gray bar). C, pretreatment with EGTA-buffered calcium-free solution inhibited DMDS-induced [Ca2+]i rise. After washing off calcium-free solution, an elevation in cytosolic free calcium was observed. Inset, 
