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The inhibitory effects of BmK IT2, a scorpion neurotoxin on rat nociceptive flexion reflex and a possible mechanism for modulating voltage-gated Na+ channels
Neuropharmacology 40 (2001) 352–357 www.elsevier.com/locate/neuropharm
The inhibitory effects of BmK IT2, a scorpion neurotoxin on rat nociceptive flexion reflex and a possible mechanism for modulating voltage-gated Na+ channels Zhi-Yong Tan a, Hang Xiao b, Xia Mao b, Cong-Ying Wang a, Zhi-Qi Zhao a, Yong-Hua Ji a,* a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China b Institute of Applied Toxicology, Nanjing Medical University, Nanjing, People’s Republic of China Received 6 April 2000; received in revised form 12 September 2000; accepted 25 September 2000
Abstract Buthus martensi Karsch IT2 (BmK IT2), a scorpion neurotoxin, was found to display a biphasic inhibitory effect on the C component of the rat nociceptive flexion reflex by subcutaneous injection in vivo, and also on the total Na+ currents of rat dorsal root ganglion neurons using whole-cell patch clamping. BmK IT2 blocked the tetrodotoxin-resistant (TTX-R) component of the Na+ currents with a degree of selectivity. The partial block of the TTX-R Na+ currents, brought about by 0.01 µg/µl BmK IT2, reversed less rapidly and completely than the partial block of the tetrodotoxin-sensitive (TTX-S) current brought about by the same concentration of BmK IT2. These results suggest that the inhibition of the rat nociceptive flexion reflex by BmK IT2 may be attributed to modulation of the different voltage-gated Na+ channels. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Scorpion neurotoxin; BmK IT2; Nociceptive flexion reflex; Voltage-gated Na+ channels
1. Introduction Scorpion neurotoxins composed of 60–70 amino acids can generally be classified as α- or β-mammalian-selective types or as excitatory or depressant insect-selective types according to their biological activity in vivo, including pharmacological and electrophysiological characteristics. Most of them have been demonstrated to be voltage-gated Na+ channel-specific ligands (MartinEauclaire and Couraud, 1995; Zlotkin et al., 1985, 1993). The Asian scorpion Buthus martensi Karsch (BmK) is a species distributed widely in northwestern China. Although this species is not dangerously venomous for mammals, its sting can cause a fierce pain (Baloozet, 1971). Since the Song Dynasty in China, the body of scorpion BmK has been used as a rare drug to cure various complaints, especially neurological symptoms, based on the doctrine of Chinese herbalism: ‘combat poison
* Corresponding author. Fax: +86-21-6433-2445. E-mail address: [email protected] (Y.-H. Ji).
with poison’. However, modern knowledge of the pharmacological function of the effective components in BmK venom seems to be scarce so far. Our previous studies described the structural and functional characteristics of several kinds of BmK neurotoxins (Ji et al., 1999, 1996; Ji et al., 1994a,b), in which one named as BmK IT2 (BmK insect-selective toxins 2) was structurally assigned to the group of depressant insect-selective toxins (Ji et al., 1994c). Recently, a binding assay showed that BmK IT2 could bind significantly to insect Na+ channels but not to rat brain Na+ channels, even at a high concentration (10⫺5 M). However, it is worth noting that native BmK IT2 at an applied concentration of 10⫺7 M could partially inhibit the specific binding of either BmK AS or BmK AS-1 to rat brain synaptosomes (Jia et al., 1999; Li et al., 2000). In addition, BmK AEP, a component from the same venom, which displayed high sequence homology with that of BmK IT2, has been demonstrated to be a strong antiepilepsy peptide by either intravenous or ventricular injection (Zhou et al., 1989). The results suggested that BmK IT2 is a neurotoxic polypeptide with potential
0028-3908/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 0 ) 0 0 1 6 8 - 4
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multi-pharmacological effects in both mammals and insects, and the target receptor site of BmK IT2 on mammalian brain-type Na+ channels might be different from that on insect Na+ channels. With the aim of understanding the neuropharmacology and neurotoxicology of ion channel-specific scorpion toxic ligands, in the present communication we describe the inhibitory effect of BmK IT2 on the rat peripheral nociceptive flexion reflex. The possible mechanism for BmK IT2 modulation of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat small-diameter dorsal root ganglion (DRG) neurons is discussed.
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student t-test. After the observation of FR, the rats were killed by intravenously administration of saturated KCl. 2.3. Cell preparation
2. Material and methods
Cells were isolated from the DRG of adult rats (120±12 g) according to the method described by Song et al. (1997). Rats were killed by concussion with subsequent cervical dislocation. Ganglia were dissected from the full length of the vertebral column. The trimmed ganglia were digested with collagenase type II (1.0 mg/ml) and trypsin type I (0.5 mg/ml) at 32±1°C for about 1 h and 40 min respectively. Single cells were dissociated mechanically with a series of fire-polished Pasteur pipettes, and then plated into dishes.
2.1. Materials
2.4. Whole-cell patch-clamp recording
Crude venom of the scorpion BmK was purchased from a private farm in Suzhou, Jiangsu Province, China. BmK IT2 was purified according to the procedure described by Ji et al. (1994a). Sprague-Dawley rats were from the experimental animal center of Jiang-Su Province. Collagenase (type II) and trypsin (type I) were purchased from Sigma. All other chemical reagents used were of analytic grade.
All the recordings were performed on DRG neurons of small diameter (10–25 µm) except those of TTX-S Na+ currents which were from large-diameter neurons (45–55 µm). A holding potential of ⫺70 mV was chosen. Membrane currents (1–5 M⍀) were measured with pipettes pulled from glass capillary tubes and connected to an EPC-9 amplifier operating Pulse/Pulsefit software (HEKA elektronik, Germany). The pipette solution was composed of (in mM): CsCl 120, TEACl 20, Na2ATP 5, EGTA 10, HEPES 10, MgCl2 2.5, Na2GTP 0.4. The pH of the solution was adjusted to 7.3 with CsOH. The external solution for recording from the small neurons was composed of (in mM): NaCl 140, KCl 5, CaCl2 0.01, MgCl2 1, HEPES 10, d-glucose 10. The solution was adjusted to pH 7.3–7.4 with NaOH. The external solution for recording from the large neurons was composed of (in mM): NaCl 65, TEACl 20, cholineCl 50, KCl 5, CaCl2 0.01, MgCl2 5, HEPES 5, dglucose 5. The solution was adjusted to pH 7.3–7.4 with Tris–OH. An Ag–AgCl saline bridge was used for the reference electrode. TTX-S and TTX-R Na+ currents were distinguished according to the method described by Song et al. (1997). Briefly, TTX (1 µM) was used to separate the TTX-R Na+ currents from TTX-S Na+ currents. After complete inhibition of the TTX-S portion using 1 µM TTX, the whole-cell Na+ currents were recorded in the external solution containing both drugs and 1 µM TTX, and then the cells were washed with the external solution containing 1 µM TTX. The fast kinetics enabled the TTX-S Na+ currents to be separated from the TTX-R Na+ currents. The neurons, which showed fast Na+ currents (completely inactivated at the end of a 10-ms depolarizing pulse to 0 or ⫺10 mV), were chosen for the recording of TTX-S Na+ currents. Results were presented as mean±standard error, and the statistical significance of differences between group means was assessed using a student t-test. The analyses
2.2. Recording of the C response of rat posterior biceps semitendinosus muscles The recording of the C response of rat posterior biceps semitendinosus (PBST) muscles was performed according to the method described by Bai and Zhao (1997). Rats (240±24 g) were anesthetized with urethane (1.1 g/kg, i.p.) and fixed in a stereotaxic frame. The body temperature and blood pressure of the rats were monitored and kept at physiological levels. Electromyography (EMG) recordings from rat PBST muscle were employed for the assessment of the nociceptive flexion reflex (FR). The muscle was activated by electrical stimulation (4 ms, 40 V, 15 pulses, 0.5 Hz at 3-min intervals) via a pair of stainless-steel needles inserted subcutaneously in the two toes of the ipsilateral hindpaw. The C responses of the EMG of the PBST reflex, with a latency of more than 100 ms, corresponding to C-afferent fibre-evoked responses, were integrated and averaged at 3-min intervals. A stable baseline was established for at least 20 min prior to the application of 10 µl of the drugs subcutaneously into the paw between the two hind-toes being stimulated. The effect of BmK IT2 on the C component of the nociceptive flexion reflex was expressed as percent change compared to baseline. The data were presented as mean±standard error, and the statistical significance of differences between group means were assessed using a
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of currents were achieved by using Sigma-Plot (Jandel Scientific, San Rafael, CA).
3. Results 3.1. Effect of BmK IT2 on the C component of rat FR The EMG recording found that the C responses of the rat FR could be completely abolished by a high dose of BmK IT2 (1 µg/µl) (Fig. 1A), and significantly inhibited by a low dose of BmK IT2 (0.02 µg/µl) in a biphasic manner (Fig. 1B). The time–response curves for 0.02 µg/µl BmK IT2 showed that the C responses were reduced rapidly to 47±5% at 3 min and then recovered to 75±1% at 6 min, which formed the first inhibitory phase, they then dropped again to 44±5% at 9 min and recovered gradually to 77±5% at 18 min, to give a second inhibitory phase, followed by relatively stable firing between 65% and 80% up to the 33 min (Fig. 2). A similar inhibitory curve was also recorded when 0.1 µg/µl of BmK IT2 was applied. The first inhibitory phase was recorded in the first 6 min, where the C responses were depressed to 37±2% at 3 min, and recovered to 47±4% at 6 min. The second inhibitory phase occurred from 6 to 18 min, during which the C responses were depressed again to 32±4% at 9 min, and then kept as a plateau from 9 to 18 min. Finally, the C responses dropped to 18±4% from 21 to 33 min (Fig. 2).
Fig. 2. Time–effect relationship of BmK IT2 inhibiting the C responses of the flexion reflex. The C responses of the EMG of the PBST reflex were integrated and averaged at 3-min intervals. The stable baseline was established at least 20 min prior to the application of 10 µl of drugs. The effect of BmK IT2 on the C component of the nociceptive flexion reflex was expressed as percent change compared to the baseline. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. saline). Saline induced insignificant effects recorded over the whiole time, while BmK IT2 caused a biphasic inhibitory effect at a dose of 0.02 µg/µl, and a strong inhibition at a high dose of 0.1 µg/µl without obvious recovery (n=6).
3.2. Effect of BmK IT2 on Na+ currents in rat DRG neurons
Fig. 1. Inhibitory action of BmK IT2 on the C responses of the rat flexion reflex. EMG recordings were made and integrated at 3-min intervals (0.2 Hz, 15 pulses). (A) The C responses were completely inhibited by a high dose of BmK IT2 (1 µg/µl). (B) BmK IT2 at low doses (0.02 µg/µl) inhibited the C responses in a biphasic manner. The vertical arrows indicate the time at which the BmK IT2 was given.
The membrane potential was held at ⫺70 mV on rat DRG neurons. The Na+ currents were elicited by a series of steps in potential from ⫺60 to 60 mV with a 10-mV increment. About 12.4±3.8%, 23.9±4.5% and 83.1±4.1% of TTX-R Na+ currents were blocked by the applied concentrations of 0.0002, 0.001 and 0.01 µg/µl BmK IT2, respectively. In contrast, only 8.2±2.3%, 18.0±2.5% and 34.1±5.4% of TTX-S (S) Na+ currents (recorded from the small neurons), and 3.9±1.8%, 5.7±2.3% and 17.7±3.6% of TTX-S (L) Na+ currents (recorded from the large neurons), were inhibited by the corresponding concentration of BmK IT2, respectively (Fig. 3). At a dose of 0.01 µg/µl, the inhibitory effect of BmK IT2 on TTX-R Na+ currents was found to be greater than that on TTX-S (S) and TTX-S (L) Na+ currents with a high degree of significance (Fig. 5). In addition, the inhibitory effect of BmK IT2 on TTX-S Na+ currents could be recovered to normal levels, but for TTX-R Na+ currents the recovery was only partial (Fig. 4A and B). Furthermore, inhibitory effect of BmK IT2 on TTX-S (S) Na+ currents was found to be greater than that on TTX-S (L)
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Fig. 3. Dose–effect relationship of BmK IT2 inhibiting TTX-S (S), TTX-S (L), and TTX-R peak Na+ currents. (A) control; (B) 0.0002 µg/µl BmK IT2; (C) 0.001 µg/µl BmK IT2; (D) 0.01 µg/µl BmK IT2. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. control).
Na+ currents with a high degree of significance at doses of 0.001 and 0.01 µg/µl (Fig. 5). Although BmK IT2 could inhibit both TTX-R and TTX-S Na+ currents significantly (Fig. 3), the time duration for the inhibition of the two kinds of Na+ currents was found to be different. The effect of BmK IT2 (0.01 µg/µl) on the TTX-S Na+ currents was found to be faster than that on TTX-R Na+ currents as shown in Fig. 6. The normalized TTX-S (S) peak Na+ currents were reduced immediately from 1.0±0.03 to 0.64±0.02 at 1 min, and then recovered to normal levels at 3 min with 2-min washing. The normalized TTX-S (L) peak Na+ currents were immediately reduced from 1.0±0.02 to 0.83±0.02 at 1 min, and then recovered to 0.95±0.02 at 3 min with 2-min washing. In contrast, the normalized TTX-R peak Na+ currents were reduced from 1.0±0.07 to 0.20±0.07 at 5 min with 4-min delay compared to the case of TTXS peak Na+ currents. Moreover, the recovery of TTX-R Na+ currents was very slow, reaching only 0.46±0.08 at 10 min with continuous washing. In some cases, the duration of inhibition by BmK IT2 (0.01 µg/µl) on the total Na+ currents was recorded to have a biphasic manner. The normalized total peak Na+ currents were reduced immediately to 0.57±0.06 at 1 min, quickly returned to 0.82±0.05 and 0.82±0.02 at 2 and 3 min, which formed the first inhibitory phase, and then dropped again to 0.71±0.04 at 4 min, recovering slowly from 0.8 to 0.9 within 6 to 10 min, thus forming the second inhibitory phase as shown in Fig. 6.
Fig. 4. Inhibitory effect of BmK IT2 (0.01 µg/µ) on TTX-S (S), TTX-S (L) and TTX-R peak Na+ currents. The whole-cell Na+ currents were evoked by 50-ms depolarizing steps to various levels from a ⫺70 mV holding potential. Peak Na+ currents were selected to assess the inhibition of BmK IT2 on TTX-R (A), TTX-S (S) (B) and TTX-S (L) (C) Na+ current. TTX was applied to test the purity of TTX-S currents after washing, as shown in (B).
4. Discussion Two main types of Na+ channels, termed TTX-S and TTX-R Na+ currents, have been identified in C fibers and somata of rat small-diameter DRG neurons (Rush et al., 1998; Roy and Narahashi, 1992). In the present study, BmK IT2, an insect Na+ channel-specific ligand with potential multiple pharmacological functions, for example as an anti-epileptic (Zhou et al., 1989), was found to eliminate the C component of the rat nociceptive flexion reflex in a biphasic manner. A similar biphasic inhibition by BmK IT2 of total Na+ currents, which are deemed to be composed of TTX-S and TTXR Na+ currents, was recorded in some DRG small neurons. The inhibition of TTX-R Na+ currents by BmK IT2 was found to be much more potent than the inhibition of TTX-S Na+ currents, but the TTX-S Na+ currents were inhibited more quickly than the TTX-R Na+ currents. The results thus suggest that the biphasic inhibition of the C component of the nociceptive flexion reflex by
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Fig. 5. Comparison of inhibitory effects of BmK IT2 on TTX-S (S), TTX-S (L) and TTX-R, TTX-S (S) and TTX-S (L) Na+ currents. (A) 0.01 µg/µl BmK IT2; (B) 0.001 µg/µl BmK IT2; (C) 0.0002 µg/µl BmK IT2. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05, **, P⬍0.01 TTX-S (S), TTX-S (L) vs. TTX-R; +, P⬍0.05, ++, P⬍0.01 TTX-S (L) vs. TTX-S (S)).
Fig. 6. Time–effect relationship of BmK IT2 on the TTX-S (S), TTXS (L), TTX-R and total peak Na+ currents. BmK IT2 (10 µg/ml) induced different effects on the time–response curve of TTX-S (L) (n=7), TTX-S (S) (n=4), TTX-R (n=4) and total (n=3) peak Na+ currents. Inhibitory and recovery curves of TTX-S Na+ currents were faster than those of TTX-R Na+ currents. The biphasic inhibition was observed in the curve of total current. The washing began at 1 min for TTX-S (L) and TTX-S (S) Na+ current recording, and at 2 min for TTX-R and total Na+ current recording. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. control).
BmK IT2 may be caused by the different action kinetics of BmK IT2 on the TTX-S and TTX-R Na+ currents in the C fibers. The first inhibitory phase on the C component might be caused by depression of the TTX-S Na+ channels, whereas the second phase might result mainly from inhibition of the TTX-R Na+ channels. The inhibitory course of BmK IT2 on the C component in the rat FR indicated that BmK IT2 at low doses (0.02 µg/µl) can produce a biphasic inhibitory course, but at higher doses, the biphasic nature can be reduced or even abolished (Figs. 1 and 2). This corresponded with the concentration-dependent inhibitory effects of BmK IT2 on both TTX-S and TTX-R Na+ currents (Fig. 3). By comparison, it was found that the amplitude of the second inhibitory phase was smaller than that of the first inhibitory phase in time–response curves for BmK IT2 on total Na+ currents in some DRG small neurons, while the amplitude of the second inhibitory phase was larger than that of the first inhibitory phase for the C component of the rat FR. These differences may be attributed to the presence of various Na+ channel subtypes or different resting potentials in the fibers and somata of the DRG small neurons. It is also interesting that the inhibitory effect of BmK IT2 on TTX-S (S) Na+ currents was found to be greater than that on TTX-S (L) Na+ currents with a high degree of significance (Fig. 5). The results suggest that there exist different components between the TTX-S (S) and TTX-S (L) Na+ currents, which could be partly distinguished by BmK IT2. Two cloned subtypes of TTX-R Na+ channels have been found to be differentially distributed among the DRG small neurons (Akopian et al., 1996; Dib-Haji et al., 1998), which were regulated by specific growth factors, and activated with more hyperpolarization potential after peripheral injury or inflammation (Gold et al., 1996; Tanaka et al., 1998). In addition, TTX-R Na+ currents were also found in peripheral axons of DRG small neurons (Buchanan et al., 1996), and could produce the rising phase of the action potential independently with TTX-S Na+ channels (Quasthoff et al., 1995). The results obtained in this study indicate that the inhibitory action of BmK IT2 on TTX-R Na+ currents was more potent and long-lasting than that on TTX-S Na+ currents. Since limited by the protocol with a holding potential of ⫺70 mV to record the TTX-R Na+ currents produced by NaN (novel Na channel α-subunit) Na+ channels (Cummins et al., 1999), which display hyperpolarized voltagedependence and persistent kinetics, our results focused on the inhibitory effects of BmK IT2 on the PN3/SNS (peripheral nerve type 3/the sensory neuron-specific sodium channel) and other uncloned Na+ channels. Therefore, BmK IT2 may be useful as a relatively specific polypeptide in understanding the function and dys-
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function of nociception by modulating either TTX-R or TTX-S Na+ currents.
Acknowledgements This study was supported by the National Nature Science Foundation of China (39625010), the National Basic Research Program of China (1999054001), and partially by the Shanghai–Unilever Research and Development Fund (9803) and the Chinese Academy of Sciences (Stz98306). The authors thank Li Hong-Zhao for technical assistance.
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The inhibitory effects of BmK IT2, a scorpion neurotoxin on rat nociceptive flexion reflex and a possible mechanism for modulating voltage-gated Na+ channels Zhi-Yong Tan a, Hang Xiao b, Xia Mao b, Cong-Ying Wang a, Zhi-Qi Zhao a, Yong-Hua Ji a,* a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China b Institute of Applied Toxicology, Nanjing Medical University, Nanjing, People’s Republic of China Received 6 April 2000; received in revised form 12 September 2000; accepted 25 September 2000
Abstract Buthus martensi Karsch IT2 (BmK IT2), a scorpion neurotoxin, was found to display a biphasic inhibitory effect on the C component of the rat nociceptive flexion reflex by subcutaneous injection in vivo, and also on the total Na+ currents of rat dorsal root ganglion neurons using whole-cell patch clamping. BmK IT2 blocked the tetrodotoxin-resistant (TTX-R) component of the Na+ currents with a degree of selectivity. The partial block of the TTX-R Na+ currents, brought about by 0.01 µg/µl BmK IT2, reversed less rapidly and completely than the partial block of the tetrodotoxin-sensitive (TTX-S) current brought about by the same concentration of BmK IT2. These results suggest that the inhibition of the rat nociceptive flexion reflex by BmK IT2 may be attributed to modulation of the different voltage-gated Na+ channels. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Scorpion neurotoxin; BmK IT2; Nociceptive flexion reflex; Voltage-gated Na+ channels
1. Introduction Scorpion neurotoxins composed of 60–70 amino acids can generally be classified as α- or β-mammalian-selective types or as excitatory or depressant insect-selective types according to their biological activity in vivo, including pharmacological and electrophysiological characteristics. Most of them have been demonstrated to be voltage-gated Na+ channel-specific ligands (MartinEauclaire and Couraud, 1995; Zlotkin et al., 1985, 1993). The Asian scorpion Buthus martensi Karsch (BmK) is a species distributed widely in northwestern China. Although this species is not dangerously venomous for mammals, its sting can cause a fierce pain (Baloozet, 1971). Since the Song Dynasty in China, the body of scorpion BmK has been used as a rare drug to cure various complaints, especially neurological symptoms, based on the doctrine of Chinese herbalism: ‘combat poison
* Corresponding author. Fax: +86-21-6433-2445. E-mail address: [email protected] (Y.-H. Ji).
with poison’. However, modern knowledge of the pharmacological function of the effective components in BmK venom seems to be scarce so far. Our previous studies described the structural and functional characteristics of several kinds of BmK neurotoxins (Ji et al., 1999, 1996; Ji et al., 1994a,b), in which one named as BmK IT2 (BmK insect-selective toxins 2) was structurally assigned to the group of depressant insect-selective toxins (Ji et al., 1994c). Recently, a binding assay showed that BmK IT2 could bind significantly to insect Na+ channels but not to rat brain Na+ channels, even at a high concentration (10⫺5 M). However, it is worth noting that native BmK IT2 at an applied concentration of 10⫺7 M could partially inhibit the specific binding of either BmK AS or BmK AS-1 to rat brain synaptosomes (Jia et al., 1999; Li et al., 2000). In addition, BmK AEP, a component from the same venom, which displayed high sequence homology with that of BmK IT2, has been demonstrated to be a strong antiepilepsy peptide by either intravenous or ventricular injection (Zhou et al., 1989). The results suggested that BmK IT2 is a neurotoxic polypeptide with potential
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multi-pharmacological effects in both mammals and insects, and the target receptor site of BmK IT2 on mammalian brain-type Na+ channels might be different from that on insect Na+ channels. With the aim of understanding the neuropharmacology and neurotoxicology of ion channel-specific scorpion toxic ligands, in the present communication we describe the inhibitory effect of BmK IT2 on the rat peripheral nociceptive flexion reflex. The possible mechanism for BmK IT2 modulation of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat small-diameter dorsal root ganglion (DRG) neurons is discussed.
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student t-test. After the observation of FR, the rats were killed by intravenously administration of saturated KCl. 2.3. Cell preparation
2. Material and methods
Cells were isolated from the DRG of adult rats (120±12 g) according to the method described by Song et al. (1997). Rats were killed by concussion with subsequent cervical dislocation. Ganglia were dissected from the full length of the vertebral column. The trimmed ganglia were digested with collagenase type II (1.0 mg/ml) and trypsin type I (0.5 mg/ml) at 32±1°C for about 1 h and 40 min respectively. Single cells were dissociated mechanically with a series of fire-polished Pasteur pipettes, and then plated into dishes.
2.1. Materials
2.4. Whole-cell patch-clamp recording
Crude venom of the scorpion BmK was purchased from a private farm in Suzhou, Jiangsu Province, China. BmK IT2 was purified according to the procedure described by Ji et al. (1994a). Sprague-Dawley rats were from the experimental animal center of Jiang-Su Province. Collagenase (type II) and trypsin (type I) were purchased from Sigma. All other chemical reagents used were of analytic grade.
All the recordings were performed on DRG neurons of small diameter (10–25 µm) except those of TTX-S Na+ currents which were from large-diameter neurons (45–55 µm). A holding potential of ⫺70 mV was chosen. Membrane currents (1–5 M⍀) were measured with pipettes pulled from glass capillary tubes and connected to an EPC-9 amplifier operating Pulse/Pulsefit software (HEKA elektronik, Germany). The pipette solution was composed of (in mM): CsCl 120, TEACl 20, Na2ATP 5, EGTA 10, HEPES 10, MgCl2 2.5, Na2GTP 0.4. The pH of the solution was adjusted to 7.3 with CsOH. The external solution for recording from the small neurons was composed of (in mM): NaCl 140, KCl 5, CaCl2 0.01, MgCl2 1, HEPES 10, d-glucose 10. The solution was adjusted to pH 7.3–7.4 with NaOH. The external solution for recording from the large neurons was composed of (in mM): NaCl 65, TEACl 20, cholineCl 50, KCl 5, CaCl2 0.01, MgCl2 5, HEPES 5, dglucose 5. The solution was adjusted to pH 7.3–7.4 with Tris–OH. An Ag–AgCl saline bridge was used for the reference electrode. TTX-S and TTX-R Na+ currents were distinguished according to the method described by Song et al. (1997). Briefly, TTX (1 µM) was used to separate the TTX-R Na+ currents from TTX-S Na+ currents. After complete inhibition of the TTX-S portion using 1 µM TTX, the whole-cell Na+ currents were recorded in the external solution containing both drugs and 1 µM TTX, and then the cells were washed with the external solution containing 1 µM TTX. The fast kinetics enabled the TTX-S Na+ currents to be separated from the TTX-R Na+ currents. The neurons, which showed fast Na+ currents (completely inactivated at the end of a 10-ms depolarizing pulse to 0 or ⫺10 mV), were chosen for the recording of TTX-S Na+ currents. Results were presented as mean±standard error, and the statistical significance of differences between group means was assessed using a student t-test. The analyses
2.2. Recording of the C response of rat posterior biceps semitendinosus muscles The recording of the C response of rat posterior biceps semitendinosus (PBST) muscles was performed according to the method described by Bai and Zhao (1997). Rats (240±24 g) were anesthetized with urethane (1.1 g/kg, i.p.) and fixed in a stereotaxic frame. The body temperature and blood pressure of the rats were monitored and kept at physiological levels. Electromyography (EMG) recordings from rat PBST muscle were employed for the assessment of the nociceptive flexion reflex (FR). The muscle was activated by electrical stimulation (4 ms, 40 V, 15 pulses, 0.5 Hz at 3-min intervals) via a pair of stainless-steel needles inserted subcutaneously in the two toes of the ipsilateral hindpaw. The C responses of the EMG of the PBST reflex, with a latency of more than 100 ms, corresponding to C-afferent fibre-evoked responses, were integrated and averaged at 3-min intervals. A stable baseline was established for at least 20 min prior to the application of 10 µl of the drugs subcutaneously into the paw between the two hind-toes being stimulated. The effect of BmK IT2 on the C component of the nociceptive flexion reflex was expressed as percent change compared to baseline. The data were presented as mean±standard error, and the statistical significance of differences between group means were assessed using a
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of currents were achieved by using Sigma-Plot (Jandel Scientific, San Rafael, CA).
3. Results 3.1. Effect of BmK IT2 on the C component of rat FR The EMG recording found that the C responses of the rat FR could be completely abolished by a high dose of BmK IT2 (1 µg/µl) (Fig. 1A), and significantly inhibited by a low dose of BmK IT2 (0.02 µg/µl) in a biphasic manner (Fig. 1B). The time–response curves for 0.02 µg/µl BmK IT2 showed that the C responses were reduced rapidly to 47±5% at 3 min and then recovered to 75±1% at 6 min, which formed the first inhibitory phase, they then dropped again to 44±5% at 9 min and recovered gradually to 77±5% at 18 min, to give a second inhibitory phase, followed by relatively stable firing between 65% and 80% up to the 33 min (Fig. 2). A similar inhibitory curve was also recorded when 0.1 µg/µl of BmK IT2 was applied. The first inhibitory phase was recorded in the first 6 min, where the C responses were depressed to 37±2% at 3 min, and recovered to 47±4% at 6 min. The second inhibitory phase occurred from 6 to 18 min, during which the C responses were depressed again to 32±4% at 9 min, and then kept as a plateau from 9 to 18 min. Finally, the C responses dropped to 18±4% from 21 to 33 min (Fig. 2).
Fig. 2. Time–effect relationship of BmK IT2 inhibiting the C responses of the flexion reflex. The C responses of the EMG of the PBST reflex were integrated and averaged at 3-min intervals. The stable baseline was established at least 20 min prior to the application of 10 µl of drugs. The effect of BmK IT2 on the C component of the nociceptive flexion reflex was expressed as percent change compared to the baseline. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. saline). Saline induced insignificant effects recorded over the whiole time, while BmK IT2 caused a biphasic inhibitory effect at a dose of 0.02 µg/µl, and a strong inhibition at a high dose of 0.1 µg/µl without obvious recovery (n=6).
3.2. Effect of BmK IT2 on Na+ currents in rat DRG neurons
Fig. 1. Inhibitory action of BmK IT2 on the C responses of the rat flexion reflex. EMG recordings were made and integrated at 3-min intervals (0.2 Hz, 15 pulses). (A) The C responses were completely inhibited by a high dose of BmK IT2 (1 µg/µl). (B) BmK IT2 at low doses (0.02 µg/µl) inhibited the C responses in a biphasic manner. The vertical arrows indicate the time at which the BmK IT2 was given.
The membrane potential was held at ⫺70 mV on rat DRG neurons. The Na+ currents were elicited by a series of steps in potential from ⫺60 to 60 mV with a 10-mV increment. About 12.4±3.8%, 23.9±4.5% and 83.1±4.1% of TTX-R Na+ currents were blocked by the applied concentrations of 0.0002, 0.001 and 0.01 µg/µl BmK IT2, respectively. In contrast, only 8.2±2.3%, 18.0±2.5% and 34.1±5.4% of TTX-S (S) Na+ currents (recorded from the small neurons), and 3.9±1.8%, 5.7±2.3% and 17.7±3.6% of TTX-S (L) Na+ currents (recorded from the large neurons), were inhibited by the corresponding concentration of BmK IT2, respectively (Fig. 3). At a dose of 0.01 µg/µl, the inhibitory effect of BmK IT2 on TTX-R Na+ currents was found to be greater than that on TTX-S (S) and TTX-S (L) Na+ currents with a high degree of significance (Fig. 5). In addition, the inhibitory effect of BmK IT2 on TTX-S Na+ currents could be recovered to normal levels, but for TTX-R Na+ currents the recovery was only partial (Fig. 4A and B). Furthermore, inhibitory effect of BmK IT2 on TTX-S (S) Na+ currents was found to be greater than that on TTX-S (L)
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Fig. 3. Dose–effect relationship of BmK IT2 inhibiting TTX-S (S), TTX-S (L), and TTX-R peak Na+ currents. (A) control; (B) 0.0002 µg/µl BmK IT2; (C) 0.001 µg/µl BmK IT2; (D) 0.01 µg/µl BmK IT2. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. control).
Na+ currents with a high degree of significance at doses of 0.001 and 0.01 µg/µl (Fig. 5). Although BmK IT2 could inhibit both TTX-R and TTX-S Na+ currents significantly (Fig. 3), the time duration for the inhibition of the two kinds of Na+ currents was found to be different. The effect of BmK IT2 (0.01 µg/µl) on the TTX-S Na+ currents was found to be faster than that on TTX-R Na+ currents as shown in Fig. 6. The normalized TTX-S (S) peak Na+ currents were reduced immediately from 1.0±0.03 to 0.64±0.02 at 1 min, and then recovered to normal levels at 3 min with 2-min washing. The normalized TTX-S (L) peak Na+ currents were immediately reduced from 1.0±0.02 to 0.83±0.02 at 1 min, and then recovered to 0.95±0.02 at 3 min with 2-min washing. In contrast, the normalized TTX-R peak Na+ currents were reduced from 1.0±0.07 to 0.20±0.07 at 5 min with 4-min delay compared to the case of TTXS peak Na+ currents. Moreover, the recovery of TTX-R Na+ currents was very slow, reaching only 0.46±0.08 at 10 min with continuous washing. In some cases, the duration of inhibition by BmK IT2 (0.01 µg/µl) on the total Na+ currents was recorded to have a biphasic manner. The normalized total peak Na+ currents were reduced immediately to 0.57±0.06 at 1 min, quickly returned to 0.82±0.05 and 0.82±0.02 at 2 and 3 min, which formed the first inhibitory phase, and then dropped again to 0.71±0.04 at 4 min, recovering slowly from 0.8 to 0.9 within 6 to 10 min, thus forming the second inhibitory phase as shown in Fig. 6.
Fig. 4. Inhibitory effect of BmK IT2 (0.01 µg/µ) on TTX-S (S), TTX-S (L) and TTX-R peak Na+ currents. The whole-cell Na+ currents were evoked by 50-ms depolarizing steps to various levels from a ⫺70 mV holding potential. Peak Na+ currents were selected to assess the inhibition of BmK IT2 on TTX-R (A), TTX-S (S) (B) and TTX-S (L) (C) Na+ current. TTX was applied to test the purity of TTX-S currents after washing, as shown in (B).
4. Discussion Two main types of Na+ channels, termed TTX-S and TTX-R Na+ currents, have been identified in C fibers and somata of rat small-diameter DRG neurons (Rush et al., 1998; Roy and Narahashi, 1992). In the present study, BmK IT2, an insect Na+ channel-specific ligand with potential multiple pharmacological functions, for example as an anti-epileptic (Zhou et al., 1989), was found to eliminate the C component of the rat nociceptive flexion reflex in a biphasic manner. A similar biphasic inhibition by BmK IT2 of total Na+ currents, which are deemed to be composed of TTX-S and TTXR Na+ currents, was recorded in some DRG small neurons. The inhibition of TTX-R Na+ currents by BmK IT2 was found to be much more potent than the inhibition of TTX-S Na+ currents, but the TTX-S Na+ currents were inhibited more quickly than the TTX-R Na+ currents. The results thus suggest that the biphasic inhibition of the C component of the nociceptive flexion reflex by
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Fig. 5. Comparison of inhibitory effects of BmK IT2 on TTX-S (S), TTX-S (L) and TTX-R, TTX-S (S) and TTX-S (L) Na+ currents. (A) 0.01 µg/µl BmK IT2; (B) 0.001 µg/µl BmK IT2; (C) 0.0002 µg/µl BmK IT2. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05, **, P⬍0.01 TTX-S (S), TTX-S (L) vs. TTX-R; +, P⬍0.05, ++, P⬍0.01 TTX-S (L) vs. TTX-S (S)).
Fig. 6. Time–effect relationship of BmK IT2 on the TTX-S (S), TTXS (L), TTX-R and total peak Na+ currents. BmK IT2 (10 µg/ml) induced different effects on the time–response curve of TTX-S (L) (n=7), TTX-S (S) (n=4), TTX-R (n=4) and total (n=3) peak Na+ currents. Inhibitory and recovery curves of TTX-S Na+ currents were faster than those of TTX-R Na+ currents. The biphasic inhibition was observed in the curve of total current. The washing began at 1 min for TTX-S (L) and TTX-S (S) Na+ current recording, and at 2 min for TTX-R and total Na+ current recording. The data were presented as mean±S.E.M., and the statistical significance of differences between group means was assessed using a student t-test (*, P⬍0.05; **, P⬍0.01 vs. control).
BmK IT2 may be caused by the different action kinetics of BmK IT2 on the TTX-S and TTX-R Na+ currents in the C fibers. The first inhibitory phase on the C component might be caused by depression of the TTX-S Na+ channels, whereas the second phase might result mainly from inhibition of the TTX-R Na+ channels. The inhibitory course of BmK IT2 on the C component in the rat FR indicated that BmK IT2 at low doses (0.02 µg/µl) can produce a biphasic inhibitory course, but at higher doses, the biphasic nature can be reduced or even abolished (Figs. 1 and 2). This corresponded with the concentration-dependent inhibitory effects of BmK IT2 on both TTX-S and TTX-R Na+ currents (Fig. 3). By comparison, it was found that the amplitude of the second inhibitory phase was smaller than that of the first inhibitory phase in time–response curves for BmK IT2 on total Na+ currents in some DRG small neurons, while the amplitude of the second inhibitory phase was larger than that of the first inhibitory phase for the C component of the rat FR. These differences may be attributed to the presence of various Na+ channel subtypes or different resting potentials in the fibers and somata of the DRG small neurons. It is also interesting that the inhibitory effect of BmK IT2 on TTX-S (S) Na+ currents was found to be greater than that on TTX-S (L) Na+ currents with a high degree of significance (Fig. 5). The results suggest that there exist different components between the TTX-S (S) and TTX-S (L) Na+ currents, which could be partly distinguished by BmK IT2. Two cloned subtypes of TTX-R Na+ channels have been found to be differentially distributed among the DRG small neurons (Akopian et al., 1996; Dib-Haji et al., 1998), which were regulated by specific growth factors, and activated with more hyperpolarization potential after peripheral injury or inflammation (Gold et al., 1996; Tanaka et al., 1998). In addition, TTX-R Na+ currents were also found in peripheral axons of DRG small neurons (Buchanan et al., 1996), and could produce the rising phase of the action potential independently with TTX-S Na+ channels (Quasthoff et al., 1995). The results obtained in this study indicate that the inhibitory action of BmK IT2 on TTX-R Na+ currents was more potent and long-lasting than that on TTX-S Na+ currents. Since limited by the protocol with a holding potential of ⫺70 mV to record the TTX-R Na+ currents produced by NaN (novel Na channel α-subunit) Na+ channels (Cummins et al., 1999), which display hyperpolarized voltagedependence and persistent kinetics, our results focused on the inhibitory effects of BmK IT2 on the PN3/SNS (peripheral nerve type 3/the sensory neuron-specific sodium channel) and other uncloned Na+ channels. Therefore, BmK IT2 may be useful as a relatively specific polypeptide in understanding the function and dys-
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function of nociception by modulating either TTX-R or TTX-S Na+ currents.
Acknowledgements This study was supported by the National Nature Science Foundation of China (39625010), the National Basic Research Program of China (1999054001), and partially by the Shanghai–Unilever Research and Development Fund (9803) and the Chinese Academy of Sciences (Stz98306). The authors thank Li Hong-Zhao for technical assistance.
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