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Antihyperalgesia effect of BmK IT2, a depressant insect-selective scorpion toxin in rat by peripheral administration
Brain Research Bulletin, Vol. 53, No. 3, pp. 335–338, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
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Antihyperalgesia effect of BmK IT2, a depressant insect-selective scorpion toxin in rat by peripheral administration Cong-Ying Wang, Zhi-Yong Tan, Bing Chen, Zhi-Qi Zhao and Yong-Hua Ji* Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai, People’s Republic of China [Received 6 April 2000; Revised 19 July 2000; Accepted 19 July 2000] ABSTRACT: The study was undertaken to assess the antihyperalgesia effect of BmK IT2, a sodium channel-specific ligand purified from the venom of Chinese scorpion Buthus martensi Karsch in rat by peripheral injection. The peripheral inflammation of rat was induced by carrageenan resulted in hyperalgesia to heat stimulus. The heat hyperalgesia was measured by paw withdrawal latency (PWL). PWL was increased to 272 ⴞ 18%, 217 ⴞ 19% and 186 ⴞ 16% of the control by application of 10 l BmK IT2 at the concentration of 0.1, 0.01 and 0.001 mg/ml in inflammatory rats, respectively. In contrast, PWL was enhanced to about 177 ⴞ 16%, 141 ⴞ 15% and 133 ⴞ 15% of the control at the same applied concentrations of BmK IT2 in normal rats. The results thus suggest that BmK IT2 can produce peripheral antihyperalgesia and antinociception, which might be attributed a pathway of modulating the sodium channels on nociceptor. © 2000 Elsevier Science Inc.
swelling and primary hyperalgesia. Primary hyperalgesia is characterized by a lowing of response threshold and/or an increased response to suprathreshold stimulus at the site of injury. Meanwhile, primary hyperalgesia can result in sensitization of primary nociceptors, or an increase in the excitability of peripheral nociceptors to mechanical and thermal stimulus at inflammatory site. Sensitization depends on the release, synthesis or attraction inflammatory mediators to the site of injury, ordinarily. BmK IT2 is a component classified into the group of depressant insect-selective toxin [12,15,16], which has been tested in vivo to be non-toxic to the mammal [12]. Although the binding characteristics of a few depressant-insect scorpion toxins have been investigated on insect neuronal membranes [2,32], their possible pharmacological properties, especially in mammal seem to be still sparse. For example, it is worth noticing that BmK AEP, a component from the same venom, which displays a sequence highly homologous with that of BmK IT2, has been demonstrated to be a strong anti-epileptic peptide by either intravenous or ventricular injection [33]. In addition, our latest study revealed that BmK IT2 could significantly inhibit the specific binding of BmK AS to sodium channels in the mammalian brain [18]. It is thus suggested that BmK IT2 type neurotoxic polypeptides may be a distinctive sodium channel-blocking ligand with multiple pharmacological functions whether in insect or mammal. In the present communication, we further examined the effect of BmK IT2 on inflammation induced hyperalgesia in rats, and discussed its possible mechanism.
KEY WORDS: Scorpion neurotoxin, BmK IT2, Paw withdrawal latency, Antihyperalgesia.
INTRODUCTION It has been demonstrated that most long-chain scorpion neurotoxins composed of 60-70 amino acids with four disulfide bridges are ligands of voltage-gated sodium channels in mammals, insects and other vertebrates [7,8]. According to biological specificity in vivo and pharmacological and electrophysiological activity, these scorpion neurotoxins can be divided into ␣- or -mammal neurotoxins and excitatory or depressant insect-selective neurotoxins [7,8]. The Asian scorpion Buthus martensi Karsch (BmK) is a species widely distributed in northwestern China, Mongolia and Korea. In Chinese traditional medicine, even at the present time, the bodies of scorpion BmK are used as a rare drug to cure various complaints, especially neurological symptoms based on the herbalism ‘combat poison with poison’. Although several kinds of sodium channel-specific ligands have been purified and characterized from BmK venom [12–17], the modern knowledge on the pharmacological function of effective components in BmK venom seems to be scarce so far. Inflammation is often associated with either tissue damage or nerve injury. Common signs of inflammation include redness,
MATERIALS AND METHODS Male Sprague–Dawley rats (220 –250 g) were from Shanghai experimental animals center, and kept at 21–23°C on a 12-h light/dark cycle with free food and water. Each animal was tested only once. Crude venom of scorpion BmK, collected by electric stimulation, was purchased from an individual scorpion culture farm in Suzhou, Jiangsu Province, China. BmK IT2 were purified according to the methods described by Ji et al. [12]. The purity of BmK IT2 peptide used in the study was assessed to be a single peak on mass spectrum. The paw withdrawal latency (PWL) to radiant heat was exam-
* Address for correspondence: Dr. Yong-Hua Ji, Shanghai Institute of Physiology, CAS, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China. Fax: ⫹86-21-6433-2445; E-mail: [email protected]
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ined for heat hyperalgesia of animals using previously described method [11]. Rats were placed individually in clear plastic chambers on a glass plate, the temperature of chamber was maintained at 30°C. Animals were allowed to adapt for 30 min before the experiment. Radiant heat was applied to the plantar surface of the hindpaw until the rat lifted its paw. The time from the onset of heat application to paw lifting was considered to be the PWL. Both hindpaws were tested independently with a 5-min interval between trials. The examination was performed to be the nearest 0.1 s of PWL. To prevent tissue damage, the cut-off latency was set at 20.5 s. Five baselines of PWL were made before the induction of unilateral hindpaw inflammation. Under light sevoflurane anaesthesia, 2 mg carrageenan (dissolved in 0.1 ml saline) was injected subcutaneously into the plantar surface of either hindpaw (random choice between left and right paw). The rats were recovered in 1 or 2 min after anaesthesia. BmK IT2 was injected intraplantarly at 3 h post-carrageenan. This animal model was approved by animal care committee [23]. Guidelines on ethical standards for investigation of experimental pain conscious animals were performed strictly according to Zimmermann [34]. Three groups of rats were employed in the study, first group was inflammatory rats induced with carrageenan (n ⫽ 30), the second group was normal rats (n ⫽ 30), and the third group was control which were injected with saline instead of BmK IT2 (n ⫽ 20). Ten microliters of BmK IT2 dissolved in saline was injected into plantar of hindpaw at three concentrations 0.1, 0.01 and 0.001 mg/ml. Drug effects were denoted as percentage of the control responses before administration of BmK IT2 at the time of maximal responses. Data were presented as mean ⫾ SEM. Statistical comparison was made by Student t-test for grouped or paired data. p ⬍ 0.05 was considered to be significant. RESULTS The rat hindpaw was noticeably inflamed to show redness and swelling at 3 h after the carrageenan was injected. A significant reduction in the response of PWL to heat source evidenced the development of cutaneous hyperalgesia, which could last for the entire 4-h test period. Unilateral carrageenan injection was found to be no effect on PWL for contralateral paw. The repeated testing on both hindpaws was proved no significant change in PWL throughout the test likewise. The PWL was deceased from the baseline of 9.8 ⫾ 0.3 s to 3.6⫾0.2 s in carrageenan treated hindpaw (p ⬍ 0.01), while contralateral PWL was about 9.6 ⫾ 0.4 s (P ⬎ 0.05). A total of 30 rats, which were treated with carrageenan after 3 h, were employed for BmK IT2 antihyperalgesia. PWL was determined to be 3.6 ⫾ 0.2 s before injection of BmK IT2. The PWL was increased after 10 l of BmK IT2 (0.001 mg/ml) was intraplantarly applied at 5 min. The peak of prolongation appeared at 10 –15 min in the most cases. These responses in rats seemed to be gradually recovered within 30 min (Fig. 1). Fifteen minutes after injection of BmK IT2 (10 l) at concentrations of 0.1, 0.01 and 0.001 mg/ml, the PWL was increased to 272 ⫾ 18%, 217 ⫾ 19% and 186 ⫾ 16% of the control, respectively, indicating a dose-dependent effect (p ⬍ 0.05, groups compare) (Fig. 3). Meanwhile, PWL of contralateral hindpaw was not affected by intraplanter injection of BmK IT2. BmK IT2-induced antinociception was investigated in 30 normal rats. The results showed that PWL was rapidly increased by intraplantar injection of BmK IT2 (Fig. 2). PWL was determined to be 10.1 ⫾ 0.4 s at the absence of BmK IT2, which were increased to 177 ⫾ 16%, 141 ⫾ 15% and 133 ⫾ 15% of the
FIG. 1. Time course for intraplantar injection of Buthus martensi Karsch (BmK) IT2 in inflammatory rats. Significant difference: **p ⬍ 0.01, *p ⬍ 0.05, Student t-test (Drug vs. control). Vertical bars: ⫾ SEM. 1 con (inj): The starting point of administration of BmK IT2.
control after BmK IT2 was applied at the concentrations of 0.1, 0.01 and 0.001 mg/ml, respectively determined at 15 min (Fig. 3). In addition, it was found that the contralateral hindpaw PWL was not affected by intraplantar injection of BmK IT2 likewise. In control group, 10 l of saline was injected into the hindpaw in both normal and carrageenan-treated animals. The results showed that saline could not prolong detectable PWL in rats as shown in Figs. 1 and 2. DISCUSSION Progresses in understanding the channelopathy have proved that the mutation of voltage-gated sodium channels may cause many neuro-muscle and cardiac diseases [5]. Voltage-gated sodium channels were also demonstrated to be the target of various natural toxins [21]. Therefore, these natural toxins were severed as powerful probes for clarifying and correlating structure and function of ion channels. For example, the receptor binding sites 3 and 4 on voltage-gated sodium channels have been solidly investigated using specific ligands, either ␣- or -scorpion toxins [7,8]. Two main types of sodium currents, termed tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R), have been identified in the dorsal root ganglion (DRG) neurons [3,19,24]. The fast-inactivating TTX-S sodium currents were found in all types of DRG neurons, which may be mediated by one or more of several ␣-subunits of sodium channel expressed in these neurons [1,3,26]. In contrast, the more slowly inactivating TTX-R sodium currents were preferentially expressed in a subpopulation of nociceptors [19,24]. At least six sodium channel transcription, such as SNS/PN3 and NaN3, have been well demonstrated in DRG neurons [1,4,24,25,27].
ANTIHYPERALGESIA EFFECT OF BMK IT2
FIG. 2. Time course for intraplantar injection of Buthus martensi Karsch (BmK) IT2 in the normal rats. Significant difference: *p ⬍ 0.05, Student t-test (Drug vs. control). Vertical bars: ⫾ SEM. 1con (inj): The starting point of administration of BmK IT2.
In the present study, a significant effect of BmK IT2 with intraplantar injection to PWL has been determined not only in inflammatory hindpaw, but also in normal hindpaw with dosedependent manner. The PWL prolongation of contralateral hindpaw was failed by the injection of BmK IT2, strongly suggesting that BmK IT2 might block sodium channels in peripheral nociceptor rather than that in the spinal cord. The dose-dependent effect of BmK IT2 was in favor of a guess that BmK IT2 might act specifically on the corresponding peripheral sodium channel at or around the site of injection. The results indicate that BmK IT2 may underlie the peripheral analgesia. However, the analgesia mechanism of BmK IT2 seems to be still remained a question. Although the target of BmK IT2 has been demonstrated to be voltage-gated sodium channels, which are currently considered to play important role in pain procession, it could not be excluded that the effect of BmK IT2 might involve other pathways, such as depression of other ion channels on nociceptors or sensory nerves, or that of inflammatory mediators release and synthesis at the site of injury. The effect of BmK IT2 to prolong PWL in carrageenan-treated rats seemed to be more potent than that in normal rats (Fig. 3). As for it, the several possibilities could be considered following the events: (a) inflammatory mediators can decrease the activation threshold, increase the rates of activation and inactivation, as well the magnitude of TTX-R sodium currents [6], which resulted in the decrease in threshold and the increase in number of action potentials evoked from sensitized neurons. The time of TTX-R sodium currents was mirrored with the time course of the development of hyperalgesia in response to a peripheral injection of directly acting inflammatory mediators [29]; (b) peripheral inflammation induced by carrageenan injection into rat hindpaw was associated with up-regulation of SNS sodium channel mRNA and the level of
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FIG. 3. Dose-response relation of Buthus martensi Karsch (BmK) IT2 on paw withdrawal latency. BmK IT2 were intraplantarly injected into a hindpaw in the normal and carrageenan-treated rats with the concentration of 0.1, 0.01 and 0.001 mg/ml, respectively. Drug vs. saline: *p ⬍ 0.05, Student t-test. Vertical bars: ⫾ SEM.
TTX-R sodium current in small DRG neurons [30]. In addition, peripheral inflammation induced by the complete Freund’s adjuvant could increase the expression of NaN3/SNS-2 mRNA and sodium channel immunoreactivity in DRG neurons [9,10,31]. The application of BmK IT2 may thus sensitively depress unusual TTX-R sodium currents expressed in inflammatory rats; (c) the peripheral inflammation may entail a deficiency of the perineurial barrier and an enhancement of endoneurial capillaries permeability [20,22], which might lead to easy access of BmK IT2 to nociceptors. The results obtained in the present study provided considerable evidence on an important role of BmK IT2 in antihyperalgesia. Finally, voltage-gated sodium channels in sensory neurons has been extensively demonstrated and protein kinases modulated on voltage-gated sodium channels of presynaptic brain nerve endings [28], which may prove an attractive targets for the pursuit of new analgesic or antihyperalgesic agents. The venom of Chinese scorpion BmK is an abundant resource of bioactive peptides active on voltage-gated sodium channels. In this study, intraplantar injection of BmK IT2 created peripheral antinociception in normal rats and peripheral antihyperalgesia in inflammatory rats, implying potentiality of such sodium channel-specific ligands as BmK IT2 and homologous depressant insect-selective scorpion toxins in treating pain disorders. ACKNOWLEDGEMENTS
This study was supported by the National Nature Science Foundation of China (39625010), National Basic Research Program of China
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(1999054001), partially by Shanghai-Unilever Research and Development Fund (9803) and Chinese Academy of Sciences (Stz98306). The authors are grateful to Mr. Zhang Jian-Wei for his technical assistance.
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Antihyperalgesia effect of BmK IT2, a depressant insect-selective scorpion toxin in rat by peripheral administration Cong-Ying Wang, Zhi-Yong Tan, Bing Chen, Zhi-Qi Zhao and Yong-Hua Ji* Shanghai Institute of Physiology, Chinese Academy of Sciences, Shanghai, People’s Republic of China [Received 6 April 2000; Revised 19 July 2000; Accepted 19 July 2000] ABSTRACT: The study was undertaken to assess the antihyperalgesia effect of BmK IT2, a sodium channel-specific ligand purified from the venom of Chinese scorpion Buthus martensi Karsch in rat by peripheral injection. The peripheral inflammation of rat was induced by carrageenan resulted in hyperalgesia to heat stimulus. The heat hyperalgesia was measured by paw withdrawal latency (PWL). PWL was increased to 272 ⴞ 18%, 217 ⴞ 19% and 186 ⴞ 16% of the control by application of 10 l BmK IT2 at the concentration of 0.1, 0.01 and 0.001 mg/ml in inflammatory rats, respectively. In contrast, PWL was enhanced to about 177 ⴞ 16%, 141 ⴞ 15% and 133 ⴞ 15% of the control at the same applied concentrations of BmK IT2 in normal rats. The results thus suggest that BmK IT2 can produce peripheral antihyperalgesia and antinociception, which might be attributed a pathway of modulating the sodium channels on nociceptor. © 2000 Elsevier Science Inc.
swelling and primary hyperalgesia. Primary hyperalgesia is characterized by a lowing of response threshold and/or an increased response to suprathreshold stimulus at the site of injury. Meanwhile, primary hyperalgesia can result in sensitization of primary nociceptors, or an increase in the excitability of peripheral nociceptors to mechanical and thermal stimulus at inflammatory site. Sensitization depends on the release, synthesis or attraction inflammatory mediators to the site of injury, ordinarily. BmK IT2 is a component classified into the group of depressant insect-selective toxin [12,15,16], which has been tested in vivo to be non-toxic to the mammal [12]. Although the binding characteristics of a few depressant-insect scorpion toxins have been investigated on insect neuronal membranes [2,32], their possible pharmacological properties, especially in mammal seem to be still sparse. For example, it is worth noticing that BmK AEP, a component from the same venom, which displays a sequence highly homologous with that of BmK IT2, has been demonstrated to be a strong anti-epileptic peptide by either intravenous or ventricular injection [33]. In addition, our latest study revealed that BmK IT2 could significantly inhibit the specific binding of BmK AS to sodium channels in the mammalian brain [18]. It is thus suggested that BmK IT2 type neurotoxic polypeptides may be a distinctive sodium channel-blocking ligand with multiple pharmacological functions whether in insect or mammal. In the present communication, we further examined the effect of BmK IT2 on inflammation induced hyperalgesia in rats, and discussed its possible mechanism.
KEY WORDS: Scorpion neurotoxin, BmK IT2, Paw withdrawal latency, Antihyperalgesia.
INTRODUCTION It has been demonstrated that most long-chain scorpion neurotoxins composed of 60-70 amino acids with four disulfide bridges are ligands of voltage-gated sodium channels in mammals, insects and other vertebrates [7,8]. According to biological specificity in vivo and pharmacological and electrophysiological activity, these scorpion neurotoxins can be divided into ␣- or -mammal neurotoxins and excitatory or depressant insect-selective neurotoxins [7,8]. The Asian scorpion Buthus martensi Karsch (BmK) is a species widely distributed in northwestern China, Mongolia and Korea. In Chinese traditional medicine, even at the present time, the bodies of scorpion BmK are used as a rare drug to cure various complaints, especially neurological symptoms based on the herbalism ‘combat poison with poison’. Although several kinds of sodium channel-specific ligands have been purified and characterized from BmK venom [12–17], the modern knowledge on the pharmacological function of effective components in BmK venom seems to be scarce so far. Inflammation is often associated with either tissue damage or nerve injury. Common signs of inflammation include redness,
MATERIALS AND METHODS Male Sprague–Dawley rats (220 –250 g) were from Shanghai experimental animals center, and kept at 21–23°C on a 12-h light/dark cycle with free food and water. Each animal was tested only once. Crude venom of scorpion BmK, collected by electric stimulation, was purchased from an individual scorpion culture farm in Suzhou, Jiangsu Province, China. BmK IT2 were purified according to the methods described by Ji et al. [12]. The purity of BmK IT2 peptide used in the study was assessed to be a single peak on mass spectrum. The paw withdrawal latency (PWL) to radiant heat was exam-
* Address for correspondence: Dr. Yong-Hua Ji, Shanghai Institute of Physiology, CAS, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China. Fax: ⫹86-21-6433-2445; E-mail: [email protected]
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ined for heat hyperalgesia of animals using previously described method [11]. Rats were placed individually in clear plastic chambers on a glass plate, the temperature of chamber was maintained at 30°C. Animals were allowed to adapt for 30 min before the experiment. Radiant heat was applied to the plantar surface of the hindpaw until the rat lifted its paw. The time from the onset of heat application to paw lifting was considered to be the PWL. Both hindpaws were tested independently with a 5-min interval between trials. The examination was performed to be the nearest 0.1 s of PWL. To prevent tissue damage, the cut-off latency was set at 20.5 s. Five baselines of PWL were made before the induction of unilateral hindpaw inflammation. Under light sevoflurane anaesthesia, 2 mg carrageenan (dissolved in 0.1 ml saline) was injected subcutaneously into the plantar surface of either hindpaw (random choice between left and right paw). The rats were recovered in 1 or 2 min after anaesthesia. BmK IT2 was injected intraplantarly at 3 h post-carrageenan. This animal model was approved by animal care committee [23]. Guidelines on ethical standards for investigation of experimental pain conscious animals were performed strictly according to Zimmermann [34]. Three groups of rats were employed in the study, first group was inflammatory rats induced with carrageenan (n ⫽ 30), the second group was normal rats (n ⫽ 30), and the third group was control which were injected with saline instead of BmK IT2 (n ⫽ 20). Ten microliters of BmK IT2 dissolved in saline was injected into plantar of hindpaw at three concentrations 0.1, 0.01 and 0.001 mg/ml. Drug effects were denoted as percentage of the control responses before administration of BmK IT2 at the time of maximal responses. Data were presented as mean ⫾ SEM. Statistical comparison was made by Student t-test for grouped or paired data. p ⬍ 0.05 was considered to be significant. RESULTS The rat hindpaw was noticeably inflamed to show redness and swelling at 3 h after the carrageenan was injected. A significant reduction in the response of PWL to heat source evidenced the development of cutaneous hyperalgesia, which could last for the entire 4-h test period. Unilateral carrageenan injection was found to be no effect on PWL for contralateral paw. The repeated testing on both hindpaws was proved no significant change in PWL throughout the test likewise. The PWL was deceased from the baseline of 9.8 ⫾ 0.3 s to 3.6⫾0.2 s in carrageenan treated hindpaw (p ⬍ 0.01), while contralateral PWL was about 9.6 ⫾ 0.4 s (P ⬎ 0.05). A total of 30 rats, which were treated with carrageenan after 3 h, were employed for BmK IT2 antihyperalgesia. PWL was determined to be 3.6 ⫾ 0.2 s before injection of BmK IT2. The PWL was increased after 10 l of BmK IT2 (0.001 mg/ml) was intraplantarly applied at 5 min. The peak of prolongation appeared at 10 –15 min in the most cases. These responses in rats seemed to be gradually recovered within 30 min (Fig. 1). Fifteen minutes after injection of BmK IT2 (10 l) at concentrations of 0.1, 0.01 and 0.001 mg/ml, the PWL was increased to 272 ⫾ 18%, 217 ⫾ 19% and 186 ⫾ 16% of the control, respectively, indicating a dose-dependent effect (p ⬍ 0.05, groups compare) (Fig. 3). Meanwhile, PWL of contralateral hindpaw was not affected by intraplanter injection of BmK IT2. BmK IT2-induced antinociception was investigated in 30 normal rats. The results showed that PWL was rapidly increased by intraplantar injection of BmK IT2 (Fig. 2). PWL was determined to be 10.1 ⫾ 0.4 s at the absence of BmK IT2, which were increased to 177 ⫾ 16%, 141 ⫾ 15% and 133 ⫾ 15% of the
FIG. 1. Time course for intraplantar injection of Buthus martensi Karsch (BmK) IT2 in inflammatory rats. Significant difference: **p ⬍ 0.01, *p ⬍ 0.05, Student t-test (Drug vs. control). Vertical bars: ⫾ SEM. 1 con (inj): The starting point of administration of BmK IT2.
control after BmK IT2 was applied at the concentrations of 0.1, 0.01 and 0.001 mg/ml, respectively determined at 15 min (Fig. 3). In addition, it was found that the contralateral hindpaw PWL was not affected by intraplantar injection of BmK IT2 likewise. In control group, 10 l of saline was injected into the hindpaw in both normal and carrageenan-treated animals. The results showed that saline could not prolong detectable PWL in rats as shown in Figs. 1 and 2. DISCUSSION Progresses in understanding the channelopathy have proved that the mutation of voltage-gated sodium channels may cause many neuro-muscle and cardiac diseases [5]. Voltage-gated sodium channels were also demonstrated to be the target of various natural toxins [21]. Therefore, these natural toxins were severed as powerful probes for clarifying and correlating structure and function of ion channels. For example, the receptor binding sites 3 and 4 on voltage-gated sodium channels have been solidly investigated using specific ligands, either ␣- or -scorpion toxins [7,8]. Two main types of sodium currents, termed tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R), have been identified in the dorsal root ganglion (DRG) neurons [3,19,24]. The fast-inactivating TTX-S sodium currents were found in all types of DRG neurons, which may be mediated by one or more of several ␣-subunits of sodium channel expressed in these neurons [1,3,26]. In contrast, the more slowly inactivating TTX-R sodium currents were preferentially expressed in a subpopulation of nociceptors [19,24]. At least six sodium channel transcription, such as SNS/PN3 and NaN3, have been well demonstrated in DRG neurons [1,4,24,25,27].
ANTIHYPERALGESIA EFFECT OF BMK IT2
FIG. 2. Time course for intraplantar injection of Buthus martensi Karsch (BmK) IT2 in the normal rats. Significant difference: *p ⬍ 0.05, Student t-test (Drug vs. control). Vertical bars: ⫾ SEM. 1con (inj): The starting point of administration of BmK IT2.
In the present study, a significant effect of BmK IT2 with intraplantar injection to PWL has been determined not only in inflammatory hindpaw, but also in normal hindpaw with dosedependent manner. The PWL prolongation of contralateral hindpaw was failed by the injection of BmK IT2, strongly suggesting that BmK IT2 might block sodium channels in peripheral nociceptor rather than that in the spinal cord. The dose-dependent effect of BmK IT2 was in favor of a guess that BmK IT2 might act specifically on the corresponding peripheral sodium channel at or around the site of injection. The results indicate that BmK IT2 may underlie the peripheral analgesia. However, the analgesia mechanism of BmK IT2 seems to be still remained a question. Although the target of BmK IT2 has been demonstrated to be voltage-gated sodium channels, which are currently considered to play important role in pain procession, it could not be excluded that the effect of BmK IT2 might involve other pathways, such as depression of other ion channels on nociceptors or sensory nerves, or that of inflammatory mediators release and synthesis at the site of injury. The effect of BmK IT2 to prolong PWL in carrageenan-treated rats seemed to be more potent than that in normal rats (Fig. 3). As for it, the several possibilities could be considered following the events: (a) inflammatory mediators can decrease the activation threshold, increase the rates of activation and inactivation, as well the magnitude of TTX-R sodium currents [6], which resulted in the decrease in threshold and the increase in number of action potentials evoked from sensitized neurons. The time of TTX-R sodium currents was mirrored with the time course of the development of hyperalgesia in response to a peripheral injection of directly acting inflammatory mediators [29]; (b) peripheral inflammation induced by carrageenan injection into rat hindpaw was associated with up-regulation of SNS sodium channel mRNA and the level of
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FIG. 3. Dose-response relation of Buthus martensi Karsch (BmK) IT2 on paw withdrawal latency. BmK IT2 were intraplantarly injected into a hindpaw in the normal and carrageenan-treated rats with the concentration of 0.1, 0.01 and 0.001 mg/ml, respectively. Drug vs. saline: *p ⬍ 0.05, Student t-test. Vertical bars: ⫾ SEM.
TTX-R sodium current in small DRG neurons [30]. In addition, peripheral inflammation induced by the complete Freund’s adjuvant could increase the expression of NaN3/SNS-2 mRNA and sodium channel immunoreactivity in DRG neurons [9,10,31]. The application of BmK IT2 may thus sensitively depress unusual TTX-R sodium currents expressed in inflammatory rats; (c) the peripheral inflammation may entail a deficiency of the perineurial barrier and an enhancement of endoneurial capillaries permeability [20,22], which might lead to easy access of BmK IT2 to nociceptors. The results obtained in the present study provided considerable evidence on an important role of BmK IT2 in antihyperalgesia. Finally, voltage-gated sodium channels in sensory neurons has been extensively demonstrated and protein kinases modulated on voltage-gated sodium channels of presynaptic brain nerve endings [28], which may prove an attractive targets for the pursuit of new analgesic or antihyperalgesic agents. The venom of Chinese scorpion BmK is an abundant resource of bioactive peptides active on voltage-gated sodium channels. In this study, intraplantar injection of BmK IT2 created peripheral antinociception in normal rats and peripheral antihyperalgesia in inflammatory rats, implying potentiality of such sodium channel-specific ligands as BmK IT2 and homologous depressant insect-selective scorpion toxins in treating pain disorders. ACKNOWLEDGEMENTS
This study was supported by the National Nature Science Foundation of China (39625010), National Basic Research Program of China
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(1999054001), partially by Shanghai-Unilever Research and Development Fund (9803) and Chinese Academy of Sciences (Stz98306). The authors are grateful to Mr. Zhang Jian-Wei for his technical assistance.
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