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No tolerance to the antinociceptive action of calcitonin in rats and mice
Neuroscience Letters 359 (2004) 5–8 www.elsevier.com/locate/neulet
No tolerance to the antinociceptive action of calcitonin in rats and mice Rachida Aboufatimaa, Abderrahman Chaita,*, Abderrahim Dalala, Abdelmajid Zyada, Renaud de Beaurepaireb a
De´partement de Biologie, Faculte´ des Sciences Semlalia BP: 2390, 40 000 Marrakech, Morocco b Laboratoire de Psychopharmacologie, Hoˆpital Paul-Guiraud, 94806, Villejuif, France
Received 21 July 2003; received in revised form 5 September 2003; accepted 9 September 2003
Abstract The involvement of a central opioid mechanism in the antinociceptive effect of calcitonin is still a matter of controversy. Since a major characteristic of the effects of opioids is tolerance to repeated treatments, we investigated the effects of acute and chronic (over 7 days) calcitonin injections on pain sensitivity in rats and mice. We examine the effect of single and repeated intraventricular (0.15 UI) and intraperitoneal (2.5, 5 and 20 IU/kg) injection of salmon calcitonin using respectively a tail flick test in rats and the writhing test in mice. The results showed that repeated injection of calcitonin produces a stronger antinociceptive effect than the single injection effect. The antalgic effect, evaluated in the writhing test, is dose-dependent. In both tail-immersion and writhing tests, repeated administration of calcitonin produced a long-lasting antinociceptive effect. These data suggest therefore that the tolerance does not develop after repeated treatment with calcitonin in both rat and mouse. These results support the hypothesis that an important component of the antinociceptive effects of calcitonin is not mediated by an opioid mechanism. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Pain; Tail-immersion test; Writhing test; Rats; Mice
Calcitonin (CT) is a polypeptide hormone secreted by the C cells of the thyroid gland. CT is primarily involved in the regulation of calcium homeostasis. The use of this hormone in osteoarticular disorders, principally in Paget’s disease, was accompanied by an expected analgesic effect [2]. Since then, the analgesic effect of CT has been widely demonstrated in laboratory animals and in humans [3,5]. When injected centrally, CT reduces pain sensation in the rat [19] and inhibits spinal nociceptive transmission in the cat [17]. Recently, we have shown that CT impairs conditioning behavior when injected into the cerebral ventricle and impairs contextual fear conditioning when injected into the periaqueductal gray of the rat [1]. It is generally believed that the antinociceptive effects of CT are mediated by the brain because central injections of CT are more affective than peripheral injections [8], and that very low doses of CT injected into selective brain sites have antinociceptive effects [16]. The mechanism by which CT has antinociceptive effects is not known. Some authors have * Corresponding author. Tel.: þ 212-44-43-46-49; fax: þ 212-44-43-7412. E-mail address: [email protected] (A. Chait).
proposed that CT has an antinociceptive effect through an opioid mechanism [16]. Opiates may be involved in CTinduced analgesia because CT administration increases endogenous opiate release. Martin et al. [16] have shown that naloxone can reverse the effects of CT when the writhing test is used. Recently, Goicoechea et al. [11] have shown that the antinociceptive effects of opiates were enhanced by CT pretreatment. However, a major characteristic of opiate analgesia is that tolerance develops after repeated opiate administration, and some authors have reported that no tolerance develops during repeated CT treatments [6]. Moreover, Braga et al. [5] found that CTinduced analgesia is not altered by naloxone. We, therefore, designed a study to specifically investigate whether tolerance develops during repeated CT administration. We studied the antinociceptive effects of single and of repeated CT administration in both rats and mice. Male Sprague– Dawley rats weighing 250– 300 g and male mice (inbred University strain) weighing 25 – 50 g were used for the experiments. All animals were kept in a room maintained on a 12 h light/dark cycle and temperature controlled (25 8C). The animals had free access to food and
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.022
6
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
water throughout all the experiments. Two pain sensitivity tests were used: a tail-immersion test in rats, and a writhing test in mice. CT was administered through intracerebroventricular (ICV) injections in rats and intraperitoneal (IP) injections in mice. In all the experiments, each animal was only used once. All groups of animals consisted of ten to 12 subjects. Injections and experiments were always started the same hour of the day, at 10 a.m. Repeated injections consisted of a unique injection repeated every day, because we have previously shown that a unique injection of CT (IP or ICV) is effective during 24 h [9]. The doses and rates of administration we used are in the range used by other authors [6]. For the tail-immersion test, the rats were anesthetized with ketamine (1.7 ml/kg) and implanted stereotaxically with a cannula that descended into the lateral ventricle and was fixed on the skull (coordinates: 1.3 mm posterior to the bregma, lateral 1.6 mm from midline, deep 3.2 mm from the dura). All animals were given 10 days to recover after surgery. During this period of time, the animals were handled daily. Synthetic salmon CT was dissolved in 1% gelatin in normal saline. On the day of the experiment, 43 ng of CT (0.15 UI), or saline, was injected into the lateral ventricle (volume of injection: 2.5 ml) through an injection cannula (0.15 mm inner diameter). In the tail-immersion test, the posterior half of the rat’s tail was dropped into a container filled with hot water maintained at a constant temperature (55 8C). The latency to tail withdrawal was recorded (using a chronometer) before the injection, and 30 min, 1 h, 2 h, 3 h, and 4 h after the injection. In Experiment 1 (single injection of CT), 11 groups of animals were used. One group had no treatment, five groups received saline, and five groups received CT. Each group of treated animals (saline or CT) was tested at a different period of time after the injection: 30 min, 1 h, 2 h, 3 h, and 4 h. In Experiment 2 (repeated injection of CT), the animals were injected daily for 6 or 7 days and were tested on day 7. Seventeen groups of animals were used. Two groups of animals had no treatment. Five groups of animals were treated with saline for the 7 days (control group). Five groups of animals were injected with saline or CT for 6 days, and on the day of the testing (day 7) they had no treatment. Five groups of animals were injected with saline or CT for the 7 days. On day 7, the animals were tested as in Experiment 1. Immediately after the sessions, the rats were anesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline followed by a 10% formalin solution. The brains were extracted, fixed in 10% formalin for 2 days, and cut at 80 mm. Localization of the cannula tips was determined according to the atlas of Paxinos and Watson. The writing test in mice was used as described by Hayashi and Takemori [13]. The mice are injected IP with an acetic acid solution to produce the typical reaction (a
wave of contraction of the abdominal musculature and extension of hindlimbs). The mice are then placed in a transparent plastic container, and the number of writhes is recorded during a 10 min period, starting 5 min after the acetic acid administration. Salmon CT or saline was injected IP (0.725, 1.45 and 5.8 mg/kg of CT (which respectively correspond to 2.5, 5 and 20 IU/kg) in 0.2 ml) 50 min before the test. In Experiment 1 (single injections of CT), four groups of animals were used. One group received saline injection, and the three other groups were injected respectively with 0.725, 1.45 and 5.8 mg/kg of CT. In Experiment 2 (repeated injections of CT), seven groups of mice were used. One group was treated with saline during the 7 days of the experiment. Three groups were treated with CT (one group for each dose) for 6 days, and with saline on day 7. Three groups of mice were treated with CT (one group for each dose) during the 7 days. The mean ^ SEM response was calculated, and comparisons between the experimental groups were made using Student’s t-test (***P , 0:001, **P , 0:01). For the tail-immersion test in Experiment 1, the latency to tail withdrawal was significantly increased (P , 0:001) after acute ICV injections of CT at all the testing time intervals (Fig. 1A). The antinociceptive effect was present until the third hour, and decreased thereafter. In Experiment 2, the latency to tail withdrawal was significantly increased (P , 0:001) on the 7th day after repeated ICV injections of CT (Fig. 1B). When CT was injected on the 7th day, the analgesia occurred at all the testing intervals; the antinociceptive effect was the most important the first hour after the injection, and then it decreased progressively, but remained highly significant at the fourth hour. When CT was injected for 6 days and saline on the 7th day, the tail withdrawal latency was also increased at all the time intervals, showing that the antinociceptive effect of CT was still present 24 h after the last injection (data not shown). For the writhing test in Experiment 1, the IP injection of CT produced a dose-dependent decrease in the number of stretches induced by the acid acetic administration: number of stretches in control mice: 57.6 ^ 1.35; number of stretches in treated mice 60 min after the injection: 51.4 ^ 0.90, 40.2 ^ 1.45 and 28.7 ^ 0.7, corresponding respectively to the 2.5, 5 and 20 IU/kg dosages (Fig. 2A). In Experiment 2, repeated IP injections of CT produced a dose-dependent decrease of the acetic acid-induced hyperalgesia. When CT was given on day 7, a significant and dose-dependent analgesia occurred for all the dosages (Fig. 2B). When saline was injected on the 7th day, the antinociceptive effect of CT was still present (Fig. 2C). It is well known that CT induces analgesia in pathologies of diverse etiology: osteoarticular disorders, Paget’s disease [2], phantom limb pain [14], or reflex sympathetic dystrophy [10] among others. It is often used when other treatments fail. It has also been previously demonstrated
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
Fig. 1. Effect of a single (A) and a repeated (B) intraventricular injection of salmon calcitonin (43 ng/rat) or saline on pain sensitivity measured by the latency (in seconds) to tail immersion into hot water. Each value represents the mean latencies (^SEM) for a testing group of ten rats. Significant differences between calcitonin and saline injected group: ***P , 0:001.
[18] that CT induces dose-dependent analgesia in animals. The mechanism underlying this effect is not completely known, but the involvement of the serotonergic system appears to be important because when the integrity of the serotonergic pathways or its function are disturbed, the analgesic effect is reduced or even disappears [7]. Furthermore, CT is able to increase the in vitro release of 5-HT in spinal medulla [4], although in vivo treatment did not modify the level of 5-HT. The results of the present study show that, in rats and mice, no tolerance to the antinociceptive effects of CT occurs after repeated administration. The results confirm that intracerebral administration of CT has an antinociceptive effect on the tail-immersion test in rats, and that peripheral injections of CT have a dose-dependent effect on the writhing test in mice [16]. The results also show that, when CT is administered repeatedly over 6 days, the antinociceptive effect of CT is still present 24 h after the last injection (in groups of animals treated for 6 days with CT, and with saline on the 7th day). These results indicate that
7
Fig. 2. Effects of single (A) and repeated (B,C) intraperitoneal injections of three doses of salmon calcitonin (2.5, 5, and 20 IU/kg), with calcitonin (B) and saline (C) injected on the testing day, in mice on the number of stretches. Ctrl, controls. Each value represents the mean values (^SEM) for a testing group of ten mice. Significant differences between calcitonin and saline injected group: ***P , 0:001.
CT has a long duration of action, which is in accordance with the report by Umeno et al. [20]. The mechanism of the antinociceptive action of CT is not well understood. CT activates raphe serotonergic neurons, and the antinociceptive effects of CT can be modulated by lesions of the serotonergic systems and by serotonergic antagonists. Such interactions with the serotonergic systems have led several authors to propose that the antinociceptive action of CT is related to its activating effect on serotonin [7]. However, the antinociceptive action of CT can also be modulated by noradrenergic agents [12,18]. In fact, such interactions of CT with different neurotransmitter systems do not necessarily mean that the modulation of nociceptive thresholds by CT is directly related to these interactions. These effects may simply be independent additive or subtractive actions on other pain regulation mechanisms. Several research works have demonstrated that CT and opiates have a certain number of specific interactions. CT increases the peripheral release of beta-endorphin [15] and enhances the opioid actions on k, d and m receptors in isolated peripheral tissues [16]. Central injections of CT also increase opiate-like material in the spinal cord. Braga et al. [5] have shown that the antinociceptive effects of CT
8
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
were not blocked by naloxone, but other authors have observed that naloxone partially blocks the effects of CT [16]. Therefore, it would seem that the antinociceptive effects of CT are related, closely or partially, to the ability of CT to interact with the opiate systems. However, tolerance is a major characteristic of repeated administration of opiates, and our results show that no tolerance develops after repeated administration of CT. Therefore, even though CT interacts with opioid systems, these interactions do not seem to be important in the antinociceptive effects of CT. In conclusion, we propose that CT exerts a primary antinociceptive effect that does not have an action through an opioid mechanism. However, CT and certain opioid systems may act in synergy to increase nociceptive thresholds.
[8]
[9]
[10]
[11]
[12]
[13]
References [14] [1] R. Aboufatima, A. Chait, A. Dalal, R. de Beaurepaire, Calcitonin microinjection into the periaqueductal gray impairs contextual fear conditioning in the rat, Neurosci. Lett. 275 (1999) 101–104. [2] O.L.M. Bijvoet, A.P. Hjansen, Thyrocalcitonin in Paget’s disease (letter), Lancet 2 (1967) 471–472. [3] J. Blanchard, E. Menk, S. Ramamurthy, Subarachnoid and epidural calcitonin in patients with pain due to metastatic cancer, J. Pain Symptom Manage. 5 (1990) 42 –45. [4] S.M. Bourgoin, A. Pohl, F. Mauborgne, M. Cesselin, M. Hamon, Direct stimulatory effect of calcitonin on (H)5-hydroxytryptamine release from the rat spinal cord, Eur. J. Pharmacol. 156 (1988) 13– 23. [5] P.C. Braga, Calcitonin and its antinociceptive activity: animal and human investigations 1975 – 1992, Agents Actions 41 (1994) 121–131. [6] G. Clementi, C. Valero, A. Prato, A. Caruso, F. Patti, S. Patane, F. Drago, Effects of calcitonin on morphine tolerance and withdrawal syndrome in morphine physically dependent rats, Eur. J. Pharmacol. 163 (1989) 175 –179. [7] M.I. Colado, M.J. Ormazabal, C. Goicoechea, F. Lopez, M.J. Alfaro, M.I. Martin, Involvement of central serotonergic pathway in analgesia
[15]
[16]
[17]
[18]
[19]
[20]
elicited by salmon calcitonin in the mouse, Eur. J. Pharmacol. 252 (1994) 291 –297. W.J. Freed, R. de Beaurepaire, Calcitonin as an anorectic agent: localization of the brain and to the hypothalamus, Psychopharmacol. Bull. 20 (1984) 456– 458. T. Fujita, Therapy of osteoporosis in Japan, in: C. Christiansen, K. Overgaard (Eds.), Osteoporosis, Osteopress, Copenhagen, 1990, pp. 2107–2112. C. Gobelet, M. Waldburger, J.L. Meier, The effect of adding calcitonin to physical treatment on reflex sympathetic dystrophy, Pain 48 (1992) 171–175. C. Goicoechea, M.J. Ormazabal, M.J. Alfaro, M.I. Martin, Effect of salmon-calcitonin on the analgesic effect of selective mu, delta and kappa opioid agonists in mice, Neurosci. Lett. 262 (1999) 25–28. F. Guidobono, C. Netti, F. Pagani, V. Sibilia, A. Pecile, A. Candeletti, S. Ferri, Relationship of analgesia induced by centrally injected calcitonin to CNS serotonergic system, Neuropeptides 8 (1986) 259 –271. G. Hayashi, A.E. Takermori, The type of analgesic-receptor interaction involved in certain analgesic assays, Eur. J. Pharmacol. 16 (1971) 63– 66. H. Jager, C. Maier, Calcitonin in phantom limb pain: a double blind study, Pain 48 (1992) 21 –27. L. Laurian, Z. Oberman, E. Graf, S. Gilad, E. Hoerer, R. Simantove, Calcitonin-induced increase in ACTH, b-endorphin and cortisol secretion, Horm. Metab. Res. 18 (1986) 268– 271. M.I. Martin, M.J. Alfaro, C. Goicoechea, M.I. Colado, In vitro study of the interaction of salmon calcitonin with m, d and k opioid agonists, Naunyn-Schmiedeberg’s Arch. Pharmacol. 347 (1993) 324– 328. C.R. Morton, B. Maisch, M. Zimmermann, Calcitonin: brainstem microinjection but not systemic administration inhibits spinal nociceptive transmission in the cat, Brain Res. 372 (1) (1986) 149 –154. M.J. Ormazabal, C. Goicoechea, E. Sanchez, M. Isabel, Salmon calcitonin potentiates the analgesia induced by antidepressants, Pharmacol. Biochem. Behav. 68 (2001) 125 –133. A. Pecile, S. Ferri, P.C. Braga, V.R. Olgiati, Effects of intracerebroventricular calcitonin in the conscious rabbit, Experientia 31 (1975) 332 –333. H. Umeno, T. Nagazawa, N. Ymazaki, Y. Kuraichi, Antinociceptive effects of repeated systemic injections of calcitonin in formalininduced hyperalgesic rats, Pharmacol. Behav. 55 (1996) 151–156.
No tolerance to the antinociceptive action of calcitonin in rats and mice Rachida Aboufatimaa, Abderrahman Chaita,*, Abderrahim Dalala, Abdelmajid Zyada, Renaud de Beaurepaireb a
De´partement de Biologie, Faculte´ des Sciences Semlalia BP: 2390, 40 000 Marrakech, Morocco b Laboratoire de Psychopharmacologie, Hoˆpital Paul-Guiraud, 94806, Villejuif, France
Received 21 July 2003; received in revised form 5 September 2003; accepted 9 September 2003
Abstract The involvement of a central opioid mechanism in the antinociceptive effect of calcitonin is still a matter of controversy. Since a major characteristic of the effects of opioids is tolerance to repeated treatments, we investigated the effects of acute and chronic (over 7 days) calcitonin injections on pain sensitivity in rats and mice. We examine the effect of single and repeated intraventricular (0.15 UI) and intraperitoneal (2.5, 5 and 20 IU/kg) injection of salmon calcitonin using respectively a tail flick test in rats and the writhing test in mice. The results showed that repeated injection of calcitonin produces a stronger antinociceptive effect than the single injection effect. The antalgic effect, evaluated in the writhing test, is dose-dependent. In both tail-immersion and writhing tests, repeated administration of calcitonin produced a long-lasting antinociceptive effect. These data suggest therefore that the tolerance does not develop after repeated treatment with calcitonin in both rat and mouse. These results support the hypothesis that an important component of the antinociceptive effects of calcitonin is not mediated by an opioid mechanism. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Pain; Tail-immersion test; Writhing test; Rats; Mice
Calcitonin (CT) is a polypeptide hormone secreted by the C cells of the thyroid gland. CT is primarily involved in the regulation of calcium homeostasis. The use of this hormone in osteoarticular disorders, principally in Paget’s disease, was accompanied by an expected analgesic effect [2]. Since then, the analgesic effect of CT has been widely demonstrated in laboratory animals and in humans [3,5]. When injected centrally, CT reduces pain sensation in the rat [19] and inhibits spinal nociceptive transmission in the cat [17]. Recently, we have shown that CT impairs conditioning behavior when injected into the cerebral ventricle and impairs contextual fear conditioning when injected into the periaqueductal gray of the rat [1]. It is generally believed that the antinociceptive effects of CT are mediated by the brain because central injections of CT are more affective than peripheral injections [8], and that very low doses of CT injected into selective brain sites have antinociceptive effects [16]. The mechanism by which CT has antinociceptive effects is not known. Some authors have * Corresponding author. Tel.: þ 212-44-43-46-49; fax: þ 212-44-43-7412. E-mail address: [email protected] (A. Chait).
proposed that CT has an antinociceptive effect through an opioid mechanism [16]. Opiates may be involved in CTinduced analgesia because CT administration increases endogenous opiate release. Martin et al. [16] have shown that naloxone can reverse the effects of CT when the writhing test is used. Recently, Goicoechea et al. [11] have shown that the antinociceptive effects of opiates were enhanced by CT pretreatment. However, a major characteristic of opiate analgesia is that tolerance develops after repeated opiate administration, and some authors have reported that no tolerance develops during repeated CT treatments [6]. Moreover, Braga et al. [5] found that CTinduced analgesia is not altered by naloxone. We, therefore, designed a study to specifically investigate whether tolerance develops during repeated CT administration. We studied the antinociceptive effects of single and of repeated CT administration in both rats and mice. Male Sprague– Dawley rats weighing 250– 300 g and male mice (inbred University strain) weighing 25 – 50 g were used for the experiments. All animals were kept in a room maintained on a 12 h light/dark cycle and temperature controlled (25 8C). The animals had free access to food and
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.022
6
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
water throughout all the experiments. Two pain sensitivity tests were used: a tail-immersion test in rats, and a writhing test in mice. CT was administered through intracerebroventricular (ICV) injections in rats and intraperitoneal (IP) injections in mice. In all the experiments, each animal was only used once. All groups of animals consisted of ten to 12 subjects. Injections and experiments were always started the same hour of the day, at 10 a.m. Repeated injections consisted of a unique injection repeated every day, because we have previously shown that a unique injection of CT (IP or ICV) is effective during 24 h [9]. The doses and rates of administration we used are in the range used by other authors [6]. For the tail-immersion test, the rats were anesthetized with ketamine (1.7 ml/kg) and implanted stereotaxically with a cannula that descended into the lateral ventricle and was fixed on the skull (coordinates: 1.3 mm posterior to the bregma, lateral 1.6 mm from midline, deep 3.2 mm from the dura). All animals were given 10 days to recover after surgery. During this period of time, the animals were handled daily. Synthetic salmon CT was dissolved in 1% gelatin in normal saline. On the day of the experiment, 43 ng of CT (0.15 UI), or saline, was injected into the lateral ventricle (volume of injection: 2.5 ml) through an injection cannula (0.15 mm inner diameter). In the tail-immersion test, the posterior half of the rat’s tail was dropped into a container filled with hot water maintained at a constant temperature (55 8C). The latency to tail withdrawal was recorded (using a chronometer) before the injection, and 30 min, 1 h, 2 h, 3 h, and 4 h after the injection. In Experiment 1 (single injection of CT), 11 groups of animals were used. One group had no treatment, five groups received saline, and five groups received CT. Each group of treated animals (saline or CT) was tested at a different period of time after the injection: 30 min, 1 h, 2 h, 3 h, and 4 h. In Experiment 2 (repeated injection of CT), the animals were injected daily for 6 or 7 days and were tested on day 7. Seventeen groups of animals were used. Two groups of animals had no treatment. Five groups of animals were treated with saline for the 7 days (control group). Five groups of animals were injected with saline or CT for 6 days, and on the day of the testing (day 7) they had no treatment. Five groups of animals were injected with saline or CT for the 7 days. On day 7, the animals were tested as in Experiment 1. Immediately after the sessions, the rats were anesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline followed by a 10% formalin solution. The brains were extracted, fixed in 10% formalin for 2 days, and cut at 80 mm. Localization of the cannula tips was determined according to the atlas of Paxinos and Watson. The writing test in mice was used as described by Hayashi and Takemori [13]. The mice are injected IP with an acetic acid solution to produce the typical reaction (a
wave of contraction of the abdominal musculature and extension of hindlimbs). The mice are then placed in a transparent plastic container, and the number of writhes is recorded during a 10 min period, starting 5 min after the acetic acid administration. Salmon CT or saline was injected IP (0.725, 1.45 and 5.8 mg/kg of CT (which respectively correspond to 2.5, 5 and 20 IU/kg) in 0.2 ml) 50 min before the test. In Experiment 1 (single injections of CT), four groups of animals were used. One group received saline injection, and the three other groups were injected respectively with 0.725, 1.45 and 5.8 mg/kg of CT. In Experiment 2 (repeated injections of CT), seven groups of mice were used. One group was treated with saline during the 7 days of the experiment. Three groups were treated with CT (one group for each dose) for 6 days, and with saline on day 7. Three groups of mice were treated with CT (one group for each dose) during the 7 days. The mean ^ SEM response was calculated, and comparisons between the experimental groups were made using Student’s t-test (***P , 0:001, **P , 0:01). For the tail-immersion test in Experiment 1, the latency to tail withdrawal was significantly increased (P , 0:001) after acute ICV injections of CT at all the testing time intervals (Fig. 1A). The antinociceptive effect was present until the third hour, and decreased thereafter. In Experiment 2, the latency to tail withdrawal was significantly increased (P , 0:001) on the 7th day after repeated ICV injections of CT (Fig. 1B). When CT was injected on the 7th day, the analgesia occurred at all the testing intervals; the antinociceptive effect was the most important the first hour after the injection, and then it decreased progressively, but remained highly significant at the fourth hour. When CT was injected for 6 days and saline on the 7th day, the tail withdrawal latency was also increased at all the time intervals, showing that the antinociceptive effect of CT was still present 24 h after the last injection (data not shown). For the writhing test in Experiment 1, the IP injection of CT produced a dose-dependent decrease in the number of stretches induced by the acid acetic administration: number of stretches in control mice: 57.6 ^ 1.35; number of stretches in treated mice 60 min after the injection: 51.4 ^ 0.90, 40.2 ^ 1.45 and 28.7 ^ 0.7, corresponding respectively to the 2.5, 5 and 20 IU/kg dosages (Fig. 2A). In Experiment 2, repeated IP injections of CT produced a dose-dependent decrease of the acetic acid-induced hyperalgesia. When CT was given on day 7, a significant and dose-dependent analgesia occurred for all the dosages (Fig. 2B). When saline was injected on the 7th day, the antinociceptive effect of CT was still present (Fig. 2C). It is well known that CT induces analgesia in pathologies of diverse etiology: osteoarticular disorders, Paget’s disease [2], phantom limb pain [14], or reflex sympathetic dystrophy [10] among others. It is often used when other treatments fail. It has also been previously demonstrated
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
Fig. 1. Effect of a single (A) and a repeated (B) intraventricular injection of salmon calcitonin (43 ng/rat) or saline on pain sensitivity measured by the latency (in seconds) to tail immersion into hot water. Each value represents the mean latencies (^SEM) for a testing group of ten rats. Significant differences between calcitonin and saline injected group: ***P , 0:001.
[18] that CT induces dose-dependent analgesia in animals. The mechanism underlying this effect is not completely known, but the involvement of the serotonergic system appears to be important because when the integrity of the serotonergic pathways or its function are disturbed, the analgesic effect is reduced or even disappears [7]. Furthermore, CT is able to increase the in vitro release of 5-HT in spinal medulla [4], although in vivo treatment did not modify the level of 5-HT. The results of the present study show that, in rats and mice, no tolerance to the antinociceptive effects of CT occurs after repeated administration. The results confirm that intracerebral administration of CT has an antinociceptive effect on the tail-immersion test in rats, and that peripheral injections of CT have a dose-dependent effect on the writhing test in mice [16]. The results also show that, when CT is administered repeatedly over 6 days, the antinociceptive effect of CT is still present 24 h after the last injection (in groups of animals treated for 6 days with CT, and with saline on the 7th day). These results indicate that
7
Fig. 2. Effects of single (A) and repeated (B,C) intraperitoneal injections of three doses of salmon calcitonin (2.5, 5, and 20 IU/kg), with calcitonin (B) and saline (C) injected on the testing day, in mice on the number of stretches. Ctrl, controls. Each value represents the mean values (^SEM) for a testing group of ten mice. Significant differences between calcitonin and saline injected group: ***P , 0:001.
CT has a long duration of action, which is in accordance with the report by Umeno et al. [20]. The mechanism of the antinociceptive action of CT is not well understood. CT activates raphe serotonergic neurons, and the antinociceptive effects of CT can be modulated by lesions of the serotonergic systems and by serotonergic antagonists. Such interactions with the serotonergic systems have led several authors to propose that the antinociceptive action of CT is related to its activating effect on serotonin [7]. However, the antinociceptive action of CT can also be modulated by noradrenergic agents [12,18]. In fact, such interactions of CT with different neurotransmitter systems do not necessarily mean that the modulation of nociceptive thresholds by CT is directly related to these interactions. These effects may simply be independent additive or subtractive actions on other pain regulation mechanisms. Several research works have demonstrated that CT and opiates have a certain number of specific interactions. CT increases the peripheral release of beta-endorphin [15] and enhances the opioid actions on k, d and m receptors in isolated peripheral tissues [16]. Central injections of CT also increase opiate-like material in the spinal cord. Braga et al. [5] have shown that the antinociceptive effects of CT
8
R. Aboufatima et al. / Neuroscience Letters 359 (2004) 5–8
were not blocked by naloxone, but other authors have observed that naloxone partially blocks the effects of CT [16]. Therefore, it would seem that the antinociceptive effects of CT are related, closely or partially, to the ability of CT to interact with the opiate systems. However, tolerance is a major characteristic of repeated administration of opiates, and our results show that no tolerance develops after repeated administration of CT. Therefore, even though CT interacts with opioid systems, these interactions do not seem to be important in the antinociceptive effects of CT. In conclusion, we propose that CT exerts a primary antinociceptive effect that does not have an action through an opioid mechanism. However, CT and certain opioid systems may act in synergy to increase nociceptive thresholds.
[8]
[9]
[10]
[11]
[12]
[13]
References [14] [1] R. Aboufatima, A. Chait, A. Dalal, R. de Beaurepaire, Calcitonin microinjection into the periaqueductal gray impairs contextual fear conditioning in the rat, Neurosci. Lett. 275 (1999) 101–104. [2] O.L.M. Bijvoet, A.P. Hjansen, Thyrocalcitonin in Paget’s disease (letter), Lancet 2 (1967) 471–472. [3] J. Blanchard, E. Menk, S. Ramamurthy, Subarachnoid and epidural calcitonin in patients with pain due to metastatic cancer, J. Pain Symptom Manage. 5 (1990) 42 –45. [4] S.M. Bourgoin, A. Pohl, F. Mauborgne, M. Cesselin, M. Hamon, Direct stimulatory effect of calcitonin on (H)5-hydroxytryptamine release from the rat spinal cord, Eur. J. Pharmacol. 156 (1988) 13– 23. [5] P.C. Braga, Calcitonin and its antinociceptive activity: animal and human investigations 1975 – 1992, Agents Actions 41 (1994) 121–131. [6] G. Clementi, C. Valero, A. Prato, A. Caruso, F. Patti, S. Patane, F. Drago, Effects of calcitonin on morphine tolerance and withdrawal syndrome in morphine physically dependent rats, Eur. J. Pharmacol. 163 (1989) 175 –179. [7] M.I. Colado, M.J. Ormazabal, C. Goicoechea, F. Lopez, M.J. Alfaro, M.I. Martin, Involvement of central serotonergic pathway in analgesia
[15]
[16]
[17]
[18]
[19]
[20]
elicited by salmon calcitonin in the mouse, Eur. J. Pharmacol. 252 (1994) 291 –297. W.J. Freed, R. de Beaurepaire, Calcitonin as an anorectic agent: localization of the brain and to the hypothalamus, Psychopharmacol. Bull. 20 (1984) 456– 458. T. Fujita, Therapy of osteoporosis in Japan, in: C. Christiansen, K. Overgaard (Eds.), Osteoporosis, Osteopress, Copenhagen, 1990, pp. 2107–2112. C. Gobelet, M. Waldburger, J.L. Meier, The effect of adding calcitonin to physical treatment on reflex sympathetic dystrophy, Pain 48 (1992) 171–175. C. Goicoechea, M.J. Ormazabal, M.J. Alfaro, M.I. Martin, Effect of salmon-calcitonin on the analgesic effect of selective mu, delta and kappa opioid agonists in mice, Neurosci. Lett. 262 (1999) 25–28. F. Guidobono, C. Netti, F. Pagani, V. Sibilia, A. Pecile, A. Candeletti, S. Ferri, Relationship of analgesia induced by centrally injected calcitonin to CNS serotonergic system, Neuropeptides 8 (1986) 259 –271. G. Hayashi, A.E. Takermori, The type of analgesic-receptor interaction involved in certain analgesic assays, Eur. J. Pharmacol. 16 (1971) 63– 66. H. Jager, C. Maier, Calcitonin in phantom limb pain: a double blind study, Pain 48 (1992) 21 –27. L. Laurian, Z. Oberman, E. Graf, S. Gilad, E. Hoerer, R. Simantove, Calcitonin-induced increase in ACTH, b-endorphin and cortisol secretion, Horm. Metab. Res. 18 (1986) 268– 271. M.I. Martin, M.J. Alfaro, C. Goicoechea, M.I. Colado, In vitro study of the interaction of salmon calcitonin with m, d and k opioid agonists, Naunyn-Schmiedeberg’s Arch. Pharmacol. 347 (1993) 324– 328. C.R. Morton, B. Maisch, M. Zimmermann, Calcitonin: brainstem microinjection but not systemic administration inhibits spinal nociceptive transmission in the cat, Brain Res. 372 (1) (1986) 149 –154. M.J. Ormazabal, C. Goicoechea, E. Sanchez, M. Isabel, Salmon calcitonin potentiates the analgesia induced by antidepressants, Pharmacol. Biochem. Behav. 68 (2001) 125 –133. A. Pecile, S. Ferri, P.C. Braga, V.R. Olgiati, Effects of intracerebroventricular calcitonin in the conscious rabbit, Experientia 31 (1975) 332 –333. H. Umeno, T. Nagazawa, N. Ymazaki, Y. Kuraichi, Antinociceptive effects of repeated systemic injections of calcitonin in formalininduced hyperalgesic rats, Pharmacol. Behav. 55 (1996) 151–156.