- Email: [email protected]
Stimulation of mu and delta opioid receptors induces hyperalgesia while stimulation of kappa receptors induces antinociception in the hot plate test in the naked mole-rat (Heterocephalus glaber)
Brain Research Bulletin 71 (2006) 60–68
Stimulation of mu and delta opioid receptors induces hyperalgesia while stimulation of kappa receptors induces antinociception in the hot plate test in the naked mole-rat (Heterocephalus glaber) Philemon Kipkemoi Towett a,∗ , Titus Ikusya Kanui a , Francis D. Juma b,1 a
Department of Veterinary Anatomy and Physiology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya b Department of Clinical Pharmacology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Received 27 January 2006; received in revised form 20 July 2006; accepted 1 August 2006 Available online 23 August 2006
Abstract The antinociceptive effects of highly selective mu (DAMGO), delta (DPDPE) and kappa (U-50488 and U-69593) opioid agonists were evaluated following intraperitoneal (i.p.) administration in the naked mole-rat. A hot plate test set at 60 ◦ C was used as a nociceptive test and the latency to the stamping of the right hind paw (response latency) was used as the end-point. DAMGO (5–10 mg/kg) and DPDPE (2.5–5 mg/kg) caused a naloxone-reversible significant decrease in the mean response latency. Subcutaneous injection of naloxonazine (20 mg/kg) 24 h prior to the administration of DAMGO (5 mg/kg) also blocked the reduction in the response latency observed when DAMGO was injected alone. On the contrary, U-50488 (2.5–5 mg/kg) or U-69593 (0.08 or 0.1 mg/kg) caused a naloxone-reversible significant increase in the mean response latency. These results showed that activation of mu or delta receptors caused hyperalgesia, whereas activation of kappa receptors caused antinociception in the hot plate test in naked mole-rat. This suggests that mu and delta receptors modulate thermal pain in a different way than kappa receptors in the naked mole-rat. It is not possible at the moment to point out how they modulate thermal pain as little is known about the neuropharmacology of the naked mole-rat. © 2006 Elsevier Inc. All rights reserved. Keywords: Antinociception; Hyperalgesia; Opioid systems; Hot plate test
1. Introduction Opioids have been and are still being used for the treatment of various types of pain in both humans and animals. The effects of opioids are mediated through opioid receptors that are widely distributed in the central nervous system. The role of opioid receptors in pain modulation has been investigated in a wide range of animal species and it appears that their mechanisms of actions differ in different types of pain and also in different animal species. Studies performed in the naked mole-rat suggested that the opioid systems of this subterranean rodent could be quite different from those of other rodents [21,53]. The authors reported
∗
Corresponding author. Tel.: +254 20 4448648. E-mail addresses: [email protected], [email protected] (P.K. Towett). 1 Tel.: +254 20 726300. 0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.08.001
that acute i.p. administration of morphine or pethidine caused a naloxone-reversible increase in pain sensitivity in the hot plate test set at 60 ◦ C in the naked mole-rat. This was referred to as a hyperalgesia. Hyperalgesia, tolerance and physical dependence usually occur following repeated opioid administration [24,26,31,55,57]. One of the causes of opioid tolerance is an increase in the break down of opioid peptides by aminopeptidases [25]. There is ample evidence indicating that hyperalgesia and other central hyperactive states are mediated by N-methyld-aspartate receptors in the NS [26,27,29,31]. Considering that the animals used in the published reports [21,53] had not been injected with opioids before, the data were very unique and interesting. Furthermore similar opioids have been reported to cause analgesia in other rodents such as the rat and mice in the hot plate test [1,19,56]. Studies performed in another subterranean rodent, the root-rat mole-rat (Tachyoryctes splendens), also showed that morphine caused hyperalgesia in the hot plate test set at 60 ◦ C [54]. These studies suggest that the nociceptive systems of mole-rats can be activated by exogenous
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
opioids. The exact role of opioid systems of mole-rats in thermal nociception is unclear and therefore further research is required to provide more information. Naked mole-rats have very unique physiology and anatomy. They are cold-blooded and are virtually blind. They have long incisors for digging, chewing and carrying objects and young. Naked mole-rats depend heavily on tactile senses to navigate their environment and this is facilitated by the presence of unique body vibrissae in rows along the trunk [12]. The two structures i.e. the incisors and the few hair in the skin, have large representation in the somatosensory cortex [6,12,17]. The skin of a naked mole-rat is highly folded and it lacks subcutaneous fat. Park et al. [36] have shown that the skin of a naked mole-rat has abundant A delta fibres but it lacks C-fibres immunoreactive for substance P and calcitonin gene-related peptides (CGRP). SP and CGRP are implicated in a number of functions such as nociception [34] and inflammation [8], vasodilation [5] and tissue maintenance [30]. The absence of SP and CGRP in the skin of the naked mole-rat raises questions on how this primitive rodent responds to injury and nociception. It is, however, reported that the naked mole-rat has several nonpeptidergic C-fibres in the skin and hair follicles [36]. A beta lanceolate endings containing SP and CGRP and lectinbinding C-fibres are also plenty in the skin and in the trigerminal and dorsal root ganglions of the naked mole-rat [36]. Such fibres have been implicated in nociception [43] and are perhaps very important in controlling the same in this primitive rodent. The opioids used in the two studies published [21,53] in the naked mole-rats act predominantly on mu receptors. To the best of our understanding, the roles of other opioid receptors (delta and kappa) on thermal nociception in the naked mole-rat is not known. The present study was aimed at providing data on the roles of the three classical opioid receptors on thermal nociception in the naked mole-rat. The data collected was hoped to provide solutions for the apparent peculiarities of the opioid systems of the naked mole-rat. To achieve this objective, receptor-selective mu (DAMGO), delta (DPDPE) and kappa (U50488 and U-69593) opioid agonists were used in the study. The selected drugs are receptor-selective and their analgesic effects in a wide range of nociceptive tests have been demonstrated in other rodents [10,11,13,39,48–51,59] but not in mole-rats. Naloxone and naloxonazine were used as antagonists in the present study. This was a more systematic study to be done in the naked mole-rat in our laboratory. The data obtained showed a clear physiological difference between kappa agonists and the other two opioid agonists (mu or delta) on thermal nociception in the naked mole-rat. 2. Materials and methods 2.1. Animals Adult male and female naked mole-rats (Heterocephalus glaber), weighing 20–40 g, were used in the experiments. They were trapped at Kathekani in Makueni District and transported by road to a laboratory in Chiromo Campus of the University of Nairobi, Kenya. The animals were kept in colonies of 50–100 in plastic lidded cages measuring 70 cm × 50 cm × 20 cm. Each cage was sub-
61
divided into two sub-compartments, with an open entrance joining them. The naked mole-rats used one sub-compartment as a toilet and the other as a bedroom. The bedding consisted of untreated wood shavings mixed with sand. The bedding was changed twice in a week. The cages were kept in a sound and a vibration proof room. Conditions almost similar to those in the wild were adopted in the room housing the mole-rats. The room temperature was maintained at between 28 and 31 ◦ C by infrared lamps (250 W) and/or electric heaters. Light was maintained at 0/24 h light/dark cycle except during the course of the experiments. In order to avoid problems of dry skin, the humidity in the room was kept at between 60% and 80%. In the wild, naked mole-rats feed on a varied diet consisting of roots, bulbs, and tubers. To maintain healthy colonies in the laboratory a varied diet consisting of fresh carrots, sweet potatoes, carrot tops and freshly cut grass was provided ad libitum. No water was provided since the naked mole-rats do not take it from either the bottles or the Petri dish. The animals can survive forever without water so long as they are fed on fresh food. The naked molerats were allowed at least one month to acclimate to the laboratory conditions before they were used for the experiments. The animals were also examined for fitness before being used for experiments. Touch reflex, locomotion and response to sounds or vibrations were tested prior to each experiment. The animals were randomly selected and each animal was used only once and the experiments were done blindly. International and national ethical guidelines regarding the use of conscious animals for pain research were adhered to during the course of the experiments. A committee of the Kenya Society for Protection and Care of Animals (KSPCA) approved the experimental procedures used in the study.
2.2. Drugs All the opioid agents used in the experiments were bought from Research Biochemicals International (RBI, Natick, USA). These were d-Ala2 -NMePhe4 Gly-ol5 -enkephalin (DAMGO), d-Pen2 -d-Pen5 -enkephalin (DPDPE), trans3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (U-50488), (5␣,7␣,8)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspirol[4,5]dec-8-yl]-benzeneacetamide (U-69593), naloxone hydrochloride, and naloxonazine hydrobromide. DAMGO, DPDPE, U-50488 and naloxone were dissolved in physiological saline (0.9% NaCl). U-69593 and naloxonazine were dissolved in dilute hydrochloric acid in saline. Fresh preparations of all drugs were always used and the volume injected was always 50 l. All the precautions regarding handling and stability of the drugs as recommended by the manufacturer were followed strictly.
2.3. Nociceptive and antinociceptive testing The nociceptive test used was a hot plate test (30 cm × 30 cm × 30 cm; iit Inc. Woodland Hills, CA, Model 35D analgesy-meter) set at 60 ◦ C. The surface temperature was continuously monitored with a digital thermometer. A naked mole-rat was gently placed on the hot plate and the latency to the stamping of the right hind paw (response latency) was recorded in seconds. The response latency was recorded 30 min after administration of vehicle or drug. To avoid thermal injury to the paws, an animal that failed to step its paws within 60 s was removed from the plate and assigned a value of 60 s. The reason for using the hot plate test was because this was a follow-up study of what is published [21,53]. The test was also adapted from a standard test used in rats and mice. Investigations using other nociceptive tests are being carried out in our laboratory. To investigate antinociceptive effects, DAMGO (0.5–10 mg/kg), DPDPE (1–5 mg/kg), U-50488 (1–10 mg/kg), and U-69593 (0.025–0.1 mg/kg) were administered intraperitoneally 30 min before nociceptive testing. The effects of naloxone and/or naloxonazine on the mean responses caused by the opioid drugs were also investigated. DAMGO (5 mg/kg), DPDPE (2.5 mg/kg), U-50488 (5 or 10 mg/kg) or U-69593 (0.08 or 0.1 mg/kg) was each administered in combination with naloxone (5 mg/kg). Naloxonazine (20 mg/kg) was administered subcutaneously 24 h prior to intraperitoneal administration of DAMGO (5 mg/kg). Naloxonazine is a slowly absorbed mu-1 receptor antagonist [28]. The dosages and the times for drug administration were based on pilot dose–response
62
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
experiments. Control animals for DAMGO and DPDPE were injected with physiological saline while those for U-50488 and U-69593 were injected with physiological saline plus dilute HCl.
2.4. Statistics The response latencies were analyzed using one-way analysis of variance (ANOVA) and least significant difference where applicable. The level of significance was set at 5% (p < 0.05). The results are presented as means ± S.E.M.
3. Results 3.1. Effects of DAMGO The hot plate baseline latencies (controls) for each of the experiment were determined by administering saline or a vehicle. The mean response latency for controls was 23.65 ± 0.4 s whereas for DAMGO 0.5, 1, 5 and 10 mg/kg they were 21.28 ± 1.2, 23.60 ± 0.3, 13.51 ± 0.5 and 12.53 ± 0.3 s, respectively. Intraperitoneal administration of DAMGO (0.5, 5 or 10 mg/kg) caused a statistically significant reduction in the mean response latency (p < 0.05, Fig. 1A). There was no statistical significant difference between controls and the animals injected with DAMGO (1 mg/kg: p > 0.5, Fig. 1A). When DAMGO (5 mg/kg) and naloxone (5 mg/kg) were administered simultaneously a response latency of 25.49 ± 0.4 s was recorded. When the mean response latency for the combined treatment (25.49 ± 0.4 s) was compared with that for DAMGO (5 mg/kg: 13.51 ± 0.5 s) alone, a statistically significant difference was recorded (p < 0.05; Fig. 1B). Naloxone (2 mg/kg) did not block the effect of DAMGO (5 mg/kg) (data not shown). Subcutaneous administration of naloxonazine (20 mg/kg) 24 h before DAMGO (5 mg/kg) was administered intraperitoneally caused a mean response latency of 31.28 ± 0.7 s. To find out whether naloxonazine blocked or not the effect of DAMGO on the response latency, the mean response latency for the combined treatment (31.28 ± 0.7 s) and that for DAMGO (5 mg/kg) alone (13.51 ± 0.5 s) were compared. A statistically significant difference was noted (p < 0.05; Fig. 1C). The number of animals in these experiments ranges from 8 to 10 in each group. 3.2. Effects of DPDPE The controls for DPDPE had a mean response latency of 24.11 ± 0.4 s. Intraperitoneal administration of DPDPE (1, 2.5 or 5 mg/kg) caused mean response latencies of 23.71 ± 0.9, 15.06 ± 0.3 or 12.47 ± 0.5 s, respectively. The mean response latency for DPDPE (1 mg/kg) was not statistically significant when it was compared with that for controls (p > 0.5; Fig. 2A). The mean response latency for DPDPE (2.5 or 5 mg/kg) was significant when compared with that for controls (p < 0.05; Fig. 2A). Simultaneous intraperitoneal administration of DPDPE (2.5 mg/kg) and naloxone (5 mg/kg) caused a response latency of 22.42 ± 0.5 s, which when statistically compared with that for DPDPE (2.5 mg/kg) alone (15.06 ± 0.3 s) was significant (p < 0.05; Fig. 2B). The number of animals in these experiments was 10 in each treatment group.
3.3. Effects of U-50488 and U-69593 The mean response latency for controls of U-50488 was 31.57 ± 0.7 s. Intraperitoneal administration of 1, 2.5, 5 or 10 mg/kg of U-50488 caused mean response latencies of 31.49 ± 0.6, 40.97 ± 0.9, 45.67 ± 1.8, or 60.0 ± 0.0 s, respectively. The mean response latencies for U-50488 (2.5, 5 or 10 mg/kg) were statistically significant (p < 0.05; Fig. 3A) when each one of them was compared with that for controls whereas that for U-50488 (1 mg/kg) was not (p > 0.05). Simultaneous administration of U-50488 (5 or 10 mg/kg) and naloxone (5 mg/kg) caused mean response latencies of 34.05 ± 0.6 or 35.82 ± 0.7 s, respectively. When each of the means of the combined treatment was compared with that for U-50488 (5 or 10 mg/kg) alone (45.67 ± 1.8 or 60 0 s) a statistically significant difference was noted (p < 0.05; Fig. 3B). In each treatment group n was 9–12. The response latency for the controls of U-69593 was 33.76 ± 1.6 s. Intraperitoneal administration of U-69593 (0.025, 0.08 or 0.1 mg/kg) caused mean response latencies of 33.94 ± 1.3, 41.30 ± 0.5, or 42.32 ± 1.2 s, respectively. There was no statistically significant difference between the mean response latency for controls and that for the group injected with 0.025 mg/kg of U-69593 (p > 0.05; Fig. 4A). On comparing the response latencies for U-69593 (0.08 or 0.1 mg/kg) with that for controls, a statistically significant difference was noted (p < 0.05; Fig. 4A). Simultaneous intraperitoneal administration of U-69593 (0.08 or 0.1 mg/kg) and naloxone (5 mg/kg) caused mean response latencies of 30.51 ± 0.7 or 34.17 ± 1 s, respectively. The response latency for each of the combined treatment was compared with that of U-69593 (0.08 or 0.1 mg/kg) alone and a statistically significant difference was recorded for these comparisons (p < 0.05; Fig. 4B). In each treatment group n was 9–10. 4. Discussion The hot plate test is a supraspinally mediated nociceptive test commonly used to study pain mechanisms. Naked mole-rats show clear quantifiable behaviour when tested on the hot plate set at 60 ◦ C [21,53]. In the current study, DAMGO (mu) or DPDPE (delta) caused a decrease in the response latency when they were injected intraperitoneally. In the hot plate test, antinociception is usually indicated by an increase in the response latency but not a decrease as recorded in the present study. A decrease in the response latency suggests that the nociceptive system of the animal is more sensitive to the noxious stimulation than before the injection of the drug. This phenomenon is referred to as hyperalgesia. The hyperalgesia caused by either DAMGO or DPDPE was blocked by naloxone, administered simultaneously with the agonist (DAMGO or DPDPE), suggesting the involvement of opioid receptors. Naloxonazine, a mu-1 antagonist, also blocked the effect of DAMGO, suggesting that mu-1 opioid receptors were involved in the decrease in the response latency observed following the administration of DAMGO. DAMGO or DPDPE, at doses that caused the recorded apparent hyperalgesia did not cause a change in the normal behaviour
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
63
Fig. 1. (A) Effects of i.p. administration of vehicle (0) or DAMGO (0.5, 1, 5 or 10 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 8–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 5 mg/kg of naloxone (Nal 5), 5 mg/kg of DAMGO (DAM 5), or a combination of 5 mg/kg of DAMGO and 5 mg/kg of naloxone (DAM 5/Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (C) Effects of i.p. administration of vehicle (0), a combination 20 mg/kg of naloxonazine and saline (N-zine 20/saline), 5 mg/kg of DAMGO (DAM 5), or 20 mg/kg of naloxonazine followed 24 h later by 5 mg/kg of DAMGO (DAM 5/N-zine 20) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
64
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
Fig. 2. (A) Effects of i.p. administration of vehicle (0) or DPDPE (1, 2.5 or 5 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). B. Effects of i.p. administration of vehicle (0), 5 mg/kg of naloxone (Nal 5), 2.5 mg/kg of DPDPE (DP 2.5), or a combination of 2.5 mg/kg of DPDPE and 5 mg/kg of naloxone (DP 2.5/Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
Fig. 3. (A) Effects of i.p. administration of vehicle (0) or U-50488 (1, 2.5, 5 or 10 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 10–12 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 5 or 10 mg/kg of U-50488 (U505 or U-5010) alone or each in combination with 5 mg/kg of naloxone (Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–12 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
of the naked mole-rat. This implied that the hyperkinesis and hyperexcitation, which had earlier been incriminated as the possible causes of morphine- or pethidine-induced apparent hyperalgesia [21,53], could not be responsible for the opioidinduced hyperalgesia in the naked mole-rat. In the same species of mole-rat, antinociception, in presence of hyperkinesis and
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
Fig. 4. (A) Effects of i.p. administration of vehicle (0) or U-69593 (0.025, 0.08 or 0.1 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 0.08 or 0.1 mg/kg of U-69593 (U-69.0.08 or U-69. 0.1) alone or each in combination with 5 mg/kg of naloxone (Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
65
hyperexcitation, has been reported after systemic administration of morphine [22] or codeine [23] in the formalin test. This suggests that thermal and chemical pains have different mechanisms and are modulated differently by the opioid systems of the naked mole-rat. In rats and cats, overactivation of opioid receptors has been observed to cause non-opioid receptor mediated hyperalgesia [60]. Overactivation of opioid receptors occurs when high doses are administered directly into the central nervous system. There are four differences between the report by Yaksh et al. [60] and the current data. Firstly, the hyperalgesia in the present study was naloxone-reversible, suggesting that it was opioid mediated. Secondly, the doses of DAMGO or DPDPE that caused hyperalgesia were not high because lower doses had no significant effect on nociception. Thirdly, the routes of administration are different in that in the present study drugs were injected intraperitoneally whereas in Yaksh’s report [60] drugs were injected spinally and fourthly the species of the animals used in the two studies are also different. The animals used in the study (naked mole-rats) have very unique physiology and anatomy. They are the only known coldblooded mammals. The skin of the naked mole-rat is rather unique in that it lacks C-fibres immunoreactive for substance P and calcitonin gene-related peptides (CGRP) [36]. SP and CGRP are implicated in a number of actions including nociception [34] and inflammation [8]. Because of the absence of SP and CGRP, the naked mole-rat has been described as painless animals. However, the current results and the published ones [21–23,53] clearly indicated that naked mole-rats have antinociceptive systems. These systems are perhaps SP-independent. It also appears from the same published reports that naked molerats have higher nociceptive thresholds. Hot plate temperatures below 60 ◦ C do not elicit significant reactions in the mole-rat and this may suggest that the animal is painless. A Similar observation was noted with the formalin test [23]. High nociceptive threshold in the naked mole-rat could be due to the lack of SP in the skin. The presence of abundant A␦ fibres, several nonpeptidergic C-fibres, A lanceolate endings containing SP and CGRP and many lectin-binding C-fibres [36] in the skin of the naked mole-rat may suggest that these fibres are crucial in nociception in this rodent. Mu opioids are potent, effective analgesics used for the treatment of acute and chronic pain syndromes. Delta agonists can also relieve pain but have not been used so much for this purpose. Experiments have shown that DAMGO and DPDPE have good antinociceptive effects in various nociceptive tests and in different animal species such as mice [32,44,48,49] and amphibians [46,47]. Analgesic sensitivity, especially to opioids, in animals can be influenced by a number of factors including genetics. For instance, inbred CXBK mouse-strain is deficient in mu opioid receptors and mu agonists such as morphine or DAMGO are not effective in producing antinociception after systemic or supraspinal administration [2,7,40,58]. Naked mole-rats live in large colonies with one breeding female served by many males. The possibility of inbreeding in the colony cannot be ruled out and this may contribute to the data observed in the previous and current studies. A naloxone-reversible hyperalgesia follow-
66
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
ing systemic administration of morphine and pethidine has also been observed in the root-rat mole-rat [54], where a possibility of inbreeding exists. Opiates can cause hyperthermic and other excitatory effects in animals [20]. Increased nociception is expected whenever there is an increase in skin temperature [52]. The naked molerat is a poikilothermic mammal and its skin lacks SP and CGRP which play a role in vasodilation and blood flow [5]. Effects of DAMGO and DPDPE on blood flow and body temperature in the naked mole-rat is not known at the moment. In rats, DAMGO, DPDPE and U-50488H can modify body temperature in different ways [45] and this may have an impact on antinociception. Perhaps mu and delta opioid agonists have an influence on the body temperature and blood flow in the naked mole-rat. Body temperature and blood flow are of great significance to a naked mole-rat, considering that it inhabits areas with relatively high (>30 ◦ C) ambient temperatures. In contrast to DAMGO and DPDPE, administration of U50488 or U-69593 caused an increase in the mean response latency and this was a clear antinociceptive effect. The antinociceptive effect of the two kappa agonists was blocked by naloxone. U-50488 and U-69593 are highly selective for kappa receptors and their antinociceptive actions have been demonstrated in a variety of assays, including thermal, pressure and irritant tests in mice and rats [18,38,59] and also in amphibians [46,47]. The data reported in the present study is in line with these reports. However, this data is the first to demonstrate the antinociceptive effects of kappa agonists in the hot plate test in the naked mole-rat. The mean response latencies for DAMGO and DPDPE control groups were insignificant but significantly different as compared to those of kappa opioids. Dilute HCl was added to physiological saline before it was injected to kappa control groups and this may have contributed to the difference observed. The antinociceptive doses of the two kappa agonists used were relatively lower than those used in mice and rats [59]. This may suggest the abundance of kappa receptors in the central nervous system of the naked mole-rat. In rats, high doses (>10 mg/kg) of U-50488 have been reported to cause no side effects [16]. In the naked mole-rat, doses of U-50488 greater than 5 mg/kg and U-69593 greater than 0.1 mg/kg appeared to be very high because they caused side effects. Although U-50488 and U-69593 are both kappa agonists, it appeared, on the basis of the dose range used, that U-69593 was more potent in causing antinociception than U-50488, and this could be due to differences in structure [4]. It is interesting that kappa agonists caused antinociception while mu or delta agonists caused hyperalgesia in the hot plate test. At the receptor level, mu and delta agonists increase potassium conductance, whereas kappa agonists directly inhibit the entry of calcium through voltage-dependent calcium channels [33,41]. The end result of these mechanisms will be inhibition of the release of neurotransmitter substance or release of inhibitory neurotransmitters. The onward transmission of nociceptive signal will be blocked. The heterogeneity of G-protein sub-units may influence the downstream effector mechanisms that follow the coupling of an opioid agonist to the G-protein, and this may explain the differences, in terms of functions, between kappa
and other opioid receptors [15]. Perhaps this is the case for the kappa and mu or delta agonists in the naked mole-rat. The difference between the effects of kappa agonists (U50488 and U-69593) and those of mu or delta agonists on thermal pain could probably also be due to differences in neurochemical pain pathways or their rates of metabolism. Opioids interact in various ways with central neurotransmitters to cause a wide variety of actions including antinociception [35]. It has been shown that the analgesic effects of U-50488 but not morphine could be blocked by pretreatment with the serotonin depletors [59], suggesting the importance of serotonergic pathways in kappainduced antinociception. Whether this is also true in the naked mole-rat is not known at the moment because no attempt has been made to investigate the relationship between the various neurotransmitter systems and thermal nociception in the naked mole-rat. Such a study would shed a lot of light on nociception and antinociception in the naked mole-rat. Opioid peptides are broken down in the body by pepdidases. Puromycin sensitive aminopeptidase and aminopeptidase N are two aminopeptidases that have high affinity for enkephalins and dynorphin [42]. Enzymatic break down of opioids in the body can produce neuroexcitatory metabolites such as morphine-3-glucuronide, which is very potent in causing hyperalgesia and myoclonus in patients treated by high doses of opioids [60]. DAMGO and DPDPE are enkephalin-related peptides whereas U-50488 and U-69593 are dynorphin-related peptides. Although human puromycin-sensitive aminopeptidase can metabolize both enkephalins and dynorphins [14], this may not be the case in the naked mole-rat. Perhaps, in the naked molerat, the activity of aminopeptidases for mu or delta opioids is so high that DAMGO and DPDPE are quickly metabolized into hyperalgesic products. Chronic administration of opiates results in tolerance and physical dependence [55], and also an increase in pain sensitivity [26,31]. Tolerance and physical dependence are as a result of decrease in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the nervous system [55]. An increase in the catabolism of opioid peptides in the brain may also cause tolerance and physical dependence [25]. Activation of N-methyl-d-aspartate receptors in the CNS is implicated in the opioid-induced hyperalgesia [61] because NMDA receptor antagonists can block it [3,27,29,37]. Mu-opioids activate NMDA receptors by reducing mg2+ block via protein kinase C activation and this leads to an increase in intracellular ca2+ concentrations, which subsequently enhances glutamate activity [9,31]. Glutamate is one of the excitatory amino acids that are involved in pain transmission in the spinal cord. The naked molerats used in the current experiments received only one injection of an opioid drug. The increase in pain sensitivity after DAMGO or DPDPE was therefore not expected. Our data therefore suggest that DAMGO and DPDPE are pronociceptive at least in the hot plate test set at 60 ◦ C in the naked mole-rat. The mechanisms underlying the observed hyperalgesia are not known at the moment. Perhaps NMDA receptors are involved. This needs to be investigated. Although the exact mechanism of action of the three opioids used in the current study cannot be presented here, the
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
data collected suggest the existence of the three classical opioid receptors in the naked mole-rat and their roles on thermal nociception. A lot of work is required to establish the main cause of the difference between kappa and mu or delta receptors in pain control. Perhaps the use of receptor-selective antagonists may clearly demonstrate why kappa agonists caused antinociception while mu or delta agonists caused an apparent hyperalgesia in the hot plate test in the naked mole-rat. Effects of receptor-selective opioids on pain induced by non-thermal stimuli also need to be investigated in the naked mole-rat. Acknowledgements We thank the German Academic Exchange Programme (DAAD) for supporting this study. We do also extend our gratitude to the technical staff in the department for their technical support. References [1] S.I. Ankier, New hot plate tests to quantify antinociceptive and narcotic antagonists activities, Eur. J. Pharmacol. 27 (1974) 1–4. [2] A. Baron, L. Shuster, B.E. Elefterhiou, D.W. Bailey, Opiate receptors in mice: genetic differences, Life Sci. 17 (1975) 633–640. [3] A.Y. Bespalov, E.E. Zvartau, P.M. Beardsley, Opioid-NMDA receptor interactions may clarify conditioned (associative) components of opioid analgesic tolerance, Neurosci. Biobehav. Rev. 25 (2001) 343–353. [4] S.J. Boyle, K.G. Meecham, J.C. Hunter, J. Hughes, [3H]-CI-977: a highly selective ligand for the k-opioid receptor in both guinea-pig and rat forebrain, Mol. Neuropharmacol. 1 (1990) 23–29. [5] S.D. Brain, T.J. Williams, Substance P regulates the vasodilator activity of calcitonin gene-related peptide, Nature 335 (1988) 73–75. [6] K.C. Catania, M.C. Remple, Somatosensory cortex dominated by the representation of the teeth in the naked mole-rat brain, Proc. Natl. Acad. Sci. USA 99 (2002) 5692–5697. [7] A. Chang, D.W. Emmel, G.C. Rossi, G.W. Pasternak, Methadone analgesia in morphine-insensitive CXBK mice, Eur. J. Pharmacol. 351 (1998) 189–191. [8] L.F. Chapman, Mechanisms of the flare reaction in human skin, J. Invest. Dermatol. 69 (1977) 88–97. [9] L. Chen, L.Y. Huang, Protein kinase C reduces mg2+ block of NMDAreceptor channels as mechanism of modulation, Nature 356 (1992) 521–523. [10] A.D. Corbett, M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson, Tyr-d-Pen-Gly-Phe-l-Pen and Tyr-d-Pen-Gly-Phe-d-Pen are selective ligands for the delta-binding site, Br. J. Pharmacol. 80 (1983) 669–678. [11] A.D. Corbett, M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson, L.E. Robson, Selectivities of opioid peptide analogues as agonists and antagonists at the delta receptor, Br. J. Pharmacol. 83 (1984) 271–279. [12] S.D. Crish, F.L. Rice, T.J. Park, C.M. Comer, Somatosensory organization and behaviour in naked mole-rat. I. Vibrissa-like body hairs comprise a sensory array that mediates orientation to tactile stimuli, Brain Behav. Evol. 62 (2003) 141–151. [13] A.H. Dickenson, A.F. Sullivan, Subcutaneous formalin-induced activity of dorsal horn neurons in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain 30 (1987) 349–360. [14] J.R. Gibson, J.R. McDermont, B. Lauffart, D. Mantle, Specificity of action of human brain alanylaminopeptidase on leu-enkephalin and dynorphinrelated peptides, Neuropeptides 13 (1989) 259–262. [15] T.J. Grudt, J.T. Williams, Kappa-opioid receptors also increase potassium conductance, Proc. Natl. Acad. Sci. USA 90 (1993) 11429–11432. [16] A.G. Hayes, M. Skingle, M.B. Tyers, Reversal by beta-funaltrexamine of the antonociceptive effect of opioid agonists in the rat, Br. J. Pharmacol. 88 (1986) 867–872.
67
[17] E.C. Henry, M.S. Remple, M.J. O’rian, K.C. Catania, Organization of somatosensory cortical areas in the naked mole-rat (Heterocephalus glaber), J. Comp. Neurol. 495 (2006) 434–452. [18] P.J. Horan, P.R. De Costa, K. Rice, R.C. Haaseth, V.J. Hruby, F. Porreca, Differential antagonism by bremazocine and U-69593-induced antinociception by quadazocine: further evidence for functional evidence of opioid kappa receptor multiplicity in the mouse, J. Pharmacol. Exp. Ther. 266 (1993) 926–933. [19] S. Hunskaar, O.-G. Berge, K. Hole, A modified hot plate test sensitive to mild analgesics, Behav. Brain Res. 21 (1986) 101–108. [20] D.E. Jorenby, R.E. Keesey, T.M. Baker, Characterization of morphine’s excitatory effects, Behav. Neurosci. 102 (1988) 975–985. [21] T.I. Kanui, K. Hole, Morphine induces aggression but not analgesia in the naked mole-rat (Heterocephalus glaber), Comp. Biochem. Physiol. 96C (1990) 131–132. [22] T.I. Kanui, F. Karim, P.K. Towett, The formalin test in the mole-rat (Heterocephalus glaber): analgesic effects of morphine, nefopam and paracetamol, Brain Res. 600 (1993) 123–126. [23] F. Karim, T.I. Kanui, S. Mbugua, Effects of codeine, naproxen and dexamethasone on formalin-induced pain in the naked mole rat, Neuroreport 4 (1993) 25–28. [24] I. Kassin, B.T. Brown, C.A. Robinson, E.L. Bradley Jr., Acute tolerance in morphine analgesia: continuous infusion and single injection in rats, Anesthesiology 74 (1991) 166–171. [25] G. Larrinaga, J. Gill, J.J. Meana, F. Ruiz, L.F. Callado, J. Irazusta, Aminopeptidase activity in the postmortem brain of human heroin addicts, Neurochem. Int. 46 (2005) 213–219. [26] J.-P. Laulin, E. Celerier, A. Larcher, M. Le Moal, G. Simonnet, Opiate tolerance to daily heroin administration: an apparent phenomenon associated with enhanced pain sensitivity, Neuroscience 89 (1999) 631–636. [27] J.-P. Laulin, P. Maurette, J.-B. Corcuff, C. Rivat, M. Chauvin, G. Simonnet, The role of ketamine in preventing fentanyl-induced hyperlalgesia and subsequent acute morphine tolerance, Anesth. Analog. 94 (2002) 1263–1269. [28] G.S. Ling, R. Simantov, J.A. Clark, G.W. Pasternak, Naloxonazine actions in vivo, Eur. J. Pharmacol. 129 (1986) 33–38. [29] H. Machelska, D. Lobuz, R. Przewlocki, B. Przewlocka, Inhibition of nitric oxide synthase enhances antinociception mediated by mu, delta, and kappa opioid receptors in acute and prolonged pain in the rat spinal cord, J. Pharmacol. Exp. Ther. 282 (1997) 977–984. [30] C.A. Maggi, Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves, Prog. Neurobiol. 45 (1995) 1–98. [31] J. Mao, D.D. Price, D.J. Mayer, Mechanisms of hyperalgesia and morphine tolerance: a current view of their interactions, Pain 62 (1995) 259–274. [32] A. Mattia, T. Vanderah, H.I. Mosberg, F. Porreca, Lack of antinociceptive cross-tolerance between [d-Pen2, d-Pen5]enkephalin and [dAla2]deltorphin II in mice: evidence for receptor subtypes, J. Pharmacol. Exp. Ther. 258 (1991) 583–5878. [33] I. McFadzean, The ionic mechanisms underlying opioid actions, Neuropeptides 11 (1988) 173–180. [34] R. Oku, M. Satoh, N. Fujii, A. Otaka, H. Yajima, H. Takagi, Calcitonin gene-related peptide promotes nociception by potentiating the release of substance P from the spinal dorsal horn in rats, Brain Res. 403 (1987) 350–354. [35] G.A. Olson, R.D. Olson, A.J. Kastin, Review of endogenous opiates: 1991, Peptides 13 (1992) 1247–1287. [36] T.J. Park, C. Comer, A. Carol, Y. Lu, S.H. Hong, F.L. Rice, Somatosensory organization and behaviour in naked mole-rats. II. Peripheral structures, innervation, and selective lack of neuropeptides associated with thermoregulation and pain, J. Comp. Neurol. 465 (2003) 104–120. [37] C.G. Parsons, NMDA receptors as targets for drug action in neuropathies, Eur. J. Pharmacol. 429 (2001) 71–78. [38] M.F. Piercey, R.A. Lhati, L.A. Schroeder, U-50488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse, Life Sci. 31 (1982) 1197–1200. [39] F. Porreca, J.S. Heyman, J.I. Mosberg, J.R. Omnass, J.L. Vaught, Role of mu and delta receptors in the supraspinal and spinal analgesic effects of
68
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68 [d-Pen2 , d-Pen5 ]enkephalin in the mouse, J. Pharmacol. Exp. Ther. 241 (1987) 393–400. R.B. Raffa, R.P. Martinez, F. Porreca, Lack of antinociceptive efficacy of intracerebroventricular [d-Ala2 ,Glu4 ]deltorphin, but not [d-Pen2, dPen5]enkephalin, in the mu-opioid receptor deficient CXBK mouse, Eur. J. Pharmacol. 216 (1992) 453–456. W. Rosenthal, J. Hescheler, W. Trautwein, G. Schultz, Control of voltagedependent Ca2+ channels by G-protein coupled receptors, FASEB J. 2 (1988) 2784–2790. A. Safavi, L.B. Hersh, Degradation of dynorphin-related peptides by the puromycin sensitive aminopeptidase and aminopeptidase M, J. Neurochem. 65 (1995) 389–395. J.D. Silverman, L. Kruger, Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers, J. Neurocytol. 19 (1990) 789–801. M. Sofuoglu, P.S. Portoghese, A.E. Takemori, Differential antagonism of delta opioid agonists by naltrindole (NTI) and its benzofuran analogue (NTB) in mice: evidence for delta opioid receptor subtypes, J. Pharmacol. Exp. Ther. 257 (1991) 676–680. R.L. Spencer, V.J. Hruby, T.F. Burks, Body temperature response profiles for selective mu, delta, and kappa opioid agonists in restrained and unrestrained rats, J. Pharmacol. Exp. Ther. 246 (1988) 92–101. C.W. Stevens, Relative analgesic potency of mu, delta and kappa opioids after spinal administration in amphibians, J. Pharmacol. Exp. Ther. 276 (1996) 440–448. C.W. Stevens, A.J. Klopp, J.A. Facello, Analgesic potency of mu and kappa opioids after systemic administration in amphibians, J. Pharmacol. Exp. Ther. 269 (1994) 1086–1093. H.H. Suh, L.F. Tseng, Intrathecal beta-funaltrexamine antagonizes intracerebroventricular beta-endorphin—but not morphine-induced analgesia in mice, J. Pharmacol. Exp. Ther. 245 (1988) 587–593. H.H. Suh, L.F. Tseng, Different types of opioid receptors mediating analgesia induced by morphine, DAMGO, DPDPE, DADLE and betaendorphin in mice, Naunyn-Schmiedeberg’s Arch. Pharmacol. 342 (1990) 67–71.
[50] H.H. Suh, J.M. Fujimoto, L.F. Tseng, Differential mechanisms mediating beta-endorphin- and morphine-induced analgesia in mice, Eur. J. Pharmacol. 168 (1989) 61–70. [51] A.E. Takemori, B.Y. Ho, J.S. Naeseth, P.S. Portoghese, Norbinaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246 (1988) 255–258. [52] A. Tjolsen, O.-G. Berge, P.K. Eide, O.J. Broth, K. Hole, Apparent hyperalgesia after lesions of descending serotonergic pathways is due to increased tail skin temperature, Pain 33 (1988) 225–231. [53] P.K. Towett, T.I. Kanui, Effects of pethidine, acetylsalicylic acid, and indomethacin on pain and behavior in the mole-rat, Pharmacol. Biochem. Behav. 45 (1993) 153–159. [54] P.K. Towett, T.I. Kanui, Hyperalgesia following administration of morphine and pethidine in root rat (Tachyoryctes splendens), J. Vet. Pharmacol. Ther. 18 (1995) 68–71. [55] K.A. Trujillo, H. Akil, Opiate tolerance and dependence: recent findings and synthesis, New Biol. 10 (1991) 915–923. [56] M.B. Tyers, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69 (1980) 503–512. [57] T.D. Vanderah, N.M.H. Suenaga, M.H. Ossipov Jr., T.P. Malan, J. Lai, F. Porreca, Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance, J. Neurosci. 21 (2001) 279–286. [58] J.L. Vaught, J.R. Mathiesen, R.B. Raffa, Examination of the involvement of the mu and delta opioid receptors in analgesia using the mu-receptor deficient CXBK mouse, J. Pharmacol. Exp. Ther. 245 (1988) 13–16. [59] P.F. VonVoigtlander, R.A. Lahti, J.H. Ludens, U-50,488: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther. 224 (1983) 7–12. [60] T.L. Yaksh, G.J. Harty, B.M. Onfofrio, High doses of spinal morphine produce a non-opiate receptor mediated hyperesthesia: clinical and theoretic implications, Anesthesiology 64 (1986) 590–597. [61] S. Zhou, L. Bonesera, S.M. Carlton, Peripheral administration of NMDA, AMPA or KA results in pain behaviours in rats, Reuroreport 7 (1996) 895–900.
Stimulation of mu and delta opioid receptors induces hyperalgesia while stimulation of kappa receptors induces antinociception in the hot plate test in the naked mole-rat (Heterocephalus glaber) Philemon Kipkemoi Towett a,∗ , Titus Ikusya Kanui a , Francis D. Juma b,1 a
Department of Veterinary Anatomy and Physiology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya b Department of Clinical Pharmacology, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Received 27 January 2006; received in revised form 20 July 2006; accepted 1 August 2006 Available online 23 August 2006
Abstract The antinociceptive effects of highly selective mu (DAMGO), delta (DPDPE) and kappa (U-50488 and U-69593) opioid agonists were evaluated following intraperitoneal (i.p.) administration in the naked mole-rat. A hot plate test set at 60 ◦ C was used as a nociceptive test and the latency to the stamping of the right hind paw (response latency) was used as the end-point. DAMGO (5–10 mg/kg) and DPDPE (2.5–5 mg/kg) caused a naloxone-reversible significant decrease in the mean response latency. Subcutaneous injection of naloxonazine (20 mg/kg) 24 h prior to the administration of DAMGO (5 mg/kg) also blocked the reduction in the response latency observed when DAMGO was injected alone. On the contrary, U-50488 (2.5–5 mg/kg) or U-69593 (0.08 or 0.1 mg/kg) caused a naloxone-reversible significant increase in the mean response latency. These results showed that activation of mu or delta receptors caused hyperalgesia, whereas activation of kappa receptors caused antinociception in the hot plate test in naked mole-rat. This suggests that mu and delta receptors modulate thermal pain in a different way than kappa receptors in the naked mole-rat. It is not possible at the moment to point out how they modulate thermal pain as little is known about the neuropharmacology of the naked mole-rat. © 2006 Elsevier Inc. All rights reserved. Keywords: Antinociception; Hyperalgesia; Opioid systems; Hot plate test
1. Introduction Opioids have been and are still being used for the treatment of various types of pain in both humans and animals. The effects of opioids are mediated through opioid receptors that are widely distributed in the central nervous system. The role of opioid receptors in pain modulation has been investigated in a wide range of animal species and it appears that their mechanisms of actions differ in different types of pain and also in different animal species. Studies performed in the naked mole-rat suggested that the opioid systems of this subterranean rodent could be quite different from those of other rodents [21,53]. The authors reported
∗
Corresponding author. Tel.: +254 20 4448648. E-mail addresses: [email protected], [email protected] (P.K. Towett). 1 Tel.: +254 20 726300. 0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.08.001
that acute i.p. administration of morphine or pethidine caused a naloxone-reversible increase in pain sensitivity in the hot plate test set at 60 ◦ C in the naked mole-rat. This was referred to as a hyperalgesia. Hyperalgesia, tolerance and physical dependence usually occur following repeated opioid administration [24,26,31,55,57]. One of the causes of opioid tolerance is an increase in the break down of opioid peptides by aminopeptidases [25]. There is ample evidence indicating that hyperalgesia and other central hyperactive states are mediated by N-methyld-aspartate receptors in the NS [26,27,29,31]. Considering that the animals used in the published reports [21,53] had not been injected with opioids before, the data were very unique and interesting. Furthermore similar opioids have been reported to cause analgesia in other rodents such as the rat and mice in the hot plate test [1,19,56]. Studies performed in another subterranean rodent, the root-rat mole-rat (Tachyoryctes splendens), also showed that morphine caused hyperalgesia in the hot plate test set at 60 ◦ C [54]. These studies suggest that the nociceptive systems of mole-rats can be activated by exogenous
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
opioids. The exact role of opioid systems of mole-rats in thermal nociception is unclear and therefore further research is required to provide more information. Naked mole-rats have very unique physiology and anatomy. They are cold-blooded and are virtually blind. They have long incisors for digging, chewing and carrying objects and young. Naked mole-rats depend heavily on tactile senses to navigate their environment and this is facilitated by the presence of unique body vibrissae in rows along the trunk [12]. The two structures i.e. the incisors and the few hair in the skin, have large representation in the somatosensory cortex [6,12,17]. The skin of a naked mole-rat is highly folded and it lacks subcutaneous fat. Park et al. [36] have shown that the skin of a naked mole-rat has abundant A delta fibres but it lacks C-fibres immunoreactive for substance P and calcitonin gene-related peptides (CGRP). SP and CGRP are implicated in a number of functions such as nociception [34] and inflammation [8], vasodilation [5] and tissue maintenance [30]. The absence of SP and CGRP in the skin of the naked mole-rat raises questions on how this primitive rodent responds to injury and nociception. It is, however, reported that the naked mole-rat has several nonpeptidergic C-fibres in the skin and hair follicles [36]. A beta lanceolate endings containing SP and CGRP and lectinbinding C-fibres are also plenty in the skin and in the trigerminal and dorsal root ganglions of the naked mole-rat [36]. Such fibres have been implicated in nociception [43] and are perhaps very important in controlling the same in this primitive rodent. The opioids used in the two studies published [21,53] in the naked mole-rats act predominantly on mu receptors. To the best of our understanding, the roles of other opioid receptors (delta and kappa) on thermal nociception in the naked mole-rat is not known. The present study was aimed at providing data on the roles of the three classical opioid receptors on thermal nociception in the naked mole-rat. The data collected was hoped to provide solutions for the apparent peculiarities of the opioid systems of the naked mole-rat. To achieve this objective, receptor-selective mu (DAMGO), delta (DPDPE) and kappa (U50488 and U-69593) opioid agonists were used in the study. The selected drugs are receptor-selective and their analgesic effects in a wide range of nociceptive tests have been demonstrated in other rodents [10,11,13,39,48–51,59] but not in mole-rats. Naloxone and naloxonazine were used as antagonists in the present study. This was a more systematic study to be done in the naked mole-rat in our laboratory. The data obtained showed a clear physiological difference between kappa agonists and the other two opioid agonists (mu or delta) on thermal nociception in the naked mole-rat. 2. Materials and methods 2.1. Animals Adult male and female naked mole-rats (Heterocephalus glaber), weighing 20–40 g, were used in the experiments. They were trapped at Kathekani in Makueni District and transported by road to a laboratory in Chiromo Campus of the University of Nairobi, Kenya. The animals were kept in colonies of 50–100 in plastic lidded cages measuring 70 cm × 50 cm × 20 cm. Each cage was sub-
61
divided into two sub-compartments, with an open entrance joining them. The naked mole-rats used one sub-compartment as a toilet and the other as a bedroom. The bedding consisted of untreated wood shavings mixed with sand. The bedding was changed twice in a week. The cages were kept in a sound and a vibration proof room. Conditions almost similar to those in the wild were adopted in the room housing the mole-rats. The room temperature was maintained at between 28 and 31 ◦ C by infrared lamps (250 W) and/or electric heaters. Light was maintained at 0/24 h light/dark cycle except during the course of the experiments. In order to avoid problems of dry skin, the humidity in the room was kept at between 60% and 80%. In the wild, naked mole-rats feed on a varied diet consisting of roots, bulbs, and tubers. To maintain healthy colonies in the laboratory a varied diet consisting of fresh carrots, sweet potatoes, carrot tops and freshly cut grass was provided ad libitum. No water was provided since the naked mole-rats do not take it from either the bottles or the Petri dish. The animals can survive forever without water so long as they are fed on fresh food. The naked molerats were allowed at least one month to acclimate to the laboratory conditions before they were used for the experiments. The animals were also examined for fitness before being used for experiments. Touch reflex, locomotion and response to sounds or vibrations were tested prior to each experiment. The animals were randomly selected and each animal was used only once and the experiments were done blindly. International and national ethical guidelines regarding the use of conscious animals for pain research were adhered to during the course of the experiments. A committee of the Kenya Society for Protection and Care of Animals (KSPCA) approved the experimental procedures used in the study.
2.2. Drugs All the opioid agents used in the experiments were bought from Research Biochemicals International (RBI, Natick, USA). These were d-Ala2 -NMePhe4 Gly-ol5 -enkephalin (DAMGO), d-Pen2 -d-Pen5 -enkephalin (DPDPE), trans3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (U-50488), (5␣,7␣,8)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspirol[4,5]dec-8-yl]-benzeneacetamide (U-69593), naloxone hydrochloride, and naloxonazine hydrobromide. DAMGO, DPDPE, U-50488 and naloxone were dissolved in physiological saline (0.9% NaCl). U-69593 and naloxonazine were dissolved in dilute hydrochloric acid in saline. Fresh preparations of all drugs were always used and the volume injected was always 50 l. All the precautions regarding handling and stability of the drugs as recommended by the manufacturer were followed strictly.
2.3. Nociceptive and antinociceptive testing The nociceptive test used was a hot plate test (30 cm × 30 cm × 30 cm; iit Inc. Woodland Hills, CA, Model 35D analgesy-meter) set at 60 ◦ C. The surface temperature was continuously monitored with a digital thermometer. A naked mole-rat was gently placed on the hot plate and the latency to the stamping of the right hind paw (response latency) was recorded in seconds. The response latency was recorded 30 min after administration of vehicle or drug. To avoid thermal injury to the paws, an animal that failed to step its paws within 60 s was removed from the plate and assigned a value of 60 s. The reason for using the hot plate test was because this was a follow-up study of what is published [21,53]. The test was also adapted from a standard test used in rats and mice. Investigations using other nociceptive tests are being carried out in our laboratory. To investigate antinociceptive effects, DAMGO (0.5–10 mg/kg), DPDPE (1–5 mg/kg), U-50488 (1–10 mg/kg), and U-69593 (0.025–0.1 mg/kg) were administered intraperitoneally 30 min before nociceptive testing. The effects of naloxone and/or naloxonazine on the mean responses caused by the opioid drugs were also investigated. DAMGO (5 mg/kg), DPDPE (2.5 mg/kg), U-50488 (5 or 10 mg/kg) or U-69593 (0.08 or 0.1 mg/kg) was each administered in combination with naloxone (5 mg/kg). Naloxonazine (20 mg/kg) was administered subcutaneously 24 h prior to intraperitoneal administration of DAMGO (5 mg/kg). Naloxonazine is a slowly absorbed mu-1 receptor antagonist [28]. The dosages and the times for drug administration were based on pilot dose–response
62
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
experiments. Control animals for DAMGO and DPDPE were injected with physiological saline while those for U-50488 and U-69593 were injected with physiological saline plus dilute HCl.
2.4. Statistics The response latencies were analyzed using one-way analysis of variance (ANOVA) and least significant difference where applicable. The level of significance was set at 5% (p < 0.05). The results are presented as means ± S.E.M.
3. Results 3.1. Effects of DAMGO The hot plate baseline latencies (controls) for each of the experiment were determined by administering saline or a vehicle. The mean response latency for controls was 23.65 ± 0.4 s whereas for DAMGO 0.5, 1, 5 and 10 mg/kg they were 21.28 ± 1.2, 23.60 ± 0.3, 13.51 ± 0.5 and 12.53 ± 0.3 s, respectively. Intraperitoneal administration of DAMGO (0.5, 5 or 10 mg/kg) caused a statistically significant reduction in the mean response latency (p < 0.05, Fig. 1A). There was no statistical significant difference between controls and the animals injected with DAMGO (1 mg/kg: p > 0.5, Fig. 1A). When DAMGO (5 mg/kg) and naloxone (5 mg/kg) were administered simultaneously a response latency of 25.49 ± 0.4 s was recorded. When the mean response latency for the combined treatment (25.49 ± 0.4 s) was compared with that for DAMGO (5 mg/kg: 13.51 ± 0.5 s) alone, a statistically significant difference was recorded (p < 0.05; Fig. 1B). Naloxone (2 mg/kg) did not block the effect of DAMGO (5 mg/kg) (data not shown). Subcutaneous administration of naloxonazine (20 mg/kg) 24 h before DAMGO (5 mg/kg) was administered intraperitoneally caused a mean response latency of 31.28 ± 0.7 s. To find out whether naloxonazine blocked or not the effect of DAMGO on the response latency, the mean response latency for the combined treatment (31.28 ± 0.7 s) and that for DAMGO (5 mg/kg) alone (13.51 ± 0.5 s) were compared. A statistically significant difference was noted (p < 0.05; Fig. 1C). The number of animals in these experiments ranges from 8 to 10 in each group. 3.2. Effects of DPDPE The controls for DPDPE had a mean response latency of 24.11 ± 0.4 s. Intraperitoneal administration of DPDPE (1, 2.5 or 5 mg/kg) caused mean response latencies of 23.71 ± 0.9, 15.06 ± 0.3 or 12.47 ± 0.5 s, respectively. The mean response latency for DPDPE (1 mg/kg) was not statistically significant when it was compared with that for controls (p > 0.5; Fig. 2A). The mean response latency for DPDPE (2.5 or 5 mg/kg) was significant when compared with that for controls (p < 0.05; Fig. 2A). Simultaneous intraperitoneal administration of DPDPE (2.5 mg/kg) and naloxone (5 mg/kg) caused a response latency of 22.42 ± 0.5 s, which when statistically compared with that for DPDPE (2.5 mg/kg) alone (15.06 ± 0.3 s) was significant (p < 0.05; Fig. 2B). The number of animals in these experiments was 10 in each treatment group.
3.3. Effects of U-50488 and U-69593 The mean response latency for controls of U-50488 was 31.57 ± 0.7 s. Intraperitoneal administration of 1, 2.5, 5 or 10 mg/kg of U-50488 caused mean response latencies of 31.49 ± 0.6, 40.97 ± 0.9, 45.67 ± 1.8, or 60.0 ± 0.0 s, respectively. The mean response latencies for U-50488 (2.5, 5 or 10 mg/kg) were statistically significant (p < 0.05; Fig. 3A) when each one of them was compared with that for controls whereas that for U-50488 (1 mg/kg) was not (p > 0.05). Simultaneous administration of U-50488 (5 or 10 mg/kg) and naloxone (5 mg/kg) caused mean response latencies of 34.05 ± 0.6 or 35.82 ± 0.7 s, respectively. When each of the means of the combined treatment was compared with that for U-50488 (5 or 10 mg/kg) alone (45.67 ± 1.8 or 60 0 s) a statistically significant difference was noted (p < 0.05; Fig. 3B). In each treatment group n was 9–12. The response latency for the controls of U-69593 was 33.76 ± 1.6 s. Intraperitoneal administration of U-69593 (0.025, 0.08 or 0.1 mg/kg) caused mean response latencies of 33.94 ± 1.3, 41.30 ± 0.5, or 42.32 ± 1.2 s, respectively. There was no statistically significant difference between the mean response latency for controls and that for the group injected with 0.025 mg/kg of U-69593 (p > 0.05; Fig. 4A). On comparing the response latencies for U-69593 (0.08 or 0.1 mg/kg) with that for controls, a statistically significant difference was noted (p < 0.05; Fig. 4A). Simultaneous intraperitoneal administration of U-69593 (0.08 or 0.1 mg/kg) and naloxone (5 mg/kg) caused mean response latencies of 30.51 ± 0.7 or 34.17 ± 1 s, respectively. The response latency for each of the combined treatment was compared with that of U-69593 (0.08 or 0.1 mg/kg) alone and a statistically significant difference was recorded for these comparisons (p < 0.05; Fig. 4B). In each treatment group n was 9–10. 4. Discussion The hot plate test is a supraspinally mediated nociceptive test commonly used to study pain mechanisms. Naked mole-rats show clear quantifiable behaviour when tested on the hot plate set at 60 ◦ C [21,53]. In the current study, DAMGO (mu) or DPDPE (delta) caused a decrease in the response latency when they were injected intraperitoneally. In the hot plate test, antinociception is usually indicated by an increase in the response latency but not a decrease as recorded in the present study. A decrease in the response latency suggests that the nociceptive system of the animal is more sensitive to the noxious stimulation than before the injection of the drug. This phenomenon is referred to as hyperalgesia. The hyperalgesia caused by either DAMGO or DPDPE was blocked by naloxone, administered simultaneously with the agonist (DAMGO or DPDPE), suggesting the involvement of opioid receptors. Naloxonazine, a mu-1 antagonist, also blocked the effect of DAMGO, suggesting that mu-1 opioid receptors were involved in the decrease in the response latency observed following the administration of DAMGO. DAMGO or DPDPE, at doses that caused the recorded apparent hyperalgesia did not cause a change in the normal behaviour
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
63
Fig. 1. (A) Effects of i.p. administration of vehicle (0) or DAMGO (0.5, 1, 5 or 10 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 8–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 5 mg/kg of naloxone (Nal 5), 5 mg/kg of DAMGO (DAM 5), or a combination of 5 mg/kg of DAMGO and 5 mg/kg of naloxone (DAM 5/Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (C) Effects of i.p. administration of vehicle (0), a combination 20 mg/kg of naloxonazine and saline (N-zine 20/saline), 5 mg/kg of DAMGO (DAM 5), or 20 mg/kg of naloxonazine followed 24 h later by 5 mg/kg of DAMGO (DAM 5/N-zine 20) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
64
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
Fig. 2. (A) Effects of i.p. administration of vehicle (0) or DPDPE (1, 2.5 or 5 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). B. Effects of i.p. administration of vehicle (0), 5 mg/kg of naloxone (Nal 5), 2.5 mg/kg of DPDPE (DP 2.5), or a combination of 2.5 mg/kg of DPDPE and 5 mg/kg of naloxone (DP 2.5/Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
Fig. 3. (A) Effects of i.p. administration of vehicle (0) or U-50488 (1, 2.5, 5 or 10 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 10–12 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 5 or 10 mg/kg of U-50488 (U505 or U-5010) alone or each in combination with 5 mg/kg of naloxone (Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–12 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
of the naked mole-rat. This implied that the hyperkinesis and hyperexcitation, which had earlier been incriminated as the possible causes of morphine- or pethidine-induced apparent hyperalgesia [21,53], could not be responsible for the opioidinduced hyperalgesia in the naked mole-rat. In the same species of mole-rat, antinociception, in presence of hyperkinesis and
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
Fig. 4. (A) Effects of i.p. administration of vehicle (0) or U-69593 (0.025, 0.08 or 0.1 mg/kg) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05). (B) Effects of i.p. administration of vehicle (0), 0.08 or 0.1 mg/kg of U-69593 (U-69.0.08 or U-69. 0.1) alone or each in combination with 5 mg/kg of naloxone (Nal 5) on the hot plate response latency. Values given are means ± S.E.M., and n = 9–10 in each group. Treatment means were compared using least significance difference, subsequent to ANOVA and the level of significance was set at p ≤ 0.05. Bars with different superscripts are significantly different (p < 0.05).
65
hyperexcitation, has been reported after systemic administration of morphine [22] or codeine [23] in the formalin test. This suggests that thermal and chemical pains have different mechanisms and are modulated differently by the opioid systems of the naked mole-rat. In rats and cats, overactivation of opioid receptors has been observed to cause non-opioid receptor mediated hyperalgesia [60]. Overactivation of opioid receptors occurs when high doses are administered directly into the central nervous system. There are four differences between the report by Yaksh et al. [60] and the current data. Firstly, the hyperalgesia in the present study was naloxone-reversible, suggesting that it was opioid mediated. Secondly, the doses of DAMGO or DPDPE that caused hyperalgesia were not high because lower doses had no significant effect on nociception. Thirdly, the routes of administration are different in that in the present study drugs were injected intraperitoneally whereas in Yaksh’s report [60] drugs were injected spinally and fourthly the species of the animals used in the two studies are also different. The animals used in the study (naked mole-rats) have very unique physiology and anatomy. They are the only known coldblooded mammals. The skin of the naked mole-rat is rather unique in that it lacks C-fibres immunoreactive for substance P and calcitonin gene-related peptides (CGRP) [36]. SP and CGRP are implicated in a number of actions including nociception [34] and inflammation [8]. Because of the absence of SP and CGRP, the naked mole-rat has been described as painless animals. However, the current results and the published ones [21–23,53] clearly indicated that naked mole-rats have antinociceptive systems. These systems are perhaps SP-independent. It also appears from the same published reports that naked molerats have higher nociceptive thresholds. Hot plate temperatures below 60 ◦ C do not elicit significant reactions in the mole-rat and this may suggest that the animal is painless. A Similar observation was noted with the formalin test [23]. High nociceptive threshold in the naked mole-rat could be due to the lack of SP in the skin. The presence of abundant A␦ fibres, several nonpeptidergic C-fibres, A lanceolate endings containing SP and CGRP and many lectin-binding C-fibres [36] in the skin of the naked mole-rat may suggest that these fibres are crucial in nociception in this rodent. Mu opioids are potent, effective analgesics used for the treatment of acute and chronic pain syndromes. Delta agonists can also relieve pain but have not been used so much for this purpose. Experiments have shown that DAMGO and DPDPE have good antinociceptive effects in various nociceptive tests and in different animal species such as mice [32,44,48,49] and amphibians [46,47]. Analgesic sensitivity, especially to opioids, in animals can be influenced by a number of factors including genetics. For instance, inbred CXBK mouse-strain is deficient in mu opioid receptors and mu agonists such as morphine or DAMGO are not effective in producing antinociception after systemic or supraspinal administration [2,7,40,58]. Naked mole-rats live in large colonies with one breeding female served by many males. The possibility of inbreeding in the colony cannot be ruled out and this may contribute to the data observed in the previous and current studies. A naloxone-reversible hyperalgesia follow-
66
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
ing systemic administration of morphine and pethidine has also been observed in the root-rat mole-rat [54], where a possibility of inbreeding exists. Opiates can cause hyperthermic and other excitatory effects in animals [20]. Increased nociception is expected whenever there is an increase in skin temperature [52]. The naked molerat is a poikilothermic mammal and its skin lacks SP and CGRP which play a role in vasodilation and blood flow [5]. Effects of DAMGO and DPDPE on blood flow and body temperature in the naked mole-rat is not known at the moment. In rats, DAMGO, DPDPE and U-50488H can modify body temperature in different ways [45] and this may have an impact on antinociception. Perhaps mu and delta opioid agonists have an influence on the body temperature and blood flow in the naked mole-rat. Body temperature and blood flow are of great significance to a naked mole-rat, considering that it inhabits areas with relatively high (>30 ◦ C) ambient temperatures. In contrast to DAMGO and DPDPE, administration of U50488 or U-69593 caused an increase in the mean response latency and this was a clear antinociceptive effect. The antinociceptive effect of the two kappa agonists was blocked by naloxone. U-50488 and U-69593 are highly selective for kappa receptors and their antinociceptive actions have been demonstrated in a variety of assays, including thermal, pressure and irritant tests in mice and rats [18,38,59] and also in amphibians [46,47]. The data reported in the present study is in line with these reports. However, this data is the first to demonstrate the antinociceptive effects of kappa agonists in the hot plate test in the naked mole-rat. The mean response latencies for DAMGO and DPDPE control groups were insignificant but significantly different as compared to those of kappa opioids. Dilute HCl was added to physiological saline before it was injected to kappa control groups and this may have contributed to the difference observed. The antinociceptive doses of the two kappa agonists used were relatively lower than those used in mice and rats [59]. This may suggest the abundance of kappa receptors in the central nervous system of the naked mole-rat. In rats, high doses (>10 mg/kg) of U-50488 have been reported to cause no side effects [16]. In the naked mole-rat, doses of U-50488 greater than 5 mg/kg and U-69593 greater than 0.1 mg/kg appeared to be very high because they caused side effects. Although U-50488 and U-69593 are both kappa agonists, it appeared, on the basis of the dose range used, that U-69593 was more potent in causing antinociception than U-50488, and this could be due to differences in structure [4]. It is interesting that kappa agonists caused antinociception while mu or delta agonists caused hyperalgesia in the hot plate test. At the receptor level, mu and delta agonists increase potassium conductance, whereas kappa agonists directly inhibit the entry of calcium through voltage-dependent calcium channels [33,41]. The end result of these mechanisms will be inhibition of the release of neurotransmitter substance or release of inhibitory neurotransmitters. The onward transmission of nociceptive signal will be blocked. The heterogeneity of G-protein sub-units may influence the downstream effector mechanisms that follow the coupling of an opioid agonist to the G-protein, and this may explain the differences, in terms of functions, between kappa
and other opioid receptors [15]. Perhaps this is the case for the kappa and mu or delta agonists in the naked mole-rat. The difference between the effects of kappa agonists (U50488 and U-69593) and those of mu or delta agonists on thermal pain could probably also be due to differences in neurochemical pain pathways or their rates of metabolism. Opioids interact in various ways with central neurotransmitters to cause a wide variety of actions including antinociception [35]. It has been shown that the analgesic effects of U-50488 but not morphine could be blocked by pretreatment with the serotonin depletors [59], suggesting the importance of serotonergic pathways in kappainduced antinociception. Whether this is also true in the naked mole-rat is not known at the moment because no attempt has been made to investigate the relationship between the various neurotransmitter systems and thermal nociception in the naked mole-rat. Such a study would shed a lot of light on nociception and antinociception in the naked mole-rat. Opioid peptides are broken down in the body by pepdidases. Puromycin sensitive aminopeptidase and aminopeptidase N are two aminopeptidases that have high affinity for enkephalins and dynorphin [42]. Enzymatic break down of opioids in the body can produce neuroexcitatory metabolites such as morphine-3-glucuronide, which is very potent in causing hyperalgesia and myoclonus in patients treated by high doses of opioids [60]. DAMGO and DPDPE are enkephalin-related peptides whereas U-50488 and U-69593 are dynorphin-related peptides. Although human puromycin-sensitive aminopeptidase can metabolize both enkephalins and dynorphins [14], this may not be the case in the naked mole-rat. Perhaps, in the naked molerat, the activity of aminopeptidases for mu or delta opioids is so high that DAMGO and DPDPE are quickly metabolized into hyperalgesic products. Chronic administration of opiates results in tolerance and physical dependence [55], and also an increase in pain sensitivity [26,31]. Tolerance and physical dependence are as a result of decrease in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the nervous system [55]. An increase in the catabolism of opioid peptides in the brain may also cause tolerance and physical dependence [25]. Activation of N-methyl-d-aspartate receptors in the CNS is implicated in the opioid-induced hyperalgesia [61] because NMDA receptor antagonists can block it [3,27,29,37]. Mu-opioids activate NMDA receptors by reducing mg2+ block via protein kinase C activation and this leads to an increase in intracellular ca2+ concentrations, which subsequently enhances glutamate activity [9,31]. Glutamate is one of the excitatory amino acids that are involved in pain transmission in the spinal cord. The naked molerats used in the current experiments received only one injection of an opioid drug. The increase in pain sensitivity after DAMGO or DPDPE was therefore not expected. Our data therefore suggest that DAMGO and DPDPE are pronociceptive at least in the hot plate test set at 60 ◦ C in the naked mole-rat. The mechanisms underlying the observed hyperalgesia are not known at the moment. Perhaps NMDA receptors are involved. This needs to be investigated. Although the exact mechanism of action of the three opioids used in the current study cannot be presented here, the
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68
data collected suggest the existence of the three classical opioid receptors in the naked mole-rat and their roles on thermal nociception. A lot of work is required to establish the main cause of the difference between kappa and mu or delta receptors in pain control. Perhaps the use of receptor-selective antagonists may clearly demonstrate why kappa agonists caused antinociception while mu or delta agonists caused an apparent hyperalgesia in the hot plate test in the naked mole-rat. Effects of receptor-selective opioids on pain induced by non-thermal stimuli also need to be investigated in the naked mole-rat. Acknowledgements We thank the German Academic Exchange Programme (DAAD) for supporting this study. We do also extend our gratitude to the technical staff in the department for their technical support. References [1] S.I. Ankier, New hot plate tests to quantify antinociceptive and narcotic antagonists activities, Eur. J. Pharmacol. 27 (1974) 1–4. [2] A. Baron, L. Shuster, B.E. Elefterhiou, D.W. Bailey, Opiate receptors in mice: genetic differences, Life Sci. 17 (1975) 633–640. [3] A.Y. Bespalov, E.E. Zvartau, P.M. Beardsley, Opioid-NMDA receptor interactions may clarify conditioned (associative) components of opioid analgesic tolerance, Neurosci. Biobehav. Rev. 25 (2001) 343–353. [4] S.J. Boyle, K.G. Meecham, J.C. Hunter, J. Hughes, [3H]-CI-977: a highly selective ligand for the k-opioid receptor in both guinea-pig and rat forebrain, Mol. Neuropharmacol. 1 (1990) 23–29. [5] S.D. Brain, T.J. Williams, Substance P regulates the vasodilator activity of calcitonin gene-related peptide, Nature 335 (1988) 73–75. [6] K.C. Catania, M.C. Remple, Somatosensory cortex dominated by the representation of the teeth in the naked mole-rat brain, Proc. Natl. Acad. Sci. USA 99 (2002) 5692–5697. [7] A. Chang, D.W. Emmel, G.C. Rossi, G.W. Pasternak, Methadone analgesia in morphine-insensitive CXBK mice, Eur. J. Pharmacol. 351 (1998) 189–191. [8] L.F. Chapman, Mechanisms of the flare reaction in human skin, J. Invest. Dermatol. 69 (1977) 88–97. [9] L. Chen, L.Y. Huang, Protein kinase C reduces mg2+ block of NMDAreceptor channels as mechanism of modulation, Nature 356 (1992) 521–523. [10] A.D. Corbett, M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson, Tyr-d-Pen-Gly-Phe-l-Pen and Tyr-d-Pen-Gly-Phe-d-Pen are selective ligands for the delta-binding site, Br. J. Pharmacol. 80 (1983) 669–678. [11] A.D. Corbett, M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson, L.E. Robson, Selectivities of opioid peptide analogues as agonists and antagonists at the delta receptor, Br. J. Pharmacol. 83 (1984) 271–279. [12] S.D. Crish, F.L. Rice, T.J. Park, C.M. Comer, Somatosensory organization and behaviour in naked mole-rat. I. Vibrissa-like body hairs comprise a sensory array that mediates orientation to tactile stimuli, Brain Behav. Evol. 62 (2003) 141–151. [13] A.H. Dickenson, A.F. Sullivan, Subcutaneous formalin-induced activity of dorsal horn neurons in the rat: differential response to an intrathecal opiate administered pre or post formalin, Pain 30 (1987) 349–360. [14] J.R. Gibson, J.R. McDermont, B. Lauffart, D. Mantle, Specificity of action of human brain alanylaminopeptidase on leu-enkephalin and dynorphinrelated peptides, Neuropeptides 13 (1989) 259–262. [15] T.J. Grudt, J.T. Williams, Kappa-opioid receptors also increase potassium conductance, Proc. Natl. Acad. Sci. USA 90 (1993) 11429–11432. [16] A.G. Hayes, M. Skingle, M.B. Tyers, Reversal by beta-funaltrexamine of the antonociceptive effect of opioid agonists in the rat, Br. J. Pharmacol. 88 (1986) 867–872.
67
[17] E.C. Henry, M.S. Remple, M.J. O’rian, K.C. Catania, Organization of somatosensory cortical areas in the naked mole-rat (Heterocephalus glaber), J. Comp. Neurol. 495 (2006) 434–452. [18] P.J. Horan, P.R. De Costa, K. Rice, R.C. Haaseth, V.J. Hruby, F. Porreca, Differential antagonism by bremazocine and U-69593-induced antinociception by quadazocine: further evidence for functional evidence of opioid kappa receptor multiplicity in the mouse, J. Pharmacol. Exp. Ther. 266 (1993) 926–933. [19] S. Hunskaar, O.-G. Berge, K. Hole, A modified hot plate test sensitive to mild analgesics, Behav. Brain Res. 21 (1986) 101–108. [20] D.E. Jorenby, R.E. Keesey, T.M. Baker, Characterization of morphine’s excitatory effects, Behav. Neurosci. 102 (1988) 975–985. [21] T.I. Kanui, K. Hole, Morphine induces aggression but not analgesia in the naked mole-rat (Heterocephalus glaber), Comp. Biochem. Physiol. 96C (1990) 131–132. [22] T.I. Kanui, F. Karim, P.K. Towett, The formalin test in the mole-rat (Heterocephalus glaber): analgesic effects of morphine, nefopam and paracetamol, Brain Res. 600 (1993) 123–126. [23] F. Karim, T.I. Kanui, S. Mbugua, Effects of codeine, naproxen and dexamethasone on formalin-induced pain in the naked mole rat, Neuroreport 4 (1993) 25–28. [24] I. Kassin, B.T. Brown, C.A. Robinson, E.L. Bradley Jr., Acute tolerance in morphine analgesia: continuous infusion and single injection in rats, Anesthesiology 74 (1991) 166–171. [25] G. Larrinaga, J. Gill, J.J. Meana, F. Ruiz, L.F. Callado, J. Irazusta, Aminopeptidase activity in the postmortem brain of human heroin addicts, Neurochem. Int. 46 (2005) 213–219. [26] J.-P. Laulin, E. Celerier, A. Larcher, M. Le Moal, G. Simonnet, Opiate tolerance to daily heroin administration: an apparent phenomenon associated with enhanced pain sensitivity, Neuroscience 89 (1999) 631–636. [27] J.-P. Laulin, P. Maurette, J.-B. Corcuff, C. Rivat, M. Chauvin, G. Simonnet, The role of ketamine in preventing fentanyl-induced hyperlalgesia and subsequent acute morphine tolerance, Anesth. Analog. 94 (2002) 1263–1269. [28] G.S. Ling, R. Simantov, J.A. Clark, G.W. Pasternak, Naloxonazine actions in vivo, Eur. J. Pharmacol. 129 (1986) 33–38. [29] H. Machelska, D. Lobuz, R. Przewlocki, B. Przewlocka, Inhibition of nitric oxide synthase enhances antinociception mediated by mu, delta, and kappa opioid receptors in acute and prolonged pain in the rat spinal cord, J. Pharmacol. Exp. Ther. 282 (1997) 977–984. [30] C.A. Maggi, Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves, Prog. Neurobiol. 45 (1995) 1–98. [31] J. Mao, D.D. Price, D.J. Mayer, Mechanisms of hyperalgesia and morphine tolerance: a current view of their interactions, Pain 62 (1995) 259–274. [32] A. Mattia, T. Vanderah, H.I. Mosberg, F. Porreca, Lack of antinociceptive cross-tolerance between [d-Pen2, d-Pen5]enkephalin and [dAla2]deltorphin II in mice: evidence for receptor subtypes, J. Pharmacol. Exp. Ther. 258 (1991) 583–5878. [33] I. McFadzean, The ionic mechanisms underlying opioid actions, Neuropeptides 11 (1988) 173–180. [34] R. Oku, M. Satoh, N. Fujii, A. Otaka, H. Yajima, H. Takagi, Calcitonin gene-related peptide promotes nociception by potentiating the release of substance P from the spinal dorsal horn in rats, Brain Res. 403 (1987) 350–354. [35] G.A. Olson, R.D. Olson, A.J. Kastin, Review of endogenous opiates: 1991, Peptides 13 (1992) 1247–1287. [36] T.J. Park, C. Comer, A. Carol, Y. Lu, S.H. Hong, F.L. Rice, Somatosensory organization and behaviour in naked mole-rats. II. Peripheral structures, innervation, and selective lack of neuropeptides associated with thermoregulation and pain, J. Comp. Neurol. 465 (2003) 104–120. [37] C.G. Parsons, NMDA receptors as targets for drug action in neuropathies, Eur. J. Pharmacol. 429 (2001) 71–78. [38] M.F. Piercey, R.A. Lhati, L.A. Schroeder, U-50488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse, Life Sci. 31 (1982) 1197–1200. [39] F. Porreca, J.S. Heyman, J.I. Mosberg, J.R. Omnass, J.L. Vaught, Role of mu and delta receptors in the supraspinal and spinal analgesic effects of
68
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
P.K. Towett et al. / Brain Research Bulletin 71 (2006) 60–68 [d-Pen2 , d-Pen5 ]enkephalin in the mouse, J. Pharmacol. Exp. Ther. 241 (1987) 393–400. R.B. Raffa, R.P. Martinez, F. Porreca, Lack of antinociceptive efficacy of intracerebroventricular [d-Ala2 ,Glu4 ]deltorphin, but not [d-Pen2, dPen5]enkephalin, in the mu-opioid receptor deficient CXBK mouse, Eur. J. Pharmacol. 216 (1992) 453–456. W. Rosenthal, J. Hescheler, W. Trautwein, G. Schultz, Control of voltagedependent Ca2+ channels by G-protein coupled receptors, FASEB J. 2 (1988) 2784–2790. A. Safavi, L.B. Hersh, Degradation of dynorphin-related peptides by the puromycin sensitive aminopeptidase and aminopeptidase M, J. Neurochem. 65 (1995) 389–395. J.D. Silverman, L. Kruger, Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers, J. Neurocytol. 19 (1990) 789–801. M. Sofuoglu, P.S. Portoghese, A.E. Takemori, Differential antagonism of delta opioid agonists by naltrindole (NTI) and its benzofuran analogue (NTB) in mice: evidence for delta opioid receptor subtypes, J. Pharmacol. Exp. Ther. 257 (1991) 676–680. R.L. Spencer, V.J. Hruby, T.F. Burks, Body temperature response profiles for selective mu, delta, and kappa opioid agonists in restrained and unrestrained rats, J. Pharmacol. Exp. Ther. 246 (1988) 92–101. C.W. Stevens, Relative analgesic potency of mu, delta and kappa opioids after spinal administration in amphibians, J. Pharmacol. Exp. Ther. 276 (1996) 440–448. C.W. Stevens, A.J. Klopp, J.A. Facello, Analgesic potency of mu and kappa opioids after systemic administration in amphibians, J. Pharmacol. Exp. Ther. 269 (1994) 1086–1093. H.H. Suh, L.F. Tseng, Intrathecal beta-funaltrexamine antagonizes intracerebroventricular beta-endorphin—but not morphine-induced analgesia in mice, J. Pharmacol. Exp. Ther. 245 (1988) 587–593. H.H. Suh, L.F. Tseng, Different types of opioid receptors mediating analgesia induced by morphine, DAMGO, DPDPE, DADLE and betaendorphin in mice, Naunyn-Schmiedeberg’s Arch. Pharmacol. 342 (1990) 67–71.
[50] H.H. Suh, J.M. Fujimoto, L.F. Tseng, Differential mechanisms mediating beta-endorphin- and morphine-induced analgesia in mice, Eur. J. Pharmacol. 168 (1989) 61–70. [51] A.E. Takemori, B.Y. Ho, J.S. Naeseth, P.S. Portoghese, Norbinaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246 (1988) 255–258. [52] A. Tjolsen, O.-G. Berge, P.K. Eide, O.J. Broth, K. Hole, Apparent hyperalgesia after lesions of descending serotonergic pathways is due to increased tail skin temperature, Pain 33 (1988) 225–231. [53] P.K. Towett, T.I. Kanui, Effects of pethidine, acetylsalicylic acid, and indomethacin on pain and behavior in the mole-rat, Pharmacol. Biochem. Behav. 45 (1993) 153–159. [54] P.K. Towett, T.I. Kanui, Hyperalgesia following administration of morphine and pethidine in root rat (Tachyoryctes splendens), J. Vet. Pharmacol. Ther. 18 (1995) 68–71. [55] K.A. Trujillo, H. Akil, Opiate tolerance and dependence: recent findings and synthesis, New Biol. 10 (1991) 915–923. [56] M.B. Tyers, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69 (1980) 503–512. [57] T.D. Vanderah, N.M.H. Suenaga, M.H. Ossipov Jr., T.P. Malan, J. Lai, F. Porreca, Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance, J. Neurosci. 21 (2001) 279–286. [58] J.L. Vaught, J.R. Mathiesen, R.B. Raffa, Examination of the involvement of the mu and delta opioid receptors in analgesia using the mu-receptor deficient CXBK mouse, J. Pharmacol. Exp. Ther. 245 (1988) 13–16. [59] P.F. VonVoigtlander, R.A. Lahti, J.H. Ludens, U-50,488: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther. 224 (1983) 7–12. [60] T.L. Yaksh, G.J. Harty, B.M. Onfofrio, High doses of spinal morphine produce a non-opiate receptor mediated hyperesthesia: clinical and theoretic implications, Anesthesiology 64 (1986) 590–597. [61] S. Zhou, L. Bonesera, S.M. Carlton, Peripheral administration of NMDA, AMPA or KA results in pain behaviours in rats, Reuroreport 7 (1996) 895–900.