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Lack of tolerance and morphine-induced cross-tolerance to the analgesia of chimeric peptide of Met-enkephalin and FMRFa
peptides 29 (2008) 2266–2275
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Lack of tolerance and morphine-induced cross-tolerance to the analgesia of chimeric peptide of Met-enkephalin and FMRFa Kshitij Gupta a, Ishwar Dutt Vats a, Yogendra Kumar Gupta b, Kishwar Saleem c, Santosh Pasha a,* a
Peptide Synthesis Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India c Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi 110025, India b
article info
abstract
Article history:
Chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFa-YFa), a k-opioid recep-
Received 3 April 2008
tor specific peptide, did not induce tolerance and cross-tolerance effects to its analgesic
Received in revised form
action on day 5 after pretreatment with either YFa or morphine for 4 days. However,
18 September 2008
pretreatment with YFa for 4 days led to the development of cross-tolerance to the analgesic
Accepted 18 September 2008
effects of morphine and also 4 days of pretreatment of morphine resulted in the expression
Published on line 26 September 2008
of tolerance to its own analgesic effects. Similar expression of tolerance and cross-tolerance were also observed when YFa was compared with the k receptor agonist peptide dynorphin
Keywords:
A(1–13) [DynA(1–13)]. Cross-tolerance effects between YFa and DynA(1–13) analgesia were
Tolerance
also not observed on day 5. Interestingly, when YFa and DynA(1–13) were tested for their
Cross-tolerance
analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–
Chimeric peptide (YFa)
13) whereas YFa maintained its analgesia for 5 days. Thus, chimeric peptide YFa may serve
Dynorphin A(1–13)
as a useful probe to understand pain modulation and expression of tolerance and cross-
Analgesia
tolerance behavior with other opioids. # 2008 Elsevier Inc. All rights reserved.
1.
Introduction
NPFF/FMRFa family of neuropeptides are known to have a significant role in pain modulation as well as in opioid tolerance and dependence in the mammalian central nervous system (CNS) [17,28,15,27]. NPFF potently antagonizes morphine induced supraspinal antinociception and that of certain endogenous opioid peptides [37] suggesting that it may function as an endogenous ‘antiopioid’ agent [8,11]. However, in addition to this NPFF also displayed certain opioid like effects. Intrathecal NPFF elicited long lasting antinociception in rats [12]. There is considerable pharmacological data which
shows that NPFF/FMRFa binding sites are distinct from opioid receptor sites [27,2] suggesting that the opioid modulating effects of these peptides may be mediated through their own set of specific receptor(s)/binding sites. Methionine-enkephalin-Arg6-Phe7 (MERF) belongs to opioid family of peptides [19] and is widely distributed in the CNS of diverse mammals [26,30]. The most noticeable feature of MERF is the presence of overlapping sequence of opioid peptide Metenkephalin and antiopioid peptide FMRF within it [14], indicating its interesting behavior other than opioid. This suggests that such type of amphiactive peptides may have a possible role in pain modulation as well as in tolerance and dependence.
* Corresponding author. Fax: +91 11 27667471. E-mail addresses: [email protected], [email protected] (S. Pasha). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.09.013
peptides 29 (2008) 2266–2275
Based on various reports of MERF and on accounts of the interaction of peptides of NPFF/FMRFa family with the opioid system a chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFamide-YFa) was designed as a useful probe to explore the role of endogenous amphiactive sequences like MERF in analgesia and its modulation as well as further establishing the role of NPFF/FMRFa in mechanisms leading to opiate tolerance and dependence [15]. In our earlier study we reported that intraperitoneal (IP) administration of YFa induced a dose dependent increase in tail flick latency in mice indicating its analgesic action. The effect was naloxone reversible suggesting its analgesic action was mediated by opioid receptors. YFa potentiated morphineinduced analgesia and attenuated development of tolerance to the analgesic action of morphine. These results threw light over the role of such types of amphiactive sequences in pain modulation [15]. Further, our group recently investigated that YFa is a kappa opioid receptor specific peptide. This specificity may be due to its nature of sequence, its ability to adopt a helix conformation [16] and most importantly its analgesic action was dose dependently antagonized by k-opioid receptor antagonist norbinaltorphimine [39]. In continuation of our efforts to understand the role of YFa in pain modulation and in lack of tolerance development to opiate analgesic effects, the present study examines the tolerance and cross-tolerance effects of YFa in relation with the standard opioid morphine by measuring their analgesic effects after pretreatment with either YFa or morphine. Tolerance and cross-tolerance effects of YFa were then compared with the structurally and pharmacologically similar peptide DynA(1–13), a kappa receptor agonist [33–35,39]. Same doses of morphine (20 mg/kg) and YFa (80 mg/kg) and time period of experiment for 5 days was chosen as described earlier in tolerance studies [15]. For comparison, the dose of 80 mg/kg for DynA(1–13) was kept equal to the dose of YFa. Receptor specificity studies were also performed at this high dose of 80 mg/kg of YFa and DynA(1–13) with an aim to study the change in affinity towards opioid receptors.
2.
Materials and methods
2.1.
Chemicals
Morphine hydrochloride was obtained from AIIMS. All the flourenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt) and rink amide resin were obtained from Novabiochem, Merck. N,N0 -Diisopropyl carbodiimide (DIPCI) was procured from Sigma. Acetonitrile and trifluoroacetic acid were obtained by Merck. 2,5-Dihydroxybenzoic acid was supplied by Bruker Daltonics Flex Analysis.
2.2.
Peptide synthesis
Peptides YFa and DynA(1–13) were synthesized by the solid phase method on automated peptide synthesizer (Advanced chemtech, USA), using the standard chemistry of fluorenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt)/N,N0 -diisopropyl carbodiimide (DIPCI) activation method on rink amide resin and wang resin respectively. The
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peptides were purified by RP-C18 column (mBondapakTM 10 mm, 7.8 mm 300 mm, Waters, USA) on semi-preparative reverse phase HPLC (Waters 600, USA) with a 40 min linear gradient from 10% to 90% acetonitrile containing 0.05% trifluoroacetic acid in water. The mass analysis of the peptides was done in linear positive ion mode by MALDI-Tof-Tof (Bruker Daltonics Flex Analysis, Germany) with 2,5-dihydroxybenzoic acid as the matrix. The peptide sequence was confirmed by automated peptide sequencing (Procise 491 Applied Biosystems, USA).
2.3.
Animals
Male swiss albino mice (25–30 g) were housed in groups of 7 per cage and accustomed to the animal room (temperature, 22– 25 8C) for 5 days and provided food and water ad libitum before the experiment. The animals were handled according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India and Animal Ethical Committee of Institute of Genomics and Integrative Biology approved the study.
2.4.
Tolerance and cross-tolerance testing
In the present study, we investigated the expression of opiate tolerance and cross-tolerance effects of YFa after pretreatment with YFa, morphine and DynA(1–13). The behavioral endpoint for tolerance and cross-tolerance was analgesia which was assessed by a local radiant heat tail-flick analgesiometer following the opiate treatments [6]. A radiant heat was applied to the dorsal surface of the tail of mice to display the tail flick response. The intensity of heat stimulus in the tail flick apparatus was adjusted to elicit a reaction in control or untreated animals within 3–5 s. Mice with control latencies (predrug treatment) of greater than 5 s were not used for tail-flick measurement. The cut-off time was set at 10 s to prevent the tail skin tissue damage. Three base line trials were given to the mice, each separated by 10 min. After the baseline measurements of respective group animals, they were injected intraperitoneally (IP) with the test compounds, e.g. saline, YFa—80 mg/kg, DynA(1–13)—80 mg/kg and morphine—20 mg/kg in 0.9% saline at 10 mL/kg twice daily for 4 days at 9:00 am and 4:30 pm and at 9:00 am on day 5 as per the protocol described below and the analgesia was tested at 5 min, 15 min, 30 min, 45 min and 60 min. The analgesic response was expressed as percentage maximum possible effect (% MPE) that was calculated as [(test latency control latency)/(10 control latency)] 100. The %MPE values depicted as mean S.E.M. for each treatment group. Three groups of experiments were done, each comprised of two sets of experiments.
2.4.1. Group A: tolerance and cross-tolerance testing of YFa with morphine The first set of experiments: experiment 1(i) examination of expression of tolerance to YFa analgesia after 4 days of either YFa or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to YFa analgesia after 4 days of either morphine or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to morphine analgesia after 4 days of either YFa
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or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to morphine analgesia after 4 days of either morphine or saline pretreatment.
2.4.2. Group B: tolerance and cross-tolerance testing of DynA(1–13) with morphine The first set of experiments: experiment 1(i) examination of expression of tolerance to DynA(1–13) analgesia after 4 days of either DynA(1–13) or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to DynA(1– 13) analgesia after 4 days of either morphine or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to morphine analgesia after 4 days of either DynA(1–13) or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to morphine analgesia after 4 days of either morphine or saline pretreatment.
2.4.3. Group C: tolerance and cross-tolerance testing of YFa with DynA(1–13) The first set of experiments: experiment 1(i) examination of expression of tolerance to YFa analgesia after 4 days of either YFa or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to YFa analgesia after 4 days of either DynA(1–13) or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to DynA(1–13) analgesia after 4 days of either YFa or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to DynA(1–13) analgesia after 4 days of either DynA(1–13) or saline pretreatment.
2.5. Effect of chronic treatment of YFa and DynA(1–13) on analgesia for 5 days The role of anti-opioid (FMRFa) moiety in YFa in comparison to DynA(1–13) was determined by examining the analgesic effect of YFa at 30 min (time point at which YFa shows maximum analgesia) and DynA(1–13) at 15 min (time point at which DynA(1–13) shows maximum analgesia) after treatment in the morning and evening for 5 days. Only maximum analgesic values measured after treatment in the morning were compared for both the peptides. Peptides YFa and DynA(1– 13) at 80 mg/kg in 0.9% saline at 10 mL/kg were administered individually twice daily for 4 days at 9:00 am and 4:30 pm and at 9:00 am on day 5 to the respective groups in mice. The analgesic effect (%MPE) was determined as described in Section 2.4.
2.6.
administration of YFa and DynA(1–13). The degree of affinity of YFa and DynA(1–13) towards opioid receptors was evaluated as a function of the antagonization of the analgesic effect of both the peptides caused by all the three opioid receptor antagonists at 5 min, 15 min, 30 min, 45 min and 60 min. All the antagonists were also administered alone at 1 mg/kg as controls and tested for their analgesic effects over the same time points (data not shown). The test samples were administered IP in 0.9% saline at 10 mL/kg in mice. The analgesic effect (%MPE) after antagonists pretreatment was determined as described in Section 2.4.
2.7.
Statistical analysis
Statistical significance was determined for the values represented as analgesia (%MPE) mean S.E.M. by one-way ANOVA at each point of time in all the experiments except for the experimental Section 3.2, where data was calculated by twotailed t-test. Significance level was set at P 0.05.
3.
Results
3.1.
Tolerance and cross-tolerance testing
3.1.1. Group A: tolerance and cross-tolerance testing of YFa with morphine 3.1.1.1. Experiment 1(i) absence of tolerance to YFa analgesia. It is quite apparent from Fig. 1, the analgesia (%MPE) values 63.75 6.39 (P < 0.0001), 69.00 5.16 (P < 0.0001), 82.32 9.88 (P < 0.0023), 60.20 6.81 (P < 0.0001), 52.85 5.04 (P < 0.0001), observed for YFa pretreated animals for 4 days, showed no appreciable difference in reduction of analgesia over 60 min as compared to the analgesia (%MPE) values 68.16 5.42, 75.56 6.45, 86.27 6.81, 65.99 7.36, 54.91 6.34 observed for saline pretreated animals over the same time points on administration of YFa on day 5. The lack of tolerance was
Receptor affinity studies
To understand the observed tolerance and cross-tolerance effects of YFa with morphine and kappa receptor agonist peptide DynA(1–13), both YFa and DynA(1–13) at a high dose of 80 mg/kg each were evaluated for affinity towards m, d and k opioid receptors by using naloxonazine (NLZ)-m opioid receptor antagonist, naltrindole (NLD)-d opioid receptor antagonist and norbinaltorphimine (NBI)-k opioid receptor antagonist. All the three opioid receptor antagonists were administered IP at the dose of 1 mg/kg 5 min prior to the
Fig. 1 – Lack of tolerance to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or YFa. Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
peptides 29 (2008) 2266–2275
Fig. 2 – Cross-tolerance was not observed to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
observed. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.1.2. Experiment 1(ii) absence of cross-tolerance to YFa analgesia. The analgesia (%MPE) values 69.68 7.35 (P < 0.0001), 71.56 7.58 (P < 0.0001), 85.82 4.41 (P < 0.0001), 66.68 7.05 (P < 0.0028), 59.65 5.87 (P < 0.0001), observed for morphine pretreated animals for 4 days, displayed no apparent difference in reduction of analgesia over 60 min test times from the analgesia (%MPE) values 68.16 5.42, 75.56 6.45, 86.27 6.81, 65.99 7.36, 54.91 6.34 observed for saline pretreated animals for 4 days over the same time points on administration of YFa on day 5 (Fig. 2). The crosstolerance to the analgesic effects of YFa did not develop after morphine pretreatment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
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Fig. 3 – Cross-tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or YFa (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
67.06 6.02, 74.23 6.69, 84.1 6.73, 79.05 7.69, 56.04 6.85 observed for animals receiving the saline pretreatment for 4 days, have significantly higher analgesia (%MPE) than the analgesia values 17.26 4.43 (P < 0.0001), 39.7 8.22 (P < 0.0001), 51.22 8.9 (P < 0.0001), 39.07 8.04 (P < 0.0006), 27.43 6.3 (P < 0.0001) observed for animals receiving the morphine pretreatment for 4 days over 60 min on administration of morphine on day 5. The tolerance to the analgesic effects of morphine appeared to have developed in the present experiment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
3.1.1.3. Experiment 2(i) cross-tolerance to morphine analgesia. It is quite apparent from Fig. 3, the analgesia (%MPE) values 67.06 6.02, 74.23 6.69, 84.1 6.73, 79.05 7.69, 56.04 6.85 observed for saline pretreated animals for 4 days are significantly higher than the analgesia (%MPE) values 24.3 5.34 (P < 0.0001), 48.4 6.91 (P < 0.0001), 30.87 7.62 (P < 0.0001), 28.44 12.12 (P < 0.0028), 15.63 9 (P < 0.0001) observed for animals receiving the YFa pretreatment for 4 days over 60 min on administration of morphine on day 5. Cross-tolerance to the analgesic effects of morphine appeared to have developed after YFa pretreatment. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.1.4. Experiment 2(ii) tolerance to morphine analgesia. It is quite evident from Fig. 4, the analgesia (%MPE) values
Fig. 4 – Tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
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Fig. 5 – Lack of tolerance to the analgesic effects of DynA(1– 13) (80 mg/kg) after 4 days of IP injections of saline or DynA(1–13). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1– 13) pretreated, saline pretreated and saline treated animals.
3.1.2. Group B: tolerance and cross-tolerance testing of DynA(1–13) with morphine 3.1.2.1. Experiment 1(i) absence of tolerance to DynA(1–13) analgesia. The analgesia (%MPE) values 66.56 7.714 (P < 0.0001), 83.93 4.238 (P < 0.0001), 69.00 5.161 (P < 0.0001), 59.89 6.535 (P < 0.0001), 53.98 7.738 (P < 0.0001) observed for DynA(1–13) pretreated animals for 4 days showed no appreciable reduction in analgesia (%MPE) over 60 min from the analgesia (%MPE) values 72.2 7.483, 87.62 2.583, 75.87 4.435, 63.83 6.941, 54.11 6.622 observed for saline pretreated animals over the same test times on administration of DynA(1– 13) on day 5 (Fig. 5). The lack of tolerance was observed. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
Fig. 6 – Cross-tolerance was not observed to the analgesic effects of DynA(1–13) (80 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
58.8 5.732 observed for animals receiving the saline pretreatment for 4 days have significantly higher analgesia (%MPE) values 26.65 5.762 (P < 0.0001), 35.59 5.015 (P < 0.0001), 30.88 7.628 (P < 0.0005), 27.28 7.207 (P < 0.0001), 18.97 7.118 (P < 0.0001) observed for animals receiving the DynA(1– 13) pretreatment for 4 days over 60 min test duration on administration of morphine on day 5. Cross-tolerance to the analgesic effects of morphine appeared to have developed after DynA(1–13) pretreatment. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
3.1.2.2. Experiment 1(ii) absence of cross-tolerance to DynA(1– 13) analgesia. The analgesia (%MPE) mean S.E.M. values 69.14 8.548 (P < 0.0001), 85.42 4.783 (P < 0.0001), 71.56 7.588 (P < 0.0001), 58.57 6.002 (P < 0.0001), 52.44 6.449 (P < 0.0001) observed for morphine pretreated animals for 4 days displayed no apparent in reduction of analgesia (%MPE) over 60 min from the analgesia (%MPE) values 72.2 7.483, 87.62 2.583, 75.87 4.435, 63.83 6.941, 54.11 6.622 observed for saline pretreated animals for 4 days over the same time points on administration of DynA(1–13) on day 5 (Fig. 6). Cross-tolerance to the analgesic effects of DynA(1–13) does not develop after morphine pretreatment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
3.1.2.3. Experiment 2(ii) cross-tolerance to morphine analgesia. It is evident from Fig. 7, the analgesia (%MPE) values 65.88 5.602,
75.81 4.392,
83.63 4.539,
78.52 5.931,
Fig. 7 – Cross-tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or DynA(1– 13) (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1– 13) pretreated, saline pretreated and saline treated animals.
peptides 29 (2008) 2266–2275
Fig. 8 – Cross-tolerance was not observed to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or DynA(1–13) (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1–13) pretreated, saline pretreated and saline treated animals.
3.1.2.4. Experiment 2(ii) tolerance to morphine analgesia. Similar results were observed as explained in Section 3.1.1. Group A: experiment 2(ii) tolerance to morphine analgesia Fig. 4. 3.1.3. Group C: tolerance and cross-tolerance testing of YFa with DynA(1–13) 3.1.3.1. Experiment 1(i) absence of tolerance to YFa analgesia. Similar results were observed as explained in Section 3.1.1. Group A: experiment 1(i) absence of tolerance to YFa analgesia Fig. 1.
3.1.3.2. Experiment 1(ii) absence of cross-tolerance to YFa analgesia. The analgesia (%MPE) values 69.26 3.516 (P < 0.0001), 74.16 5.07 (P < 0.0001), 83.09 3.838 (P < 0.0018), 63.13 7.072 (P < 0.0001), 51.43 6.815 (P < 0.0001) observed for DynA(1–13) pretreated animals for 4 days displayed no appreciable reduction in analgesia (%MPE) over 60 min test times from the analgesia (%MPE) values 68.15 4.548,76.2 5.634, 85.69 6.025, 66.3 7.5, 54.17 6.822 observed for saline pretreated animals for 4 days over the same test duration on administration of YFa on day 5 (Fig. 8). Crosstolerance to the analgesic effects of YFa did not develop after DynA(1–13) pretreatment. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
3.1.3.3. Experiment 2(i) absence of cross-tolerance to DynA(1– 13) analgesia. It is evident from Fig. 9, the analgesia (%MPE) values 71.59 4.533 (P < 0.0001), 83.45 3.854 (P < 0.0018), 73.43 4.467 (P < 0.0001), 57.39 5.748 (P < 0.00), 55.36 6.169 (P < 0.0001) observed for YFa pretreated animals for 4 days displayed no apparent reduction in analgesia (%MPE) over 60 min test duration from the analgesia (%MPE) values 73.23 6.138, 86.7 2.738, 76.00 6.853, 61.48 7.982,
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Fig. 9 – Lack of cross-tolerance to the analgesic effects of DynA(1–13) (80 mg/kg) after 4 days of IP injections of saline or YFa (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
55.7 4.331 observed for saline pretreated animals for 4 days over the same time points on administration of DynA(1–13) on day 5. Cross-tolerance to the analgesic effects of DynA(1–13) did not develop after YFa pretreatment. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.3.4. Experiment 2(ii) absence of tolerance to DynA(1–13) analgesia. Similar results were observed as explained in Section 3.1.2. Group B: experiment 1(i) absence of tolerance to DynA(1–13) analgesia Fig. 5.
3.2. Effect of chronic treatment of YFa, DynA(1–13) on analgesia for 5 days The analgesia (%MPE) for YFa at 30 min for 5 days did not show any reduction in analgesia values as compared to the analgesia (%MPE) observed for DynA(1–13) at 15 min for the same time period (Fig. 10). The analgesia (%MPE) values for DynA(1–13) showed reduction in analgesia 54.71% (P < 0.01) on day 3 in comparison to YFa analgesia 82.25% on day 3. The DynA(1–13) treated animals regained analgesia values on fifth day similar to the values observed on first day.
3.3.
Receptor affinity studies
The m, d and k-receptor antagonists antagonizes the analgesia (%MPE) of YFa in various degrees in comparison to analgesia (%MPE) obtained for YFa when it was tested alone for its analgesic effects, as shown in Fig. 11. A similar experiment was done with DynA(1–13) to compare its receptor affinity with YFa (Fig. 12). Antagonization of analgesia of 80 mg/kg YFa by opioid receptor antagonists suggested the affinity of YFa towards all the opioid receptors. NLZ antagonizes the
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Fig. 10 – (&) Analgesia (%MPE) mean W S.E.M. profile of YFa at 30 min for 5 days showing lack of tolerance to analgesia of YFa. (*) Analgesia (%MPE) mean W S.E.M. profile of DynA(1–13) at 15 min for 5 days showing tolerance to its analgesia on day 3 and then regaining of analgesia on day 5. *Significant differences between YFa and DynA(1–13) analgesia.
analgesia of YFa to 21.66 3.95 (P < 0.0005), 33.92 4.89 (P < 0.0001), 37.54 4.64 (P < 0.0001), 32.06 3.35 (P < 0.0001), 17.35 3.14 (P < 0.0001); NLD antagonizes the analgesia of YFa to 54.52 4.92 (P < 0.0001), 63.83 2.60 (P < 0.0007), 70.12
Fig. 11 – Norbinaltorphimine (NBI), naltrindole (NLD) and naloxonazine (NLZ) at 1 mg/kg antagonizes the analgesic action of 80 mg/kg YFa differentially. Data are expressed as analgesia (%MPE) mean W S.E.M. @Significant differences between animals treated with NBI 5 min before YFa, animals treated with YFa alone and NBI alone treated animals. #Significant differences between animals treated with NLZ 5 min before YFa, animals treated with YFa alone and NLZ alone treated animals. *Significant differences between animals treated with NLD 5 min before YFa, animals treated with YFa alone and NLD alone treated animals.
Fig. 12 – Norbinaltorphimine (NBI), Naltrindole (NLD) and Naloxonazine (NLZ) at 1 mg/kg antagonizes the analgesic action of 80 mg/kg DynA(1–13) differentially. Data are expressed as Analgesia (%MPE) Mean W SEM. @ Significant differences between animals treated with NBI 5 min before DynA(1–13), animals treated with DynA(1–13) alone and NBI alone treated animals. # Significant differences between animals treated with NLZ 5 min before DynA(1– 13), animals treated with DynA(1–13) alone and NLZ alone treated animals. *Significant differences between animals treated with NLD 5 min before DynA(1–13), animals treated with DynA(1–13) alone and NLD alone treated animals.
3.68 (P < 0.0001), 54.00 7.82 (P < 0.0001), 44.12 5.97 (P < 0.0001); whereas NBI caused highest antagonization in analgesia of YFa to 13.71 2.34 (P < 0.0001), 20.57 2.39 (P < 0.0005), 23.82 2.23 (P < 0.0001), 21.25 3.76 (P < 0.0001), 10.79 2.47 (P < 0.0001) when compared with analgesia of YFa alone, i.e.; 68.83 3.46, 75.41 2.86, 86.69 3.64, 66.00 4.39, 55.86 3.20 over 60 min time. The data reveal that YFa at 80 mg/kg dose shows highest affinity towards k receptor, moderate affinity towards m receptor and least affinity towards d receptor. Similarly, antagonization of analgesia of DynA(1–13) at dose of 80 mg/kg by opioid receptor antagonists suggested its affinity towards all the opioid receptors similar to YFa. NLZ antagonizes the analgesia of DynA(1–13) to 33.52 2.389 (P < 0.0001), 37.54 4.646 (P < 0.0001), 29.36 4.433 (P < 0.0001), 25.89 3.007 (P < 0.0001), 17.35 3.117 (P < 0.0001); NLD antagonizes the analgesia of DynA(1–13) to 52.17 2.935 (P < 0.0001), 65.12 3.684 (P < 0.0009), 63.83 2.603 (P < 0.0001), 54.24 4.406 (P < 0.0001), 42.12 4.877 (P < 0.0001); whereas NBI caused highest antagonization in analgesia of DynA(1–13) to 28.89 4.093 (P < 0.0001), 21.73 3.194 (P < 0.0005), 19.73 4.453 (P < 0.0001), 18.44 4.1999 (P < 0.0001), 9.23 3.541 (P < 0.0001) when compared with analgesia of DynA(1–13) alone, i.e.; 74.7 5.697, 86.39 4.298, 76.25 4.676, 63.26 4.791, 54.05 5.153 over 60 min test times. The data reveal that DynA(1–13) at 80 mg/kg has highest selectivity towards k receptor, moderate towards m receptor and least towards d receptor.
peptides 29 (2008) 2266–2275
All the antagonists were also administered alone at 1 mg/ kg (IP) as controls and displayed analgesia almost at the base line levels. One-way ANOVA analysis revealed significant differences for opioid receptor antagonists pretreated animals injected 5 min before YFa and DynA(1–13) from their respective antagonists alone treated animals and YFa and DynA(1– 13) alone treated animals.
4.
Discussion
Present studies demonstrated that pretreatment with either YFa 80 mg/kg (Fig. 1) or morphine 20 mg/kg (Fig. 2) for 4 days did not induce tolerance and cross-tolerance to YFa analgesia on day 5. However, 4 days of pretreatment of YFa (IP) led to the expression of cross-tolerance to the analgesic effects of morphine (Fig. 3) and also 4 days of pretreatment of morphine resulted in the expression of tolerance to the analgesic effects of morphine on day 5 (Fig. 4). In our earlier study, YFa showed kappa opioid receptor specificity at low dose [39], therefore similar experiment was done with the standard kappa opioid receptor agonist DynA(1–13) to compare its tolerance and cross-tolerance effects with the tolerance and cross-tolerance effects of YFa and morphine. DynA(1–13)-80 mg/kg showed similar profile for tolerance and cross-tolerance expression with morphine as observed between YFa and morphine on day 5 (Figs. 5–7 and 4). The tolerance and cross-tolerance expression between YFa and DynA(1–13) were also tested at the 80 mg/kg doses. Pretreatment with either YFa or DynA(1–13) for 4 days did not induce tolerance and cross-tolerance to either YFa or DynA(1– 13) analgesia on day 5 (Figs. 1, 5, 8 and 9). However, when YFa and DynA(1–13) at 80 mg/kg were measured for analgesic effects for all 5 days after chronic treatment, DynA(1–13) showed tolerance to its analgesia at 15 min on day 3, however on day 5 analgesia was regained up to the initial value of day 1 and was also comparable to the saline pretreated animals for 4 days followed with DynA(1–13) on day 5 (Fig. 10); contrary to this YFa maintained its same analgesic effect under the chronic treatment of 5 days. The above results prompted us to understand the opioid receptor specificity of YFa and DynA(1–13) at 80 mg/kg. Our previous study showed that although YFa and DynA(1–13) are kappa opioid receptor specific at low dose [39] but at 80 mg/kg both YFa and DynA(1–13) may also show preference to other opioid receptors in addition to kappa opioid receptor. Opioids that are relatively selective to a particular receptor may interact with other receptors when administered at high doses. This could lead to possible changes in their pharmacological profile [29]. Opioid receptors antagonists, viz.; NLZ, NLD and NBI were employed to investigate the specificity of YFa and DynA(1–13) at 80 mg/kg towards the opioid receptors. All the antagonists differentially antagonize analgesia of YFa and DynA(1–13), suggesting varying degree of affinity of YFa (Fig. 11) and DynA(1–13) (Fig. 12) towards opioid receptors. NBI caused greater reduction in analgesia of YFa and DynA(1–13) in comparison to NLZ and NLD whereas NLD caused the least. This indicates that both YFa and DynA(1–13) has higher affinity towards k-opioid receptor, moderate affinity towards m receptor and lowest affinity towards d receptor. Although
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dynorphin peptides are primarily endogenous ligands of the kopioid receptors but these peptides, and especially their shorter biotransformation products such as DynA(1–13) also bind to the other opioid receptors like m and d in addition to kopioid receptor [4,5,9,42,43]. YFa has close resemblance with DynA(1–13) so it is quite possible that YFa may behave like DynA(1–13) to bind m and d opioid receptors in addition to kopioid receptor. Similarity in the receptor specificity of YFa and DynA(1–13) towards opioid receptors explains the lack of cross-tolerance to the analgesic action of YFa (Fig. 2) and DynA(1–13) (Fig. 6) on day 5 over morphine pretreated animals. It is well known that chronic morphine treatment causes internalization and then down regulation but not reactivation of the m receptor [3,24] which results in tolerance to the analgesic action of morphine. It was also demonstrated that desensitization could take place before receptor down regulation in the development of opiate tolerance [24,41]. This implies that m receptors are desensitized by morphine (m specific agonist) in 4 days but YFa or Dyn A (1–13) may have caused reactivation of the internalized/ desensitized m receptors and do not cause their down regulation on day 5. Several studies in different species have shown that dynorphin A peptides modulate opioid effects [18], may provide analgesia and prevent or reverse opiate withdrawal signs and symptoms in morphine-dependent animals and possibly also in humans [1,36,40]. It is also a well-known fact that opioid receptors may undergo agonist specific induced internalization and reactivation [21]. A number of studies reported that some agonists induce internalization and reactivation of the opioid receptors while others do not [7,13,20–23,25,38]. Dynorphin/dynorphin A(1–13) peptides are known to cause internalization and may cause reactivation of the receptors and therefore are involved in the mechanism of opiate tolerance and dependence [20,25]. Thus, YFa and DynA(1–13) may be those agonists which can induce internalization and reactivation of the opioid receptors. Thus, YFa and DynA(1–13) show their analgesic action with the reactivated mu opioid receptors as well as with kappa and delta opioid receptors with the same degree of its interaction as observed previously. Chronic pretreatment of YFa did not show tolerance to its own analgesia on day 5 (Fig. 1) and also no depreciation of the maximum analgesia at 30 min during the 5 days (Fig. 10). Chronic treatment of DynA(1–13) also did not display tolerance to its analgesia on fifth day (Fig. 5); however, significant reduction in analgesia was observed on day 3 but regained on day 5 to almost same value as was observed on day 1 (Fig. 10) and similar to saline pretreated animals after administration of DynA(1–13) on day 5 at 15 min. This fall in analgesia on third day by DynA(1–13) points to different behavior between YFa and DynA(1–13). Although, YFa and DynA(1–13) are structurally and pharmacologically similar but YFa being a chimeric peptide of opioid (Met-enkephalin) and anti-opioid (FMRFa) peptides may be causing the difference in tolerance behavior. The possible explanation for this behavior may be that YFa besides stimulating the opioid receptors is either acting as a putative antagonist at antiopioid receptor binding site or causing indirect activation of opioid receptors through binding at antiopiate or non-opioid site [15] to induce lack of tolerance to its analgesia even on day 3 at this high dose of 80 mg/kg.
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These results also corroborate our previous findings of attenuation of tolerance to morphine analgesia [15]. Other factors like reactivation after internalization of opioid receptors may be one of the added contributing factors to the lack of tolerance to YFa analgesia. But DynA(1–13) lacks such antiopioid moiety and hence interacts only with opioid receptors. Chronic treatment with 80 mg/kg DynA(1–13) causes fall in analgesia on day 3 and then analgesia was recovered on day 5 which may be due to the agonist specific induced internalization of opioid receptors on day 3 and then reactivation of opioid receptors up to day 5 to cause reversal of analgesia. Cross-tolerance was observed to morphine analgesia on day 5 after pretreatment with either YFa (Fig. 3) or DynA(1–13) (Fig. 7) for 4 days, whereas YFa and DynA(1–13) did not cause tolerance to their own analgesia on fifth day. In case of YFa there was lack of tolerance due to the antagonist action of YFa at antiopioid receptors or indirect stimulation of opioid receptors through binding at antiopioid or non-opioid sites and reactivation of receptors by YFa; whereas in case of DynA(1–13) reactivation of receptors is the plausible cause. These results revealed that morphine should show analgesia on reactivated m receptors but cross-tolerance was observed to morphine analgesia. This suggested that peptides YFa and DynA(1–13) caused some conformational changes in the m receptor which morphine was not able to recognize completely to show its full analgesia. Opioid agonists may cause conformational changes in the receptors [10,31,32]. Tolerance was observed to morphine treated animals in comparison to saline treated animals after administration of morphine on day 5 (Fig. 4). This may be attributed to the fact that chronic morphine treatments do not cause reactivation of the m receptor after desensitization rather promotes internalization and then down regulation of m receptor [3,24,41]. This could be the plausible explanation for the development of tolerance to morphine analgesia on day 5 after morphine pretreatment for 4 days.
5.
Conclusions
Lack of tolerance and morphine-induced cross-tolerance were not observed to analgesic effects of YFa on day 5 after pretreatment with either YFa or morphine, respectively and the same effects were observed with k-opioid receptor agonist DynA(1–13) in relation with morphine on day 5. Moreover, cross-tolerance to analgesic effects of YFa or DynA(1–13) were also not observed on fifth day after pretreatment with YFa or DynA(1–13), respectively. Amusingly, when YFa and DynA(1– 13) were tested for their analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–13) whereas YFa maintained its analgesia for 5 days. Therefore, chimeric peptide YFa may serve as a useful tool to understand modulation of pain and expression of the tolerance and crosstolerance effects with other opiates.
Acknowledgements This work is supported by Council of Scientific and Industrial Research (CSIR). K. Gupta is a CSIR-SRF.
references
[1] Aceto MD, Dewey WL, Chana JK, Lee NM. Dynorphin A1–13 effects in nontolerant and morphine-dependent rhesus monkeys. Eur J Pharmacol 1982;83:139–42. [2] Allard M, Geoffre S, Legendre P, Vincent JD, Simonnet G. Characterisation of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 1989;500:169–76. [3] Bhargava HN, Gulati A. Down-regulation of brain and spinal cord m-opioid receptors in morphine tolerant-dependent rats. Eur J Pharmacol 1990;190:305–11. [4] Chavkin C, Goldstein A. Dynorphin is a specific endogenous ligand of the k-opioid receptor. Science (Wash DC) 1982;215:413–5. [5] Chen Y, Mestek A, Liu J, Yu L. Molecular cloning of a rat kopioid receptor reveals sequence similarities to the m- and d-opioid receptors. Biochem J 1993;295:625–8. [6] D’Amour FE, Smith DL. A method for determining loss of pain sensation. J Pharmacol Exp Ther 1941;72:74–9. [7] Ferguson SS. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 2001;53:1–24. [8] Galina HZ, Kastin AJ. Existence of antiopiate system as illustrated by MIF-1/Tyr-MIF-1. Life Sci 1986;39:2153–9. [9] Garzon J, Sanchez-Blazquez P, Gerhart J, Loh HH, Lee NM. Dynorphin1–13: interaction with other opiate ligand bindings in vitro. Brain Res 1984;302:392–6. [10] Gether U, Kobilka BK. G protein-coupled receptors. II. Mechanism of agonist activation. J Biol Chem 1998;273:17979–82. [11] Goodman CB, Elmer GI, Yang H-YT, Lee CH, Rothman RB. Modulation of opioid receptors by anti-opioid peptides. In: Tseng LF, editor. The pharmacology of opioid peptides. Singapore: Harwood Academic Publishers, GmbH; 1995. p. 303–19. [12] Gouarderes C, Sutak M, Zajac J-M, Jhamandas K. Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat. Eur J Pharmacol 1993;237:73–81. [13] Grecksch G, Bartzsch K, Widera A, Becker A, Ho¨llt V, Koch T. Development of tolerance and sensitization to different opioid agonists in rats. Psychopharmacology 2006;186:177–84. [14] Greenberg MJ, Painter SD, Dobble KE, Naggle GT, Price DA, Lehman HK. The molluscan neurosecretory peptide FMRFamide: comparative pharmacology and relationship to the enkephalins. Fed Proc 1983;42:82–6. [15] Gupta S, Pasha S, Gupta YK, Bhardwaj DK. Chimeric peptide of met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception. Peptides 1999;20:471–8. [16] Hanif K, Gupta K, Gupta S, Gupta YK, Maiti S, Pasha S. Chimeric peptide of met-enkephalin and FMRFa: effect of chlorination on conformation and antinociception. Neuro Lett 2006;403:131–5. [17] Harrison LM, Kastin AJ, Zadina JE. Opiate tolerance and dependence: receptors, G-proteins and antiopiates. Peptides 1998;19:1603–30. [18] Hooke LP, He L, Lee NM. Dynorphin A modulates acute and chronic opioid effects. J Pharmacol Exp Ther 1995;273:292–7. [19] Inturrisi CE, Umans JG, Wolff D, Stern AS, Lewis RV, Stein S, et al. Analgesic activity of the naturally occurring heptapeptide met5enkephalin-Arg6-Phe7. Proc Natl Acad Sci USA 1980;81:5512–4. [20] Jordan AB, Cvejic S, Devi LA. Kappa opioid receptor endocytosis by Dynorphin peptides. DNA Cell Biol 2000;19(1):19–27.
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[21] Koch T, Volker H. Role of receptor internalization in opioid tolerance and dependence. Pharmacol Ther 2008;117:199– 206. [22] Koenig JA, Edwardson JM. Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci 1997;18:276–87. [23] Law PY, Erickson LJ, El-Kouhen R, Dicker L, Solberg J, Wang W, et al. Receptor density and recycling affect the rate of agonist-induced desensitization of m-opioid receptor. Mol Pharmacol 2000;58:388–98. [24] Law PY, Hom DS, Loh HH. Opiate receptor downregulation and desensitization in neuroblastoma 3 glioma NG108-15 hybrid cells are two separate cellular adaptation processes. Mol Pharmacol 1983;24:413–24. [25] Liu-Chen L-Y. Agonist-induced regulation and trafficking of k opioid receptors. Life Sci 2004;75:511–36. [26] Majane EA, Iadarola MJ, Yang H-YT. Distribution of Met5enkephalin-Arg6-Phe7 in rat spinal cord. Brain Res 1983;264:336–69. [27] Malin DH, Lake JR, Hammond MV, Fowler DE, Rogillio RB, Brown SL, et al. FMRF-NH2 like mammalian octapeptide: possible role in opiate dependence and abstinence. Peptides 1990;11:969–72. [28] Panula P, Kalso E, Nieminen M-L, Kontinen V, Brandt A, Pertovaara A. Neuropeptide FF and modulation of pain. Brain Res 1999;848:191–6. [29] Gutstein HB, Akil H. In: Hardman JG, Limbird LE, Gilmann AG, editors. Goodman & Gilmann’s The Pharmacological Basis of Therapeutics. USA: McGraw-Hill; 2001 [Chapter 23] p. 575. [30] Rossier J, Audigier Y, Ling N, Cros J, Udenfriend S. Metenkephalin- Arg6-Phe7, present in high amounts in brain of rat, cattle and man, is an opioid agonist. Nature 1980;288:88–90. [31] Salamon Z, Cowell S, Varga E, Yamamura HI, Hruby VJ, Tollin G. Plasmon resonance studies of agonist/antagonist binding to the human d-opioid receptor: new structural insights into receptor–ligand interactions. Biophys J 2000;79:2463–74. [32] Salamon Z, Hruby VJ, Tollin G, Cowell S. Binding of agonists, antagonists and inverse agonists to the human d-opioid
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Lack of tolerance and morphine-induced cross-tolerance to the analgesia of chimeric peptide of Met-enkephalin and FMRFa Kshitij Gupta a, Ishwar Dutt Vats a, Yogendra Kumar Gupta b, Kishwar Saleem c, Santosh Pasha a,* a
Peptide Synthesis Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India c Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi 110025, India b
article info
abstract
Article history:
Chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFa-YFa), a k-opioid recep-
Received 3 April 2008
tor specific peptide, did not induce tolerance and cross-tolerance effects to its analgesic
Received in revised form
action on day 5 after pretreatment with either YFa or morphine for 4 days. However,
18 September 2008
pretreatment with YFa for 4 days led to the development of cross-tolerance to the analgesic
Accepted 18 September 2008
effects of morphine and also 4 days of pretreatment of morphine resulted in the expression
Published on line 26 September 2008
of tolerance to its own analgesic effects. Similar expression of tolerance and cross-tolerance were also observed when YFa was compared with the k receptor agonist peptide dynorphin
Keywords:
A(1–13) [DynA(1–13)]. Cross-tolerance effects between YFa and DynA(1–13) analgesia were
Tolerance
also not observed on day 5. Interestingly, when YFa and DynA(1–13) were tested for their
Cross-tolerance
analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–
Chimeric peptide (YFa)
13) whereas YFa maintained its analgesia for 5 days. Thus, chimeric peptide YFa may serve
Dynorphin A(1–13)
as a useful probe to understand pain modulation and expression of tolerance and cross-
Analgesia
tolerance behavior with other opioids. # 2008 Elsevier Inc. All rights reserved.
1.
Introduction
NPFF/FMRFa family of neuropeptides are known to have a significant role in pain modulation as well as in opioid tolerance and dependence in the mammalian central nervous system (CNS) [17,28,15,27]. NPFF potently antagonizes morphine induced supraspinal antinociception and that of certain endogenous opioid peptides [37] suggesting that it may function as an endogenous ‘antiopioid’ agent [8,11]. However, in addition to this NPFF also displayed certain opioid like effects. Intrathecal NPFF elicited long lasting antinociception in rats [12]. There is considerable pharmacological data which
shows that NPFF/FMRFa binding sites are distinct from opioid receptor sites [27,2] suggesting that the opioid modulating effects of these peptides may be mediated through their own set of specific receptor(s)/binding sites. Methionine-enkephalin-Arg6-Phe7 (MERF) belongs to opioid family of peptides [19] and is widely distributed in the CNS of diverse mammals [26,30]. The most noticeable feature of MERF is the presence of overlapping sequence of opioid peptide Metenkephalin and antiopioid peptide FMRF within it [14], indicating its interesting behavior other than opioid. This suggests that such type of amphiactive peptides may have a possible role in pain modulation as well as in tolerance and dependence.
* Corresponding author. Fax: +91 11 27667471. E-mail addresses: [email protected], [email protected] (S. Pasha). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.09.013
peptides 29 (2008) 2266–2275
Based on various reports of MERF and on accounts of the interaction of peptides of NPFF/FMRFa family with the opioid system a chimeric peptide of Met-enkephalin and FMRFa (YGGFMKKKFMRFamide-YFa) was designed as a useful probe to explore the role of endogenous amphiactive sequences like MERF in analgesia and its modulation as well as further establishing the role of NPFF/FMRFa in mechanisms leading to opiate tolerance and dependence [15]. In our earlier study we reported that intraperitoneal (IP) administration of YFa induced a dose dependent increase in tail flick latency in mice indicating its analgesic action. The effect was naloxone reversible suggesting its analgesic action was mediated by opioid receptors. YFa potentiated morphineinduced analgesia and attenuated development of tolerance to the analgesic action of morphine. These results threw light over the role of such types of amphiactive sequences in pain modulation [15]. Further, our group recently investigated that YFa is a kappa opioid receptor specific peptide. This specificity may be due to its nature of sequence, its ability to adopt a helix conformation [16] and most importantly its analgesic action was dose dependently antagonized by k-opioid receptor antagonist norbinaltorphimine [39]. In continuation of our efforts to understand the role of YFa in pain modulation and in lack of tolerance development to opiate analgesic effects, the present study examines the tolerance and cross-tolerance effects of YFa in relation with the standard opioid morphine by measuring their analgesic effects after pretreatment with either YFa or morphine. Tolerance and cross-tolerance effects of YFa were then compared with the structurally and pharmacologically similar peptide DynA(1–13), a kappa receptor agonist [33–35,39]. Same doses of morphine (20 mg/kg) and YFa (80 mg/kg) and time period of experiment for 5 days was chosen as described earlier in tolerance studies [15]. For comparison, the dose of 80 mg/kg for DynA(1–13) was kept equal to the dose of YFa. Receptor specificity studies were also performed at this high dose of 80 mg/kg of YFa and DynA(1–13) with an aim to study the change in affinity towards opioid receptors.
2.
Materials and methods
2.1.
Chemicals
Morphine hydrochloride was obtained from AIIMS. All the flourenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt) and rink amide resin were obtained from Novabiochem, Merck. N,N0 -Diisopropyl carbodiimide (DIPCI) was procured from Sigma. Acetonitrile and trifluoroacetic acid were obtained by Merck. 2,5-Dihydroxybenzoic acid was supplied by Bruker Daltonics Flex Analysis.
2.2.
Peptide synthesis
Peptides YFa and DynA(1–13) were synthesized by the solid phase method on automated peptide synthesizer (Advanced chemtech, USA), using the standard chemistry of fluorenylmethoxy carbonyl (Fmoc) amino acids and 1-hydroxybenzotriazole (HOBt)/N,N0 -diisopropyl carbodiimide (DIPCI) activation method on rink amide resin and wang resin respectively. The
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peptides were purified by RP-C18 column (mBondapakTM 10 mm, 7.8 mm 300 mm, Waters, USA) on semi-preparative reverse phase HPLC (Waters 600, USA) with a 40 min linear gradient from 10% to 90% acetonitrile containing 0.05% trifluoroacetic acid in water. The mass analysis of the peptides was done in linear positive ion mode by MALDI-Tof-Tof (Bruker Daltonics Flex Analysis, Germany) with 2,5-dihydroxybenzoic acid as the matrix. The peptide sequence was confirmed by automated peptide sequencing (Procise 491 Applied Biosystems, USA).
2.3.
Animals
Male swiss albino mice (25–30 g) were housed in groups of 7 per cage and accustomed to the animal room (temperature, 22– 25 8C) for 5 days and provided food and water ad libitum before the experiment. The animals were handled according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India and Animal Ethical Committee of Institute of Genomics and Integrative Biology approved the study.
2.4.
Tolerance and cross-tolerance testing
In the present study, we investigated the expression of opiate tolerance and cross-tolerance effects of YFa after pretreatment with YFa, morphine and DynA(1–13). The behavioral endpoint for tolerance and cross-tolerance was analgesia which was assessed by a local radiant heat tail-flick analgesiometer following the opiate treatments [6]. A radiant heat was applied to the dorsal surface of the tail of mice to display the tail flick response. The intensity of heat stimulus in the tail flick apparatus was adjusted to elicit a reaction in control or untreated animals within 3–5 s. Mice with control latencies (predrug treatment) of greater than 5 s were not used for tail-flick measurement. The cut-off time was set at 10 s to prevent the tail skin tissue damage. Three base line trials were given to the mice, each separated by 10 min. After the baseline measurements of respective group animals, they were injected intraperitoneally (IP) with the test compounds, e.g. saline, YFa—80 mg/kg, DynA(1–13)—80 mg/kg and morphine—20 mg/kg in 0.9% saline at 10 mL/kg twice daily for 4 days at 9:00 am and 4:30 pm and at 9:00 am on day 5 as per the protocol described below and the analgesia was tested at 5 min, 15 min, 30 min, 45 min and 60 min. The analgesic response was expressed as percentage maximum possible effect (% MPE) that was calculated as [(test latency control latency)/(10 control latency)] 100. The %MPE values depicted as mean S.E.M. for each treatment group. Three groups of experiments were done, each comprised of two sets of experiments.
2.4.1. Group A: tolerance and cross-tolerance testing of YFa with morphine The first set of experiments: experiment 1(i) examination of expression of tolerance to YFa analgesia after 4 days of either YFa or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to YFa analgesia after 4 days of either morphine or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to morphine analgesia after 4 days of either YFa
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peptides 29 (2008) 2266–2275
or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to morphine analgesia after 4 days of either morphine or saline pretreatment.
2.4.2. Group B: tolerance and cross-tolerance testing of DynA(1–13) with morphine The first set of experiments: experiment 1(i) examination of expression of tolerance to DynA(1–13) analgesia after 4 days of either DynA(1–13) or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to DynA(1– 13) analgesia after 4 days of either morphine or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to morphine analgesia after 4 days of either DynA(1–13) or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to morphine analgesia after 4 days of either morphine or saline pretreatment.
2.4.3. Group C: tolerance and cross-tolerance testing of YFa with DynA(1–13) The first set of experiments: experiment 1(i) examination of expression of tolerance to YFa analgesia after 4 days of either YFa or saline pretreatment, and in experiment 1(ii) examination of expression of cross-tolerance to YFa analgesia after 4 days of either DynA(1–13) or saline pretreatment. The second set of experiments: experiment 2(i) examination of the expression of cross-tolerance to DynA(1–13) analgesia after 4 days of either YFa or saline pretreatment, and in experiment 2(ii) examination of the expression of tolerance to DynA(1–13) analgesia after 4 days of either DynA(1–13) or saline pretreatment.
2.5. Effect of chronic treatment of YFa and DynA(1–13) on analgesia for 5 days The role of anti-opioid (FMRFa) moiety in YFa in comparison to DynA(1–13) was determined by examining the analgesic effect of YFa at 30 min (time point at which YFa shows maximum analgesia) and DynA(1–13) at 15 min (time point at which DynA(1–13) shows maximum analgesia) after treatment in the morning and evening for 5 days. Only maximum analgesic values measured after treatment in the morning were compared for both the peptides. Peptides YFa and DynA(1– 13) at 80 mg/kg in 0.9% saline at 10 mL/kg were administered individually twice daily for 4 days at 9:00 am and 4:30 pm and at 9:00 am on day 5 to the respective groups in mice. The analgesic effect (%MPE) was determined as described in Section 2.4.
2.6.
administration of YFa and DynA(1–13). The degree of affinity of YFa and DynA(1–13) towards opioid receptors was evaluated as a function of the antagonization of the analgesic effect of both the peptides caused by all the three opioid receptor antagonists at 5 min, 15 min, 30 min, 45 min and 60 min. All the antagonists were also administered alone at 1 mg/kg as controls and tested for their analgesic effects over the same time points (data not shown). The test samples were administered IP in 0.9% saline at 10 mL/kg in mice. The analgesic effect (%MPE) after antagonists pretreatment was determined as described in Section 2.4.
2.7.
Statistical analysis
Statistical significance was determined for the values represented as analgesia (%MPE) mean S.E.M. by one-way ANOVA at each point of time in all the experiments except for the experimental Section 3.2, where data was calculated by twotailed t-test. Significance level was set at P 0.05.
3.
Results
3.1.
Tolerance and cross-tolerance testing
3.1.1. Group A: tolerance and cross-tolerance testing of YFa with morphine 3.1.1.1. Experiment 1(i) absence of tolerance to YFa analgesia. It is quite apparent from Fig. 1, the analgesia (%MPE) values 63.75 6.39 (P < 0.0001), 69.00 5.16 (P < 0.0001), 82.32 9.88 (P < 0.0023), 60.20 6.81 (P < 0.0001), 52.85 5.04 (P < 0.0001), observed for YFa pretreated animals for 4 days, showed no appreciable difference in reduction of analgesia over 60 min as compared to the analgesia (%MPE) values 68.16 5.42, 75.56 6.45, 86.27 6.81, 65.99 7.36, 54.91 6.34 observed for saline pretreated animals over the same time points on administration of YFa on day 5. The lack of tolerance was
Receptor affinity studies
To understand the observed tolerance and cross-tolerance effects of YFa with morphine and kappa receptor agonist peptide DynA(1–13), both YFa and DynA(1–13) at a high dose of 80 mg/kg each were evaluated for affinity towards m, d and k opioid receptors by using naloxonazine (NLZ)-m opioid receptor antagonist, naltrindole (NLD)-d opioid receptor antagonist and norbinaltorphimine (NBI)-k opioid receptor antagonist. All the three opioid receptor antagonists were administered IP at the dose of 1 mg/kg 5 min prior to the
Fig. 1 – Lack of tolerance to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or YFa. Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
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Fig. 2 – Cross-tolerance was not observed to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
observed. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.1.2. Experiment 1(ii) absence of cross-tolerance to YFa analgesia. The analgesia (%MPE) values 69.68 7.35 (P < 0.0001), 71.56 7.58 (P < 0.0001), 85.82 4.41 (P < 0.0001), 66.68 7.05 (P < 0.0028), 59.65 5.87 (P < 0.0001), observed for morphine pretreated animals for 4 days, displayed no apparent difference in reduction of analgesia over 60 min test times from the analgesia (%MPE) values 68.16 5.42, 75.56 6.45, 86.27 6.81, 65.99 7.36, 54.91 6.34 observed for saline pretreated animals for 4 days over the same time points on administration of YFa on day 5 (Fig. 2). The crosstolerance to the analgesic effects of YFa did not develop after morphine pretreatment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
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Fig. 3 – Cross-tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or YFa (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
67.06 6.02, 74.23 6.69, 84.1 6.73, 79.05 7.69, 56.04 6.85 observed for animals receiving the saline pretreatment for 4 days, have significantly higher analgesia (%MPE) than the analgesia values 17.26 4.43 (P < 0.0001), 39.7 8.22 (P < 0.0001), 51.22 8.9 (P < 0.0001), 39.07 8.04 (P < 0.0006), 27.43 6.3 (P < 0.0001) observed for animals receiving the morphine pretreatment for 4 days over 60 min on administration of morphine on day 5. The tolerance to the analgesic effects of morphine appeared to have developed in the present experiment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
3.1.1.3. Experiment 2(i) cross-tolerance to morphine analgesia. It is quite apparent from Fig. 3, the analgesia (%MPE) values 67.06 6.02, 74.23 6.69, 84.1 6.73, 79.05 7.69, 56.04 6.85 observed for saline pretreated animals for 4 days are significantly higher than the analgesia (%MPE) values 24.3 5.34 (P < 0.0001), 48.4 6.91 (P < 0.0001), 30.87 7.62 (P < 0.0001), 28.44 12.12 (P < 0.0028), 15.63 9 (P < 0.0001) observed for animals receiving the YFa pretreatment for 4 days over 60 min on administration of morphine on day 5. Cross-tolerance to the analgesic effects of morphine appeared to have developed after YFa pretreatment. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.1.4. Experiment 2(ii) tolerance to morphine analgesia. It is quite evident from Fig. 4, the analgesia (%MPE) values
Fig. 4 – Tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
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Fig. 5 – Lack of tolerance to the analgesic effects of DynA(1– 13) (80 mg/kg) after 4 days of IP injections of saline or DynA(1–13). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1– 13) pretreated, saline pretreated and saline treated animals.
3.1.2. Group B: tolerance and cross-tolerance testing of DynA(1–13) with morphine 3.1.2.1. Experiment 1(i) absence of tolerance to DynA(1–13) analgesia. The analgesia (%MPE) values 66.56 7.714 (P < 0.0001), 83.93 4.238 (P < 0.0001), 69.00 5.161 (P < 0.0001), 59.89 6.535 (P < 0.0001), 53.98 7.738 (P < 0.0001) observed for DynA(1–13) pretreated animals for 4 days showed no appreciable reduction in analgesia (%MPE) over 60 min from the analgesia (%MPE) values 72.2 7.483, 87.62 2.583, 75.87 4.435, 63.83 6.941, 54.11 6.622 observed for saline pretreated animals over the same test times on administration of DynA(1– 13) on day 5 (Fig. 5). The lack of tolerance was observed. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
Fig. 6 – Cross-tolerance was not observed to the analgesic effects of DynA(1–13) (80 mg/kg) after 4 days of IP injections of saline or morphine (20 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between morphine pretreated, saline pretreated and saline treated animals.
58.8 5.732 observed for animals receiving the saline pretreatment for 4 days have significantly higher analgesia (%MPE) values 26.65 5.762 (P < 0.0001), 35.59 5.015 (P < 0.0001), 30.88 7.628 (P < 0.0005), 27.28 7.207 (P < 0.0001), 18.97 7.118 (P < 0.0001) observed for animals receiving the DynA(1– 13) pretreatment for 4 days over 60 min test duration on administration of morphine on day 5. Cross-tolerance to the analgesic effects of morphine appeared to have developed after DynA(1–13) pretreatment. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
3.1.2.2. Experiment 1(ii) absence of cross-tolerance to DynA(1– 13) analgesia. The analgesia (%MPE) mean S.E.M. values 69.14 8.548 (P < 0.0001), 85.42 4.783 (P < 0.0001), 71.56 7.588 (P < 0.0001), 58.57 6.002 (P < 0.0001), 52.44 6.449 (P < 0.0001) observed for morphine pretreated animals for 4 days displayed no apparent in reduction of analgesia (%MPE) over 60 min from the analgesia (%MPE) values 72.2 7.483, 87.62 2.583, 75.87 4.435, 63.83 6.941, 54.11 6.622 observed for saline pretreated animals for 4 days over the same time points on administration of DynA(1–13) on day 5 (Fig. 6). Cross-tolerance to the analgesic effects of DynA(1–13) does not develop after morphine pretreatment. One-way ANOVA analysis revealed significant analgesia differences for morphine and saline pretreated against saline treated animals at each point of time.
3.1.2.3. Experiment 2(ii) cross-tolerance to morphine analgesia. It is evident from Fig. 7, the analgesia (%MPE) values 65.88 5.602,
75.81 4.392,
83.63 4.539,
78.52 5.931,
Fig. 7 – Cross-tolerance to the analgesic effects of morphine (20 mg/kg) after 4 days of IP injections of saline or DynA(1– 13) (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1– 13) pretreated, saline pretreated and saline treated animals.
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Fig. 8 – Cross-tolerance was not observed to the analgesic effects of YFa (80 mg/kg) after 4 days of IP injections of saline or DynA(1–13) (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between DynA(1–13) pretreated, saline pretreated and saline treated animals.
3.1.2.4. Experiment 2(ii) tolerance to morphine analgesia. Similar results were observed as explained in Section 3.1.1. Group A: experiment 2(ii) tolerance to morphine analgesia Fig. 4. 3.1.3. Group C: tolerance and cross-tolerance testing of YFa with DynA(1–13) 3.1.3.1. Experiment 1(i) absence of tolerance to YFa analgesia. Similar results were observed as explained in Section 3.1.1. Group A: experiment 1(i) absence of tolerance to YFa analgesia Fig. 1.
3.1.3.2. Experiment 1(ii) absence of cross-tolerance to YFa analgesia. The analgesia (%MPE) values 69.26 3.516 (P < 0.0001), 74.16 5.07 (P < 0.0001), 83.09 3.838 (P < 0.0018), 63.13 7.072 (P < 0.0001), 51.43 6.815 (P < 0.0001) observed for DynA(1–13) pretreated animals for 4 days displayed no appreciable reduction in analgesia (%MPE) over 60 min test times from the analgesia (%MPE) values 68.15 4.548,76.2 5.634, 85.69 6.025, 66.3 7.5, 54.17 6.822 observed for saline pretreated animals for 4 days over the same test duration on administration of YFa on day 5 (Fig. 8). Crosstolerance to the analgesic effects of YFa did not develop after DynA(1–13) pretreatment. One-way ANOVA analysis revealed significant analgesia differences for DynA(1–13) and saline pretreated against saline treated animals at each point of time.
3.1.3.3. Experiment 2(i) absence of cross-tolerance to DynA(1– 13) analgesia. It is evident from Fig. 9, the analgesia (%MPE) values 71.59 4.533 (P < 0.0001), 83.45 3.854 (P < 0.0018), 73.43 4.467 (P < 0.0001), 57.39 5.748 (P < 0.00), 55.36 6.169 (P < 0.0001) observed for YFa pretreated animals for 4 days displayed no apparent reduction in analgesia (%MPE) over 60 min test duration from the analgesia (%MPE) values 73.23 6.138, 86.7 2.738, 76.00 6.853, 61.48 7.982,
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Fig. 9 – Lack of cross-tolerance to the analgesic effects of DynA(1–13) (80 mg/kg) after 4 days of IP injections of saline or YFa (80 mg/kg). Data are expressed as analgesia (%MPE) mean W S.E.M. *Significant differences between YFa pretreated, saline pretreated and saline treated animals.
55.7 4.331 observed for saline pretreated animals for 4 days over the same time points on administration of DynA(1–13) on day 5. Cross-tolerance to the analgesic effects of DynA(1–13) did not develop after YFa pretreatment. One-way ANOVA analysis revealed significant analgesia differences for YFa and saline pretreated against saline treated animals at each point of time.
3.1.3.4. Experiment 2(ii) absence of tolerance to DynA(1–13) analgesia. Similar results were observed as explained in Section 3.1.2. Group B: experiment 1(i) absence of tolerance to DynA(1–13) analgesia Fig. 5.
3.2. Effect of chronic treatment of YFa, DynA(1–13) on analgesia for 5 days The analgesia (%MPE) for YFa at 30 min for 5 days did not show any reduction in analgesia values as compared to the analgesia (%MPE) observed for DynA(1–13) at 15 min for the same time period (Fig. 10). The analgesia (%MPE) values for DynA(1–13) showed reduction in analgesia 54.71% (P < 0.01) on day 3 in comparison to YFa analgesia 82.25% on day 3. The DynA(1–13) treated animals regained analgesia values on fifth day similar to the values observed on first day.
3.3.
Receptor affinity studies
The m, d and k-receptor antagonists antagonizes the analgesia (%MPE) of YFa in various degrees in comparison to analgesia (%MPE) obtained for YFa when it was tested alone for its analgesic effects, as shown in Fig. 11. A similar experiment was done with DynA(1–13) to compare its receptor affinity with YFa (Fig. 12). Antagonization of analgesia of 80 mg/kg YFa by opioid receptor antagonists suggested the affinity of YFa towards all the opioid receptors. NLZ antagonizes the
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Fig. 10 – (&) Analgesia (%MPE) mean W S.E.M. profile of YFa at 30 min for 5 days showing lack of tolerance to analgesia of YFa. (*) Analgesia (%MPE) mean W S.E.M. profile of DynA(1–13) at 15 min for 5 days showing tolerance to its analgesia on day 3 and then regaining of analgesia on day 5. *Significant differences between YFa and DynA(1–13) analgesia.
analgesia of YFa to 21.66 3.95 (P < 0.0005), 33.92 4.89 (P < 0.0001), 37.54 4.64 (P < 0.0001), 32.06 3.35 (P < 0.0001), 17.35 3.14 (P < 0.0001); NLD antagonizes the analgesia of YFa to 54.52 4.92 (P < 0.0001), 63.83 2.60 (P < 0.0007), 70.12
Fig. 11 – Norbinaltorphimine (NBI), naltrindole (NLD) and naloxonazine (NLZ) at 1 mg/kg antagonizes the analgesic action of 80 mg/kg YFa differentially. Data are expressed as analgesia (%MPE) mean W S.E.M. @Significant differences between animals treated with NBI 5 min before YFa, animals treated with YFa alone and NBI alone treated animals. #Significant differences between animals treated with NLZ 5 min before YFa, animals treated with YFa alone and NLZ alone treated animals. *Significant differences between animals treated with NLD 5 min before YFa, animals treated with YFa alone and NLD alone treated animals.
Fig. 12 – Norbinaltorphimine (NBI), Naltrindole (NLD) and Naloxonazine (NLZ) at 1 mg/kg antagonizes the analgesic action of 80 mg/kg DynA(1–13) differentially. Data are expressed as Analgesia (%MPE) Mean W SEM. @ Significant differences between animals treated with NBI 5 min before DynA(1–13), animals treated with DynA(1–13) alone and NBI alone treated animals. # Significant differences between animals treated with NLZ 5 min before DynA(1– 13), animals treated with DynA(1–13) alone and NLZ alone treated animals. *Significant differences between animals treated with NLD 5 min before DynA(1–13), animals treated with DynA(1–13) alone and NLD alone treated animals.
3.68 (P < 0.0001), 54.00 7.82 (P < 0.0001), 44.12 5.97 (P < 0.0001); whereas NBI caused highest antagonization in analgesia of YFa to 13.71 2.34 (P < 0.0001), 20.57 2.39 (P < 0.0005), 23.82 2.23 (P < 0.0001), 21.25 3.76 (P < 0.0001), 10.79 2.47 (P < 0.0001) when compared with analgesia of YFa alone, i.e.; 68.83 3.46, 75.41 2.86, 86.69 3.64, 66.00 4.39, 55.86 3.20 over 60 min time. The data reveal that YFa at 80 mg/kg dose shows highest affinity towards k receptor, moderate affinity towards m receptor and least affinity towards d receptor. Similarly, antagonization of analgesia of DynA(1–13) at dose of 80 mg/kg by opioid receptor antagonists suggested its affinity towards all the opioid receptors similar to YFa. NLZ antagonizes the analgesia of DynA(1–13) to 33.52 2.389 (P < 0.0001), 37.54 4.646 (P < 0.0001), 29.36 4.433 (P < 0.0001), 25.89 3.007 (P < 0.0001), 17.35 3.117 (P < 0.0001); NLD antagonizes the analgesia of DynA(1–13) to 52.17 2.935 (P < 0.0001), 65.12 3.684 (P < 0.0009), 63.83 2.603 (P < 0.0001), 54.24 4.406 (P < 0.0001), 42.12 4.877 (P < 0.0001); whereas NBI caused highest antagonization in analgesia of DynA(1–13) to 28.89 4.093 (P < 0.0001), 21.73 3.194 (P < 0.0005), 19.73 4.453 (P < 0.0001), 18.44 4.1999 (P < 0.0001), 9.23 3.541 (P < 0.0001) when compared with analgesia of DynA(1–13) alone, i.e.; 74.7 5.697, 86.39 4.298, 76.25 4.676, 63.26 4.791, 54.05 5.153 over 60 min test times. The data reveal that DynA(1–13) at 80 mg/kg has highest selectivity towards k receptor, moderate towards m receptor and least towards d receptor.
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All the antagonists were also administered alone at 1 mg/ kg (IP) as controls and displayed analgesia almost at the base line levels. One-way ANOVA analysis revealed significant differences for opioid receptor antagonists pretreated animals injected 5 min before YFa and DynA(1–13) from their respective antagonists alone treated animals and YFa and DynA(1– 13) alone treated animals.
4.
Discussion
Present studies demonstrated that pretreatment with either YFa 80 mg/kg (Fig. 1) or morphine 20 mg/kg (Fig. 2) for 4 days did not induce tolerance and cross-tolerance to YFa analgesia on day 5. However, 4 days of pretreatment of YFa (IP) led to the expression of cross-tolerance to the analgesic effects of morphine (Fig. 3) and also 4 days of pretreatment of morphine resulted in the expression of tolerance to the analgesic effects of morphine on day 5 (Fig. 4). In our earlier study, YFa showed kappa opioid receptor specificity at low dose [39], therefore similar experiment was done with the standard kappa opioid receptor agonist DynA(1–13) to compare its tolerance and cross-tolerance effects with the tolerance and cross-tolerance effects of YFa and morphine. DynA(1–13)-80 mg/kg showed similar profile for tolerance and cross-tolerance expression with morphine as observed between YFa and morphine on day 5 (Figs. 5–7 and 4). The tolerance and cross-tolerance expression between YFa and DynA(1–13) were also tested at the 80 mg/kg doses. Pretreatment with either YFa or DynA(1–13) for 4 days did not induce tolerance and cross-tolerance to either YFa or DynA(1– 13) analgesia on day 5 (Figs. 1, 5, 8 and 9). However, when YFa and DynA(1–13) at 80 mg/kg were measured for analgesic effects for all 5 days after chronic treatment, DynA(1–13) showed tolerance to its analgesia at 15 min on day 3, however on day 5 analgesia was regained up to the initial value of day 1 and was also comparable to the saline pretreated animals for 4 days followed with DynA(1–13) on day 5 (Fig. 10); contrary to this YFa maintained its same analgesic effect under the chronic treatment of 5 days. The above results prompted us to understand the opioid receptor specificity of YFa and DynA(1–13) at 80 mg/kg. Our previous study showed that although YFa and DynA(1–13) are kappa opioid receptor specific at low dose [39] but at 80 mg/kg both YFa and DynA(1–13) may also show preference to other opioid receptors in addition to kappa opioid receptor. Opioids that are relatively selective to a particular receptor may interact with other receptors when administered at high doses. This could lead to possible changes in their pharmacological profile [29]. Opioid receptors antagonists, viz.; NLZ, NLD and NBI were employed to investigate the specificity of YFa and DynA(1–13) at 80 mg/kg towards the opioid receptors. All the antagonists differentially antagonize analgesia of YFa and DynA(1–13), suggesting varying degree of affinity of YFa (Fig. 11) and DynA(1–13) (Fig. 12) towards opioid receptors. NBI caused greater reduction in analgesia of YFa and DynA(1–13) in comparison to NLZ and NLD whereas NLD caused the least. This indicates that both YFa and DynA(1–13) has higher affinity towards k-opioid receptor, moderate affinity towards m receptor and lowest affinity towards d receptor. Although
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dynorphin peptides are primarily endogenous ligands of the kopioid receptors but these peptides, and especially their shorter biotransformation products such as DynA(1–13) also bind to the other opioid receptors like m and d in addition to kopioid receptor [4,5,9,42,43]. YFa has close resemblance with DynA(1–13) so it is quite possible that YFa may behave like DynA(1–13) to bind m and d opioid receptors in addition to kopioid receptor. Similarity in the receptor specificity of YFa and DynA(1–13) towards opioid receptors explains the lack of cross-tolerance to the analgesic action of YFa (Fig. 2) and DynA(1–13) (Fig. 6) on day 5 over morphine pretreated animals. It is well known that chronic morphine treatment causes internalization and then down regulation but not reactivation of the m receptor [3,24] which results in tolerance to the analgesic action of morphine. It was also demonstrated that desensitization could take place before receptor down regulation in the development of opiate tolerance [24,41]. This implies that m receptors are desensitized by morphine (m specific agonist) in 4 days but YFa or Dyn A (1–13) may have caused reactivation of the internalized/ desensitized m receptors and do not cause their down regulation on day 5. Several studies in different species have shown that dynorphin A peptides modulate opioid effects [18], may provide analgesia and prevent or reverse opiate withdrawal signs and symptoms in morphine-dependent animals and possibly also in humans [1,36,40]. It is also a well-known fact that opioid receptors may undergo agonist specific induced internalization and reactivation [21]. A number of studies reported that some agonists induce internalization and reactivation of the opioid receptors while others do not [7,13,20–23,25,38]. Dynorphin/dynorphin A(1–13) peptides are known to cause internalization and may cause reactivation of the receptors and therefore are involved in the mechanism of opiate tolerance and dependence [20,25]. Thus, YFa and DynA(1–13) may be those agonists which can induce internalization and reactivation of the opioid receptors. Thus, YFa and DynA(1–13) show their analgesic action with the reactivated mu opioid receptors as well as with kappa and delta opioid receptors with the same degree of its interaction as observed previously. Chronic pretreatment of YFa did not show tolerance to its own analgesia on day 5 (Fig. 1) and also no depreciation of the maximum analgesia at 30 min during the 5 days (Fig. 10). Chronic treatment of DynA(1–13) also did not display tolerance to its analgesia on fifth day (Fig. 5); however, significant reduction in analgesia was observed on day 3 but regained on day 5 to almost same value as was observed on day 1 (Fig. 10) and similar to saline pretreated animals after administration of DynA(1–13) on day 5 at 15 min. This fall in analgesia on third day by DynA(1–13) points to different behavior between YFa and DynA(1–13). Although, YFa and DynA(1–13) are structurally and pharmacologically similar but YFa being a chimeric peptide of opioid (Met-enkephalin) and anti-opioid (FMRFa) peptides may be causing the difference in tolerance behavior. The possible explanation for this behavior may be that YFa besides stimulating the opioid receptors is either acting as a putative antagonist at antiopioid receptor binding site or causing indirect activation of opioid receptors through binding at antiopiate or non-opioid site [15] to induce lack of tolerance to its analgesia even on day 3 at this high dose of 80 mg/kg.
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These results also corroborate our previous findings of attenuation of tolerance to morphine analgesia [15]. Other factors like reactivation after internalization of opioid receptors may be one of the added contributing factors to the lack of tolerance to YFa analgesia. But DynA(1–13) lacks such antiopioid moiety and hence interacts only with opioid receptors. Chronic treatment with 80 mg/kg DynA(1–13) causes fall in analgesia on day 3 and then analgesia was recovered on day 5 which may be due to the agonist specific induced internalization of opioid receptors on day 3 and then reactivation of opioid receptors up to day 5 to cause reversal of analgesia. Cross-tolerance was observed to morphine analgesia on day 5 after pretreatment with either YFa (Fig. 3) or DynA(1–13) (Fig. 7) for 4 days, whereas YFa and DynA(1–13) did not cause tolerance to their own analgesia on fifth day. In case of YFa there was lack of tolerance due to the antagonist action of YFa at antiopioid receptors or indirect stimulation of opioid receptors through binding at antiopioid or non-opioid sites and reactivation of receptors by YFa; whereas in case of DynA(1–13) reactivation of receptors is the plausible cause. These results revealed that morphine should show analgesia on reactivated m receptors but cross-tolerance was observed to morphine analgesia. This suggested that peptides YFa and DynA(1–13) caused some conformational changes in the m receptor which morphine was not able to recognize completely to show its full analgesia. Opioid agonists may cause conformational changes in the receptors [10,31,32]. Tolerance was observed to morphine treated animals in comparison to saline treated animals after administration of morphine on day 5 (Fig. 4). This may be attributed to the fact that chronic morphine treatments do not cause reactivation of the m receptor after desensitization rather promotes internalization and then down regulation of m receptor [3,24,41]. This could be the plausible explanation for the development of tolerance to morphine analgesia on day 5 after morphine pretreatment for 4 days.
5.
Conclusions
Lack of tolerance and morphine-induced cross-tolerance were not observed to analgesic effects of YFa on day 5 after pretreatment with either YFa or morphine, respectively and the same effects were observed with k-opioid receptor agonist DynA(1–13) in relation with morphine on day 5. Moreover, cross-tolerance to analgesic effects of YFa or DynA(1–13) were also not observed on fifth day after pretreatment with YFa or DynA(1–13), respectively. Amusingly, when YFa and DynA(1– 13) were tested for their analgesic effects for 5 days, reduction in analgesia on day 3 was observed in case of DynA(1–13) whereas YFa maintained its analgesia for 5 days. Therefore, chimeric peptide YFa may serve as a useful tool to understand modulation of pain and expression of the tolerance and crosstolerance effects with other opiates.
Acknowledgements This work is supported by Council of Scientific and Industrial Research (CSIR). K. Gupta is a CSIR-SRF.
references
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