| Background:Three imidazoline receptor subtypes have been described to date:I1, I2and I3. I2receptors are widely distributed in the area postrema, interpeduncular and arcuate nuclei, pineal gland and ependymal. Previous studies have demonstrated their involvement in pain, depression and neuroprotection. These findings suggest that I2receptors might be a potential therapeutic target for certain brain disorders. Medicinal chemistry efforts have identified several highly selective I2receptor ligands via receptor binding assays. However, no report exists that compares the in vivo pharmacological mechanisms among these compounds. Therefore, we have used a drug discrimination procedure to investigate the nature of the discriminative stimulus effects of I2receptor ligands, compare their similarities and differences, and explore their pharmacological mechanism, which will provide valuable information for further studying the I2receptor functionality.Although there have been reports that the I2receptor ligand, CR4056, has anti-hyperalgesic effects, it is unknown whether other I2receptor ligands can reduce inflammatory and neuropathic pain. Here, we primarily compared the anti-hyperalgesic effects among different I2receptor ligands and found that repeated treatment with I2receptor ligands did not produce antinociceptive tolerance and cross-tolercance. Moreover, I2receptor ligands were effective at attenuating the affective component of pain. These results suggest that the I2receptors represent a potential target for chronic and neuropathic pain conditions. In addition, we further found that I2receptor ligands have unique pharmacologic mechanism compared with other analgesics using the delay discounting model. The data supported that I2receptor ligands might be a novel pharmacological opportunity for the management of chronic pain.I2receptors have not been cloned and the signaling mechanism still remains unknown. Given the crosstalk between I2receptors and ? opioid receptors and the pivotal role of PKCs/ERK pathway in the morphine-induced modulation of pain, we preliminarily hypothesized whether the PKCs/ERK pathway could play a role in the mechanism of the anti-hyperalgesic effects of I2receptor ligands via molecular biological techniques.Objectives:(1) To study the discriminative stimulus effects of I2receptor ligands, compare their similarities and differences, and explore the pharmacologic mechanisms among I2receptor ligands (2-BFI, CR4056, BU224, Phenyzoline and Tracizoline).(2) To investigate whether I2receptor ligands have antinociceptive effects in models of inflammatory and neuropathic pain and whether tolerance and cross-tolerance could be produced if I2receptor ligands were used repeatedly.(3) To explore whether I2receptor ligand CR4056has a comparable effect on impulsivity as other analgesics.(4) To elucidate the molecular mechanism underlying the anti-hyperalgesic effects of I2receptors for the first time by Western blot.Methods:(1) Drug Discrimination:Each group of eight male Sprague-Dawley rats was trained to discriminate one of five I2receptor ligands (2-BFI, CR4056, BU224, Phenyzoline or Tracizoline) from vehicle in a two-level food-reinforced drug discrimination procedure. Once these rats were well trained, they were then used to conduct tests of stimulus generalization, including I2receptor ligands (2-BFI, CR4056, BU224, Phenyzoline, Tracizoline, Idazoxan and RS45041), other ligands of neurotransmitter systems (?-opioid receptor agonists Morphine and Methadone, the NMDA receptor antagonist Ketamine, the indirect dopamine receptor agonist Methamphetamine, a2-adrenoceptor agonist Clonidine and the monoamine oxidase inhibitor Harmane). When pretreated with other antagonists of neurotransmitter systems (a2-adrenoceptor antagonist Yohimbine, ?-opioid receptor antagonist Naltrexone, D2receptor antagonist Haloperidol and5-HT2A receptor antagonist MDL100907), the discriminative stimulus effects of the training drug were tested again. These results from generalization tests (complete stimulus generalization, partial stimulus generalization and vehicle-like responding) could indicate whether the effects of the test drug are identical from those produced by the dose of training drug. Furthermore, combination tests (stimulus potentiation, stimulus antagonism and no effects on discrimination) were used to elucidate the mechanism of action of I2receptor ligands.(2) Pain models and pain measurements:Rats underwent CFA hindpaw injection or conducted chronic constriction injury to induce inflammatory and neuropathic pain respectively. After the treatment of I2receptor ligands acutely or repeatedly, the von Frey filiament test was used to measure mechanical hyperalgesia and the plantar test was used to measure thermal hyperalgesia in rats. Affective pain was studied by a place escape/avoidance paradigm in separate experiments.(3) Delay Discounting:Rats were divided into two groups randomly, and were trained to respond under a discrete-trial choice procedure. Response on one lever delivered one food pellet immediately and response on the other lever delivered three food pellets either immediately or after a delay. The delay to the larger reinforcer (0,5,10,20and40s) was increased within session across varied blocks. Following pretreatment with I2receptor ligand CR4056, ?-opioid receptor agonist Morphine and ?2-adrenoceptor agonist Clonidine respectively, impulsivity behavior was tested under the delay discounting procedure. Then either of two groups underwent chronic constriction injury (CCI) and the impulsivity behavior was determined again following pretreatment of CR4056, Morphine and Clonidine in different sessions.(4) Western blot:There are four groups rats, including SS group (saline s.c. on the paw and saline i.p.), CS group (CFA s.c. on the paw and saline i.p.), CB group (CFA s.c. on the paw and10mg/kg2-BFI i.p.) and SB group (saline s.c. on the paw and10mg/kg2-BFI i.p.).24h after subcutaneous injection of either CFA or saline, rats received treatment of saline or10mg/kg2-BFI intraperitoneally and decapitated30 min later. Tissue punches of hippocampus and spinal cords were harvested for determining the concentration of p-PKC, PKC, p-ERK and ERK via Western blot.Results:(1)5.6mg/kg2-BFI,10mg/kg CR4056,5.6mg/kg BU224and32mg/kg Phenyzoline can serve as a discriminative cue in a two-lever rat drug discrimination paradigm. However, all rats cannot reliably discriminate Tracizoline from saline after100training sessions.(2) Rats which acquired criteria to discriminate2-BFI from saline, were used for substitution and combination studies. Other I2receptor ligands CR4056, BU224, Tracizoline, RS45041and Idazoxan dose-dependently substituted for2-BFI, while Phenyzoline failed to substitute for2-BFI. Morphine and Methadone partially substituted for2-BFI. Idazoxan increased the discriminative stimulus effects of2-BFI, but not Tracizoline and Phenyzoline. Naltrexone, Haloperidol and MDL100907failed to alter the stimulus effects of2-BFI.(3) Rats which can discriminate CR4056from saline, were used for substitution and combination studies. Other I2receptor ligands Tracizoline, Phenyzoline, RS45041and Idazoxan dose-dependently substituted for CR4056. Other drugs that only occasioned partial or no CR4056-associated lever responding included Morphine, Clonidine, BU224,2-BFI, Harmane, Ketamine and Methamphetamine. Idazoxan, Tracizoline and BU224increased the discriminative stimulus effects of CR4056. Yohimbine, Naltrexone, Haloperidol and MDL100907failed to alter the stimulus effects of CR4056.(4) Rats which can discriminate BU224from saline, were used for substitution and combination studies. Other I2receptor ligands2-BFI, CR4056, Tracizoline, Phenyzoline, RS45041and Idazoxan dose-dependently substituted for BU224. Other drugs that only occasioned partial or no BU224-associated lever responding included Morphine, Clonidine, Ketamine and Methamphetamine. Harmane and Yohimbine occasioned increased responding on the drug lever, which is different from CR4056 and2-BFI. Tracizoline increased the discriminative stimulus effects of BU224. Idazoxan, Naltrexone, Haloperidol and MDL100907failed to alter the stimulus effects of BU224.(5) Rats which acquired criteria to discriminate Phenyzoline from saline, were used for substitution and combination studies. Other I2receptor ligands2-BFI, BU224, CR4056, Tracizoline, RS45041and Idazoxan dose-dependently substituted for Phenyzoline. Other drugs that only occasioned partial or no Phenyzoline-associated lever responding included Morphine, Clonidine, Ketamine and Methamphetamine. Harmane and Yohimbine occasioned increased responding on the Phenyzoline-associated lever, which is similar to BU224. Naltrexone, Haloperidol and MDL100907failed to alter the stimulus effects of Phenyzoline.(6) I2receptor ligands (2-BFI, BU224and Tracizoline) all dose-dependently attenuated mechanical and thermal hyperalgesia in CFA-treated rats. The anti-hyperalgesic effects of2-BFI in CFA-treated and CCI-treated rats were attenuated by the I2receptor ligand Idazoxan. The combination of2-BFI and morphine produced additive effects against mechanical hyperalgesia in CFA-treated rats. Repeated treatment (daily for7-9days) with2-BFI or CR4056did not produce antinociceptive tolerance in CFA-treated or CCI-treated rats, I2receptor ligands (2-BFI, BU224and CR4056) were all effective at attenuating place escape/avoidance behavior in CFA-treated rats.(7) Without delay, rats chose the larger reinforcer nearly exclusively. With increasing delay, rats progressively switched their choice from the larger to the smaller reinforcer in a delay-dependent manner,I2receptor ligand CR4056did not affect impulsive decision making, whereas I2receptor agonist Morphine and ?2-adrenoceptor agonist Clonidine increased impulsivity. There is no difference in discounting between both normal rats and pain rats pretreated with CR4056, Morphine and Clonidine respectively. Pain cannot alter the discounting behavior.(8) No significant differences were found between these four groups with regard to the expression level of p-PKC and PKC in both hippocampus and the lumbar cord (L4-L6) segment. Although neither p-ERK1/2nor ERK1/2expression levels were changed in the hippocampus, there is a trend that p-ERKl/2was increased in CS group compared with SS group. Interestingly, p-ERKl/2was markedly increased in CS, CB and CS groups in lumbar cord (L4-L6) segment. Moreover, ERK1/2expression was decreased significantly in SB group compared to SS group.Conclusions:(1) I2receptor ligands can serve as discriminative stimuli in rats, which demonstrated high pharmacological specificity and seemed to be primarily mediated by imidazoline I2receptors. The D2receptor,?2-adrenoceptor, ?-opioid receptor,5-HT2A receptor and NMDA receptor were not involved in the discriminative stimulus effects of I2receptor ligands. Our results indicated that I2receptor ligands have unique psychoactive properties and exert stimulant-like subjective effects.(2) These ligands share similar, but not identical, discriminative stimulus effects. These results suggest that there may be more than one binding site of I2receptors that contribute to the discriminative stimulus effects of these ligands. Idazoxan and BU224showed similar discriminative stimulus effects with other I2receptor ligands, which were in contrast to the reports in the literature using both compounds as I2receptor antagonists, and suggests that these compounds may be I2receptor partial agonists.(3) I2receptor ligands have anti-hyperalgesic effects in rat models of inflammatory and neuropathic pain and might represent a new treatment option for chronic pain.(4) Activation of PKC?/ERK1/2pathway may not be involved in the antinociception mechanism of I2receptors, which need further investigations in the future. |
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