The molecular mechanisms of the analgesic action of melatonin

Objective To analyse the potential involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of melatonin.Methods A trauma-pain model was established in Wistar rats by combining right-hind limb amputation with 50 ℃ tail-flick test.Antinociception was determined by tail-flick latency to hot waster at 50 ℃.RT-PCR was used to observe the the expression of the M1OR and KOR gene.Results Melatonin produced the antinociceptive effect in dose-dependent manner after i.p or i.c.v.administration.Injected i.c.v.to rats,naloxone(10 μg)obviously antagonized the antinociceptive effect induced by i.p.melatonin.The expression of the M1OR gene in the rat hypothalamus and the KOR gene in the hippocampus was both significantly reduced at day 3 after injury,which was parallel to the reduction of the rat pain thresholds.However,the expression of the M1OR gene in the hippocampus and the KOR gene in the hypothalamus was not changed.Treatment of trauma-pain rats with melatonin(30-120 mg·kg-1)i.p.administrated induced the up-regulation of M1OR mRNA in the hypothalamus and the KOR mRNA in the hippocampus in a concentration-dependent manner.Conclusions The present observations suggest that Melatonin-induced antinociceptive effect may partially contribute to the up-regulation of M1OR mRNA level in the hypothalamus and the KOR mRNA level in the hippocampus.     

天元克痛方和盐酸曲马多单次给药对大鼠辐射热模型的镇痛作用研究

目的:探讨天元克痛方与盐酸曲马多单次给药后,对大鼠辐射热模型的镇痛作用。方法:63只Wistar大鼠随机分为5组,分别给予无菌蒸馏水、盐酸曲马多和天元克痛方,用辐射热甩尾法测定大鼠痛阈。结果:单次给药后,盐酸曲马多组的痛阈值与空白组相比,在60、90、120min均有显著性差异;与空白组相比,天元克痛方各组基本无显著性差异。结论:盐酸曲马多组在单次给药后60min～120min能使辐射热模型大鼠的痛阈延长(与空白组相比,P<0.05或P<0.01);天元克痛方各组虽有痛阈延长现象,但对辐射热引起的短暂锐痛无明显作用(P>0.05)。     

Possible involvement of cholinergic and opioid receptor mechanisms in fluoxetine mediated antinociception response in streptozotocin-induced diabetic mice

Clinical and experimental studies have been reported that antidepressant drugs can be used as co-analgesics in the management of neuropathic pain. However, the mechanism through which they alleviate pain still remains unclear. The aim of the present study was to investigate the possible mechanism of action of fluoxetine-induced antinociceptive effect in streptozotocin-induced diabetic mice, especially the involvement of non-serotonergic neurotransmitters and their receptors. Diabetes was induced in male Laka mice with a single intraperitoneal injection of streptozotocin (200 mg/kg). Four weeks after streptozotocin, diabetic mice were tested for pain responses in the tail-immersion and hot-plate assays. Diabetic mice exhibited significant hyperalgesia as compared with control mice. Fluoxetine (10 and 20 mg/kg, i.p) injected into diabetic mice produced an antinociceptive effect in both tail-immersion and hot-plate assays. The antinociceptive effect of fluoxetine in diabetic mice was significantly lower as compared with that in control mice. Pretreatment with a muscarinic receptor antagonist, atropine (2 and 5 mg/kg, i.p) and an opioid receptor antagonist, naloxone (2 and 5 mg/kg, i.p), but not the alpha(2)-adrenoreceptor antagonist, yohimbine (2 and 5 mg/kg, i.p) reversed the antinociceptive effect of fluoxetine (20 mg/kg). These results suggest that apart from serotonin pathway, muscarinic and opioid receptors also participate in fluoxetine-induced antinociception in diabetic neuropathic pain.     

Studies on the antinociception effect of melatonin and its mechanism in the rat

The antinociception effect of melatonin（Mel） and its mechanisms were studied on the trauma- pain model in the rat. A trauma - pain model was established in Wistar rats by combining right - hind limb amputation with 50℃ tail - flick test. The effects of Mel on central sensitization were studied through measuring the changes of the expression of c - fos （ an indicator of nociceptive transmission at the spinal level ） , subtance P（SP） and brain - derived neurotrophic factor （BDNF） in the spinal cord using immunohistochemistry. In addition, the contents of nitric oxide （NO） in brain and spinal tissues were also observed. The results showed that the immunoreactivity of BDNF and c - fos in spinal dorsal horn in injuried rats began to increase at the first day,     

Possible antinociceptive mechanisms of opioid receptor antagonists in the mouse formalin test

It has been reported that opioid receptor antagonist can induce antinociception in several nociceptive tests. In the intraplantar formalin pain model, however, opioid antagonist-induced antinociception, as well as its underlying mechanism, has not been well characterized. Therefore, in the mouse formalin test, we attempted to characterize the site of action and the possible opioid receptor subtypes. We found that naltrexone (a nonselective opioid antagonist) injected intraperitoneally (i.p., 1-20 mg/kg), intrathecally (i.t., 0.1-10 microg) and intracerebroventricularly (i.c.v., 0.1-10 microg) phase. Administration of beta-funaltrexamine (beta-FNA, 10-40 mg/kg i.p., 1.25-5 microg it or i.c.v.), naltrindole (1-10 mg/kg i.p., 1.25-5 microg it or i.c.v.) and nor-binaltorphimine (nor-BNI, 1-10 mg/kg i.p., 10-40 microg it or i.c.v.), which are selective mu-, delta- and kappa-opioid antagonists, respectively, also produced antinociception during the second phase. Additionally, we examined the involvement of the descending monoaminergic systems in the naltrexone-induced antinociception in the formalin test. Pretreatment with 5,7-dihydroxytryptamine (5,7-DHT, a serotonergic neurotoxin, 20 microg i.t.), but not N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin, 20 microg i.t.), reversed the naltrexone-induced antinociception during the second phase. Our results suggest that blockade of supraspinally or spinally located opioid receptors may play roles in the regulation of antinociception during the tonic painful stage. In addition, opioid receptors localized at the neuroterminal of the descending serotonergic, but not noradrenergic, inhibitory system in the spinal cord appear to be involved in opioid antagonist-induced antinociception during the second tonic phase of the formalin test.     

Possible mechanisms of action in melatonin reversal of morphine tolerance and dependence in mice

In our earlier study, we reported the ability of melatonin to reverse the development of morphine tolerance and dependence in mice. In the present study, we attempted to analyse the possible involvement of putative melatonin receptors, central and peripheral benzodiazepine receptors and the nitric oxide (NO) system in the mechanism of melatonin reversal of morphine tolerance and dependence in mice. Co-administration of L-N(G)-nitro arginine methyl ester (L-NAME) or melatonin with morphine during the induction phase (days 1-9) delayed the development of tolerance to the anti-nociceptive action of morphine and also reversed naloxone precipitated withdrawal jumpings. L-arginine administration during the induction phase enhanced the development of tolerance to the anti-nociceptive effect of morphine but had no effect on the naloxone-precipitated withdrawal response. During the expression phase (day 10), acute administration of melatonin or L-NAME reversed, whereas L-arginine facilitated, naloxone-precipitated withdrawal jumping in morphine-tolerant mice, but none of these drugs affected the nociceptive threshold in morphine-tolerant mice. Further, co-administration of melatonin or L-NAME with L-arginine during the induction phase antagonized later the effects on the development of morphine tolerance. Also, prior administration of melatonin or L-NAME reversed the L-arginine potentiation of naloxone-precipitated withdrawal jumping in morphine tolerant mice. Among the antagonists for putative melatonin receptors studied, neither luzindole (melatonin MT1 and MT2 receptor antagonist) nor prazosin (melatonin MT3 receptor antagonist) antagonized the melatonin reversal of morphine tolerance and dependence. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK11195), a peripheral but not central benzodiazepine receptor antagonist, flumazenil, partially antagonized the melatonin reversal of naloxone-precipitated withdrawal jumping in morphine-dependent mice, but had no effect on the reversal of morphine tolerance induced by melatonin. Overall, the present observations suggest that the melatonin-induced reversal of morphine tolerance and dependence may involve its ability to suppress nitric oxide synthase (NOS) activity. Further, the melatonin-induced reversal of morphine tolerance and dependence is not mediated through its actions via putative melatonin receptors. The agonistic activity of melatonin towards peripheral benzodiazepine receptors may partially contribute to the suppression of morphine dependence but not to the reversal of tolerance to the analgesic activity of morphine.     

Anti-depressant action of melatonin in chronic forced swimming-induced behavioral despair in mice, role of peripheral benzodiazepine receptor modulation

The possible antidepressant effect of physiological and pharmacological doses of melatonin was investigated in the Porsolt forced swimming-induced behavioral despair test. The duration of immobility period of BALB/c and C57BL/6J mice during a 6-min swim test was measured at noon (11:00–12:00 h), early dark (20:00–21:00 h) and at midnight (1:00–2:00 h), respectively. The circadian time cycle did not alter the duration of immobility in either strains of mice. Similarly, exogenously administered melatonin (10–1000 μg/kg50 nM to 5 μM/mouse), a dose that could act on high affinity melatonin receptors, did not modify the duration of immobility period at any of the time intervals studied in either strains of the mice. This suggested that neither circadian variation influenced the duration of immobility period of BALB/c and C57BL/6J mice nor at physiological doses melatonin showed any anti-depressant action. Acute administration of higher doses of melatonin (2.5–10 mg/kg) failed to induce any anti-depressant activity in mice which were subjected to forced swimming test for the first time. However, daily administration of melatonin (2.5–10 mg/kg) prior to swimming test significantly reversed the increase in immobility period that was observed on chronic exposure to swimming test. This effect was comparable with the effect of GABA-benzodiazepine (BZ) receptor agonists. Similarly, like GABAergic drugs, acute administration of melatonin also showed anti-depressant activity in a mice which were exposed to chronic forced swimming test. The anti-depressant action of melatonin was sensitive to reversal by peripheral BZ receptor antagonist, PK11195. Whereas, flumazenil failed to reverse the anti-depressant action of melatonin, thereby suggesting that central BZ receptor were not involved in its action. In conclusion the study showed that at pharmacological doses melatonin has anti-depressant action in chronic forced swimming-induced despair behavior by an action involving peripheral BZ receptors.     

Possible involvement of noradrenergic descending pain-modulating pathways in the mode of antinociceptive action of flupirtine, a novel non-opioid analgesic

These experimental studies were conducted to obtain information about the antinociceptive action of flupirtine within the central nervous system. Flupirtine dose-dependently increased pain threshold in the electrostimulated pain test in mice. Its antinociceptive activity was attenuated by simultaneous administration of the noradrenergic alpha 1/alpha 2-antagonist yohimbine and alpha 2-antagonist idazoxane. By contrast, the analgesia induced by codeine or morphine was not influenced by alpha 2-adrenergic antagonists at all. A striking resemblance could be observed in the pharmaco-EEG of freely moving rats treated with clonidine and flupirtine, respectively. The present results are consistent with the hypothesis that the noradrenergic descending pain-modulating system might be involved in the antinociceptive mode of action of flupirtine.     

Possible involvement of eicosanoids in the pharmacological action of bromelain

Bromelain, a proteolytic enzyme extracted from pineapple plants, was investigated for its capacity to interfere with arachidonic acid metabolism, since prostaglandins and other eicosanoids are well-known to be involved in the pathogenesis of inflammatory diseases. Bromelain was tested for its ability to interfere with eicosanoids generation in vivo in two experimentally-induced inflammatory reactions in the rat. Also antiplatelet aggregation activity of bromelain was studied in ex vivo rat platelets. The results seem to indicate an interference of bromelain with arachidonic acid cascade, which, however, deserves further investigation to be better assessed.     

Investigation of the involvement of opioid receptors in the action of anticonvulsants

This study investigates the possible involvement of opioid receptors in the action of a variety of anticonvulsant agents. The opioid antagonist naloxone (0.3, 1 mg/kg IP) and the selective -opioid antagonist cyprodime (3 mg/kg IP) significantly inhibited the increase in electroshock seizure threshold induced by phenytoin (3 mg/kg IP) in mice. The anticonvulsant effects of ethanol (1 g/kg IP) were also significantly antagonised by naloxone (1 mg/kg IP) but not by a 0.3 mg/kg IP dose or by cyprodime (3 mg/kg IP). The results with naloxone were confirmed using higher doses of phenytoin (10 mg/kg IP) and ethanol (1.5 g/kg IP). In contrast to the above findings, naloxone (0.3, 1 mg/kg IP) had no effect on the increase in seizure threshold induced by sodium valproate (200 mg/kg IP) or dizocilpine (MK801, 0.5 mg/kg IP) and paradoxically potentiated the increase in seizure threshold produced by phenobarbitone (15 mg/kg IP); carbamazepine (10 mg/kg IP) and the benzodiazepine agonist loprazolam (1 mg/kg IP), clearly differentiating these compounds from phenytoin and ethanol. These findings suggest that the anticonvulsant effects of phenytoin and ethanol (as assessed by their ability to prevent tonic hindlimb extension in the mouse electroshock model) may be mediated, at least in part, by the release of endogenous opioids and subsequent activation of opioid receptors (, in the case of phenytoin, but non-, in the case of ethanol) although direct activity at opioid receptors cannot be precluded.     

Cadmium-induced changes in Per 1 and Per 2 gene expression in rat hypothalamus and anterior pituitary: effect of melatonin

Chronic cadmium (Cd) administration affects the circadian release of pituitary hormones in rats. To assess whether Cd modifies expression of two major clock genes, period (Per) 1 and Per 2, in the hypothalamic-pituitary unit and to what extent the changes could be prevented by melatonin, rats were exposed to CdCl(2) (5ppm in drinking water) with or without melatonin (3 microg/mL drinking water) for 1 month and were killed at two time intervals, i.e. a the beginning of the rest span (09:00h) and at the middle of the activity span (01:00h). Hypothalamic and pituitary mRNA levels encoding Per 1 and Per 2 were measured by real-time PCR analysis. Cd treatment decreased expression of hypothalamic Per 1 gene at both time intervals, of hypothalamic Per 2 gene at 01:00h, and of adenohypophysial Per 1 and Per 2 genes at 09:00h. Melatonin administration counteracted most of the effects of Cd and augmented hypothalamic Per 2, and adenohypophysial Per 1 and Per 2 gene expression. The results indicate that Cd administered chronically in the drinking water to rats affected expression of clock genes in the hypothalamic-pituitary unit, an effect prevented by melatonin.     

Spinal opioid analgesic effects are enhanced in a model of unilateral inflammation/hyperalgesia: possible involvement of noradrenergic mechanisms.

We have examined the spinal analgesic activity of opioid agonists and antagonists in a model of short term, unilateral, carrageenan-induced inflammation/hyperalgesia. Rats received a single s.c. injection of carrageenan (2-6 mg in saline) 3-24 h prior to testing hindpaw withdrawal latencies to noxious thermal stimuli. Dose-response curves for intrathecally administered agonists with mu- and/or delta-opioid activity were shifted to the left for inflamed hindpaws when compared to contralateral non-inflamed paws. The selective kappa-receptor agonist U-50,488H had no activity in this analgesic assay on either inflamed or non-inflamed paws when administered intrathecally. However, systemic administration of U-50,488H did produce significant elevations of paw withdrawal latencies in inflamed paws. The alpha 2-adrenoceptor agonist clonidine also produced dose-dependent antinociception in the paw withdrawal assay after systemic or intrathecal administration. Inflamed hindpaws were significantly more sensitive to the antinociceptive effect of morphine on inflamed hindpaws was blocked by the opioid antagonist naloxone or the alpha 2-adrenoceptor antagonist idazoxan. The effect of clonidine was only blocked by idazoxan. Antagonists alone had no significant effect on withdrawal latencies. The data indicate that the analgesic action of opioids during conditions of inflammation may depend on an interaction with spinal noradrenergic pathways.     

5-Methoxypsoralen increases evening sleepiness in humans: Possible involvement of the melatonin secretion

Summary The acute and short-term effect of 5-methoxypsoralen (5-MOP) on daytime sleepiness was investigated in 12 healthy subjects according to a double-blind cross-over design. 5-MOP (40 mg/day) or placebo was orally administered (one week each) at 16.00 h. 5-MOP significantly increases daytime sleppiness 4 to 5 h after administration without affecting the subsequent sleep efficiency and duration. We found a positive correlation between sleepiness and melatonin levels.Our findings suggest that the melatonin secretion may play a role in the effect of 5-MOP on sleepiness in humans.     

Possible mechanisms underlying the mitogenic action of heptachlor in rat hepatocytes

The worldwide use of the organochlorine pesticide heptachlor has led to widespread contamination in the environment. Like many other organochlorine pesticides, heptachlor is considered to pose a threat to human health. It has been shown that heptachlor is a tumor-promoting agent, but the mechanisms involved still remain unclear. The negative response of heptachlor in in vitro genotoxicity test suggests that this pesticide displays its carcinogenicity through epigenetic pathways. With the growing evidence that proliferation accounts for the tumor-promoting effects of many agents, the purpose of this work was to investigate the mechanisms involved in the mitogenic activity of heptachlor in quiescent rat hepatocytes and to understand the properties of this compound as a tumor promoter in the liver. Heptachlor triggered significant proliferation in quiescent rat hepatocytes. Two mechanisms were delineated to support the mitogenic effect in the hepatocyte: activation of key kinases in signaling pathways and inhibition of apoptosis. Exposure to heptachlor led to activation of protein kinase C mitogenactivated protein kinases. Moreover, these results indicate that like many tumor promoters, heptachlor strongly inhibited TGFbeta-induced apoptosis and cytochrome c release into the cytosol. The levels of the anti-apoptotic protein Bcl-2 were also increased in the presence of heptachlor. In conclusion, these results indicate that heptachlor alters basic cell function by interfering with key cellular signaling pathways.     

Possible mechanisms for the anti-implantation action of dicofol in albino rats

Dicofol, a chlorinated pesticide showing estrogenic activity, was administered orally to virgin pregnant rats at doses of 300, 400, 500, 600, or 700 mg/kg/day for 7 consecutive days to examine its effect on blastocyst implantation. One group of rats received estradiol-17beta (10 microg) for comparison and control animals received similar quantities of olive oil. Autopsy on day 8 revealed that the olive-oil treated rats were pregnant and had a normal number of implantations and a normal duration of diestrus. Treatment with estradiol-17beta completely inhibited implantation, significantly decreased the duration of diestrus with concomitant increase in estrus. Treatment with 300, 400, or 500 mg/kg/d dicofol neither inhibited implantation nor significantly changed diestrus. Treatment with 600 mg dicofol partially inhibited implantation and significantly decreased diestrus with concomitant increase in estrus. Treatment with 700 mg dicofol completely inhibited implantation, and the uterus showed placental scars. This group exhibited a significant decrease in diestrus with concomitant increase in estrus. In all dicofol-treated rats, no significant changes were found in body weight or organ, except for a significant decrease in the weight of the uterus in groups receiving either 700 mg dicofol or estradiol-17beta. The inhibitory effect of dicofol on implantation may be due to an imbalanced estrogen-progesterone ratio, essential for implantation. The pesticide is neither tubal locking nor causes expulsion of the blastocyst from the uterus like estradiol-17beta.     

Discriminative stimulus effects of melatonin in the rat

To provide initial information on the potential mechanisms underlying the discriminative stimulus effects of melatonin, rats were trained to discriminate melatonin (150 mg/kg, IP) from saline in a two-choice discrete-trial avoidance paradigm. Stimulus generalization curves for melatonin were steep; complete generalization with melatonin occurred at 100–150 mg/kg. Triazolam generalized completely with melatonin (n=7). Flurazepam generalized completely with melatonin in only two out of six rats; however, partial generalization was produced in the remaining four animals. The melatonin-appropriate responding produced by triazolam was antagonized completely (in six out of seven rats) by 0.3–10 mg/kg flumazenil (Ro 15–1788). In contrast, the dose of flumazenil sufficient to block completely the melatonin-like discriminative effects of triazolam failed to block the stimulus effects of the training dose of melatonin. Pentobarbital produced primarily melatonin-appropriate responding, with complete generalization with melatonin in five out of seven rats. Diphenhydramine generalized completely with melatonin in two out of seven rats; however, little or no partial generalization was observed in the remaining five rats. These results suggest that melatonin may produce its discriminative effects through sites on the GABA_A-benzodiazepine receptor complex distinct from the benzodiazepine binding sites.     

Possible opioid receptor function changes in isolated atria of the spontaneously hypertensive rat

• 1.1. A comparison of the effects of various opioid peptides on the heart rates of self-paced right atria was made, as taken from spontaneously hypertensive (SHR), Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats at 4, 8, 12 and 16 weeks of age.
• 2.2. Beta-endorphin, dynorphin, met-enkephalin, DAGO and DADLE slightly decreased the spontaneously beating rate of all rat strains and ages, at 0.1 μM. Leu-enkephalin at 0.2 μM increased the spontaneous beating rate of SHR atria, but not that of atria from normotensive strains.
• 3.3. SHR atria were somewhat more sensitive than WKY atria to norepinephrine (NE)-induced positive chronotropy, but the differences were not statistically significant.
• 4.4. In the presence of mu, delta or kappa opioid receptor agonists, SHR atrial sensitivity to NE-induced chronotropy was enhanced at all ages studied. By contrast, NE chronotropy was not significantly altered by the opioids in normotensive rat atria.
• 5.5. Based on the above results, all the three major opioid receptor subtypes (mu, delta and kappa) appear to be present in rat atria but the function of these receptors appears to be greater in SHR than in WKY and SD atria.
• 6.6. The results suggest a possible involvement of altered opioid responsiveness in atria during hypertension development in SHR but the nature of this involvement appears to be complex and is not readily understandable on the basis of the present study.

     

Pharmacokinetics,oral bioavailability and metabolism of a novel isoquinolinone-based melatonin receptor agonist in rats

1. 7-Methoxy-6-(3-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (named as IS0042) is a newly identified melatoninergic agonist which exhibits selectivity to the type 2 melatonin receptor. Here, we examined the in vitro and in vivo pharmacokinetics properties of IS0042 in rats.

2. IS0042 was considerably lipophilic with a modest aqueous solubility of 27.3 µg/mL. It was stable in simulated gastrointestinal fluid, and readily penetrated across differentiated Caco-2 cell model of intestinal barrier, suggesting good oral absorption.

3. IS0042 underwent metabolism in rat intestinal and liver microsomes with an in vitro half-life of 367.5 ± 36.6 and 17.5 ± 2.7 min, respectively. Metabolite identification suggested that the major biotransformation pathways included the cleavage of ether bond, hydroxylation and demethylation. The same metabolites were also present in blood circulation following oral administration, indicating a good correlation between in vitro and in vivo metabolism.

4. The pharmacokinetics parameters of IS0042 were evaluated after intravenous administration (10 or 25 mg/kg) and oral administration (100 mg/kg) of the drug to rats. IS0042 showed moderate clearance (0.73–1.02 L/h/kg), large volume of distribution (1.76–3.16 L/kg) and long elimination half-life (3.11–6.04 h) after intravenous administration. The absolute oral bioavailability of IS0042 was relatively low (9.8–18.6%). Overall, these results provide important parameters for the further development of this novel class of melatoninergic ligands.

     

褪黑素对梗阻性黄疸肠组织ICM-1表达的影响

目的探讨褪黑素对大鼠梗阻性黄疸后肠组织细胞间粘附分子-1(ICAM-1)表达的影响。方法将36只大鼠随机分为3组(每组12只):假手术对照组(SO)、梗阻性黄疸组(OJ)、褪黑素治疗组(MT)。观察各组小肠组织中ICAM-1的表达及小肠组织形态学改变,并测量肠绒毛高度和黏膜厚度。结果梗阻性黄疸组小肠组织ICAM-1的表达明显增强,肠绒毛高度、黏膜厚度明显降低,与假手术对照组有显著性差异(P<0.01,P<0.01);褪黑素治疗组与梗阻性黄疸组比较小肠组织ICAM-1的表达明显减弱(P<0.01),肠绒毛高度、黏膜厚度增高,肠组织结构损害减轻(P<0.01)。结论褪黑素通过抑制梗阻性黄疸后肠组织ICAM-1的表达,减轻小肠黏膜的损伤。