Evidence that ginsenosides prevent the development of opioid tolerance at the central nervous system

The analgesic effect of ginsenosides or morphine was first determined following intrathecal (i.t.) administration in rat tail-flick test. The effect of chronic i.t. co-administration of ginsenosides with morphine on the development of opioid tolerance were also examined using rat tail-flick test. Administration of ginsenosides (i.t.) produced a weak antinociception in a dose-dependent manner. Administration of morphine (i.t.) also produced antinociception in a dose-dependent manner. The ED50 was 1.20 microg (1.14-1.29 microg). However, acute i.t. co-administration of ginsenosides with morphine was not additive in antinociception. Repeated i.t. co-administration of 200 microg ginsenosides with 10 microg morphine inhibited the development of tolerance induced by 10 microg morphine in rat tail-flick test, although i.t. co-administration of 50 or 100 microg ginsenosides with morphine was without effect. In conclusion, these results indicate that i.t. administered ginsenosides produce an antinociception in rat tail-flick test and also prevent opioid tolerance caused by chronic treatment with morphine at the spinal sites.     

Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

Centrally-mediated antinociceptive action of RWJ-22757 (formerly McN-5195): involvement of spinal descending inhibitory pathways (an hypothesis)

The present studies were an attempt to examine the mechanism of action of the novel antinociceptive compound RWJ-22757, (+/-)-trans-3-(2-bromophenyl)-octahydroindolizine (McN-5195). Intracerebroventricular (i.c.v.) administration of RWJ-22757 produced dose-related antinociception in the mouse tail-flick (48 degrees C) and rat hot-plate (51 degrees C) tests (ED50 = 243.3 and 261.3 micrograms, respectively). In contrast, intrathecal (i.t.) administration was without effect. The antinociception produced by peripherally (i.p.) or centrally (i.c.v.) administered RWJ-22757 was attenuated by i.t. administration of 2 micrograms phentolamine, 5 micrograms yohimbine, or 10 micrograms methysergide. I.t. administration of naloxone, at a dose (0.5 micrograms) that significantly attenuated the antinociceptive effects of peripherally or centrally administered morphine, had no effect on RWJ-22757-induced antinociception. We conclude from these results, coupled with the overall pharmacological and neurochemical profile of RWJ-22757, that the data are consistent with the hypothesis that RWJ-22757 produces antinociception predominantly at a site or sites located supraspinally with little or no activity at the spinal level and that RWJ-22757 activates adrenergic and serotonergic descending inhibitory pathways, increasing the tonic activity of endogenous antinociceptive systems.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Involvement of spinal metabotropic glutamate receptor 5 in the development of tolerance to morphine-induced antinociception

It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine-induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double-labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine-induced antinociception in mice.     

Morphine tolerance in mice changes response of heroin from mu to delta opioid receptors

Heroin produced antinociception in the tail flick test through mu receptors in the brain of ICR and CD-1 mice, a response inhibited by 3-O-methylnaltrexone. Tolerance to morphine was produced by subcutaneous morphine pellet implantation. By the third day, the heroin response was produced through delta opioid receptors. The response was inhibited by simultaneous intracerebroventricular (i.c. v.) administration of naltrindole, a delta opioid receptor antagonist. More specifically, delta1 rather than delta2 receptors were involved because 7-benzylidenenaltrexone, a delta1 receptor antagonist, inhibited but naltriben, a delta2 antagonist, did not. Also, antinociception produced by i.c.v. heroin was inhibited by intrathecal administration of bicuculline and picrotoxin consistent with the concept that delta1 receptors in the brain mediated the antinociceptive response through descending neuronal pathways to the spinal cord to activate GABAA and GABAB receptors rather than spinal alpha2-adrenergic and serotonergic receptors activated originally by the mu agonist action in naive mice. The mu response of 6-monoacetylmorphine, a metabolite of heroin, was changed by morphine pellet implantation to a delta2 response (inhibited by naltriben but not 7-benzylidenenaltrexone). The agonist action of morphine in these morphine-tolerant mice remained mu. Thus, the opioid receptor selectivity of heroin and 6-monoacetylmorphine in the brain is changed by production of tolerance to morphine. Such a change explains how morphine tolerant mice are not cross-tolerant to heroin.     

Acute cross-tolerance to opioids in heroin delta-opioid-responding Swiss Webster mice

It is generally thought that the mu receptor actions of metabolites, 6-monoacetylmorphine (6MAM) and morphine, account for the pharmacological actions of heroin. However, upon intracerebroventricular (i.c.v.) administration in Swiss Webster mice, heroin and 6MAM act on delta receptors while morphine acts on mu receptors. Swiss Webster mice made tolerant to subcutaneous (s.c.) morphine by morphine pellet were not cross-tolerant to s.c. heroin (at 20 min in the tail flick test). Now, opioids were given in combination, s.c. (6.5 h) and i.c.v. (3 h) preceding testing the challenging agonist i.c.v. (at 10 min in the tail flick test). The combination (s.c. + i.c.v.) morphine pretreatment induced tolerance to the mu action of morphine but no cross-tolerance to the delta action of heroin, 6MAM and DPDPE and explained why morphine pelleting did not produce cross-tolerance to s.c. heroin above. Heroin plus heroin produced tolerance to delta agonists but not to mu agonists. Surprisingly, all combinations of morphine with the delta agonists produced tolerance to morphine which now acted through delta receptors (inhibited by i.c.v. naltrindole), an unusual change in receptor selectivity for morphine.     

Chimeric peptide of Met-enkephalin and FMRFa induces antinociception and attenuates development of tolerance to morphine antinociception.

A synthetic chimeric peptide of Met-enkephalin and FMRFamide (YGGFMKKKFMRFa), based on MERF was synthesized. This peptide was tested for possible antinociceptive effects using the tail flick test in mice. The effect of the chimeric peptide on morphine antinociception and development of tolerance to the antinociceptive action of morphine was also investigated. The chimeric peptide produced significant, dose-dependent antinociception (40, 60 and 90 mg/kg) in the tail flick test. Pretreatment with naloxone (5 mg/kg, IP) significantly attenuated the antinociceptive effect induced by the chimeric peptide (90 mg/kg, IP), indicating involvement of an opioidergic mechanism. In combination experiments with morphine, the antinociceptive dose of the chimeric peptide (60 mg/kg, IP) potentiated morphine (7 mg/kg, IP) antinociception. A low dose of the chimeric peptide (10 mg/kg, IP), that did not produce significant antinociception on its own, also potentiated morphine antinociception. In the tolerance studies, male albino mice received twice daily injections of morphine (20 mg/kg, IP) followed by either saline (0.1 ml) or chimeric peptide (80 mg/kg, IP) for a period of 4 days. A control group received twice daily injections of saline (0.1 ml) for the same period. When tested on Day 5, tolerance to antinociceptive action of morphine (15 mg/kg, IP) was evidenced by decreased response in chronic morphine plus saline treated mice compared to control group. Concurrent administration of chimeric peptide (80 mg/kg, IP) with morphine significantly attenuated the development of tolerance to the antinociceptive action of morphine. The preliminary results of this study demonstrate that peripherally administered chimeric peptide can produce dose dependent, naloxone reversible, antinociception; potentiate morphine antinociception and attenuate morphine tolerance, indicating a possible role of these type of amphiactive sequences in antinociception and its modulation. These chimeric peptides may also prove to be useful tools for further ascertaining the role of FMRFa family of peptides in mechanisms leading to opiate tolerance and dependence.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

Lack of antinociceptive cross-tolerance between intracerebroventricularly administered beta-endorphin and morphine or DPDPE in mice

Antinociceptive tolerance and cross-tolerance to intracerebroventricular (i.c.v.) beta-endorphin, morphine, and DPDPE (D-Pen2-D-Pen5-enkephalin) induced by a prior i.c.v. administration of beta-endorphin, morphine and DPDPE, respectively, were studied in mice. Acute tolerance was induced by i.c.v. pretreatment with beta-endorphin (0.58 nmol), morphine (6 nmol) and DPDPE (31 nmol) for 120, 180 and 75 min, respectively. Various doses of beta-endorphin, morphine or DPDPE were then injected. The tail-flick and hot-plate tests were used as antinociceptive tests. Pretreatment of mice with beta-endorphin i.c.v. reduced inhibition of the tail-flick and hot-plate responses to i.c.v. administered beta-endorphin, but not morphine and DPDPE. Pretreatment of mice with morphine i.c.v. reduced inhibition of the tail-flick and hot-plate responses to morphine but not beta-endorphin. Pretreatment of mice with DPDPE reduced inhibition of the tail-flick and hot-plate responses to DPDPE but not beta-endorphin. The results indicate that one injection of beta-endorphin, morphine or DPDPE induces acute antinociceptive tolerance to its own distinctive opioid receptor and does not induce cross-tolerance to other opioid agonists with different opioid receptor specificities. The data support the hypothesis that beta-endorphin, morphine and DPDPE produce antinociception by stimulating specific epsilon, mu- and delta-opioid receptors, respectively.     

Involvement of kappa/dynorphin system in the development of tolerance to nicotine-induced antinociception

The aim of the present study was to explore the possible role of kappa/dynorphin system in the development of tolerance to nicotine antinociception in mice. First, we observed that kappa-opioid receptor (KOP-r) participates in the acute spinal antinociception produced by nicotine (3 and 5 mg/kg, s.c.) since the pre-treatment with the selective kappa antagonist nor-binaltorphimine (3 mg/kg, i.p.) attenuated this response in the tail-immersion test but not in the hot-plate test nor in locomotor responses. Possible changes in the expression of KOP-r were investigated in tolerant mice to nicotine antinociception by using autoradiography of [³H]CI-977 binding. The density of KOP-r decreased in the spinal cord of tolerant mice. In addition, bi-directional cross-tolerance between nicotine (3 and 5 mg/kg, s.c.) and the selective kappa agonist U50,488H (10 mg/kg, s.c.) was found in the tail-immersion test. Recent evidences indicate that an up-regulation of dynorphin levels in the spinal cord and subsequent activation of NMDA receptors participate in the development of tolerance to opioid and cannabinoid antinociception. In this study, dynorphin content in the lumbar spinal cord was similar in control and nicotine tolerant mice. Furthermore, the administration of the NMDA antagonist MK-801 (0.03 and 0.01 mg/kg, i.p.) before each daily nicotine injection did not modify the development of nicotine tolerance. In summary, these data indicate that KOP-r is directly involved in the development of tolerance to nicotine antinociception by a mechanism independent from dynorphin and NMDA receptors.     

Antagonism of phosphoramidon-induced antinociception in mice by mu- but not kappa-opioid receptor blockers

Intracerebroventricular (i.c.v.) administration of the neutral endopeptidase 24.11-inhibitor phosphoramidon evoked a dose-dependent antinociceptive effect in the mouse acetic acid abdominal constriction test. The present study was conducted to identify the opioid receptor subtype(s) that mediate phosphoramidon antinociception in this paradigm. Mice were pretreated with different opioid antagonists prior to being challenged with phosphoramidon, i.c.v., the mu-opioid agonist sufentanil, s.c., or the kappa-opioid agonist U-50,488H, s.c. Naltrexone significantly attenuated phosphoramidon-induced antinociception at an i.c.v. dose that also blocked both sufentanil and U-50,488H. The mu-opioid antagonist beta-funaltrexamine (beta-FNA) blocked phosphoramidon and sufentanil at an i.c.v. dose that did not block U-50,488H. The kappa-opioid antagonist nor-binaltorphimine (nor-BNI) produced dose-related effects. A low dose (10 microg) of nor-BNI had no effect on either phosphoramidon or sufentanil but did reduce U-50,488H antinociception. A higher dose (30 microg) of nor-BNI blocked phosphoramidon, sufentanil, and U-50,488H, suggesting a loss of kappa-opioid receptor selectivity at this dose. These findings suggest that mu- but not kappa-opioid receptors mediate phosphoramidon-induced antinociception in the abdominal constriction test.     

Modulation of acute morphine tolerance by corticotropin-releasing factor and dynorphin A in the mouse spinal cord.

Previously, we have demonstrated that intrathecally (i.t.) administered corticotropin-releasing factor (CRF) in mice produces stimulus-specific antinociception and modulation of morphine-induced antinociception by mechanisms involving spinal kappa opioid receptors. Recently, we also have found that CRF releases immunoreactive dynorphin A, a putative endogenous kappa opioid receptor agonist, from superfused mice spinal cords in vitro. Dynorphin A administered intracerebroventricularlly (i.c.v.) to mice has been shown to modulate the expression of morphine tolerance. In the present study, the possible modulatory effects of i.t. administered CRF as well as dynorphin A on morphine tolerance were studied in an acute tolerance model. Subcutaneous administration of 100 mg/kg of morphine sulfate (MS) to mice caused an acute tolerance to morphine-induced antinociception. The antinociceptive ED50 of MS was increased from 4.4 mg/kg (naive mice) to 17.9 mg/kg (4 hours after the injection of 100 mg/kg MS). To study the modulatory effects of spinally administered CRF and dynorphin A on the expression of morphine tolerance, CRF and dynorphin A were injected i.t. at 15 min and 5 min, respectively, before testing the tolerant mice by the tail-flick assay. The antinociceptive ED50 of MS in tolerant mice was decreased to 8.8 mg/kg and 7.1 mg/kg, respectively, after i.t. administration of CRF (0.1 nmol) and dynorphin A (0.2 nmol). In contrast, 0.5 nmol of alpha-helical CRF (9-41), a CRF antagonist and 0.4 nmol of norbinaltorphimine, a highly selective kappa opioid receptor antagonist, when administered i.t. at 15 min before the tail-flick test in tolerant mice, increased the antinociceptive ED50 of MS to 56.6 mg/kg and 88.8 mg/kg, respectively. These data confirmed the modulatory effect of dynorphin A on morphine tolerance and suggested that CRF, which releases dynorphin A in several central nervous system regions, also plays a modulatory role in the expression of morphine tolerance.     

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats

Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4-7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.     

Spinal δ2 but not δ1 opioid receptors are involved in intracereboventricular β-endorphin-induced antinociception in the mouse

The antinociception induced by β-endorphin given intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the release of Met-enkephalin and subsequent stimulation of δ receptors in the spinal cord for antinociception. The present study was designed to determine what type of opioid receptor, δ1 or δ2, in the spinal cord is involved in i.c.v. β-endorphin-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. NTB (0.2–20 nmol) and NTI0 (0.22–2.2 nmol),selective δ2 receptor antagonists, given intrathecally (i.t.) dose-dependently attenuated i.c.v. β-endorphin-induced inhibition of the tail-flick response. On the other hand, BNTX (0.02–2.2 nmol), a selective δ1 receptor antagonist, given i.t., did not block i.c.v. β-endorphin-induced antinociception. The tail-flick inhibition induced by DAMGO, a μ receptor agonist, or U50,488H, a к receptor agonist, was not blocked by i.t. BNTX, NTB or NTI. It is concluded that δ2 but not δ1 receptors in the spinal cord are involved in i.c.v. β-endorphin-induced antinociception.     

Possible involvement of mu1-opioid receptors in the fentanyl- or morphine-induced antinociception at supraspinal and spinal sites

Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. The present study was designed to investigate the mechanisms of fentanyl- or morphine-induced antinociception at both supraspinal and spinal sites. In the mouse tail-flick test, the antinociceptive effects induced by both fentanyl and morphine were blocked by either the mu1-opioid receptor antagonist naloxonazine or the mu1/mu2-opioid receptor antagonist beta-funaltrexamine (beta-FNA) after s.c., i.c.v. or i.t. injection. In contrast, both fentanyl and morphine given i.c.v. or i.t. failed to produce antinociception in mu1-deficient CXBK mice. These findings indicate that like morphine, the antinociception induced by fentanyl may be mediated predominantly through mu1-opioid receptors at both supraspinal and spinal sites in mice. We also determined the ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl- or morphine-induced antinociception in mice. The ED50 values for s.c.-, i.c.v.- and i.t.-administered fentanyl-induced antinociception were 73.7, 18.5 and 1.2-fold lower than that of morphine, respectively. The present data clearly suggest the usefulness of peripheral treatment with fentanyl for the control of pain.     

The involvement of midbrain astrocyte in the development of morphine tolerance

Aims

Systemic administration of opiate analgesics such as morphine remains the most effective treatment for alleviating severe pain across a range of conditions including acute pain. However, chronic or repeated administration of opiate analgesics results in the development of analgesic tolerance. Glial cells such as microglia and astrocytes are known to release various inflammatory cytokines and neurotrophic factors leading to regulation of neuronal function. Recently, glial cells were reported to play important roles in the development of analgesic tolerance to morphine. Here, we focused on the involvement of midbrain glial cells, particularly astrocytes, in the development of analgesic tolerance to morphine.

Main methods

Mice were treated with morphine (10 mg/kg, s.c.) or vehicle once a day for 5 days. Pentoxifylline (an inhibitor of glial activation; 20 mg/kg, i.p. or 50 and 100 μg/mouse, i.c.v.) was administered 30 min before morphine treatment. Flavopiridol (a cyclin-dependent kinase inhibitor; 5 nmol/mouse, i.c.v.) was administered 10 min before and 10 h after morphine treatment. The analgesic effect of morphine was measured using the tail flick method.

Key findings

The development of analgesic tolerance to morphine was gradually observed during daily treatment of morphine for 5 days in mice. On days 1 and 3 after repeated morphine treatment, astrocyte marker glial fibrillary acidic protein expression levels were significantly increased, as determined by western blot analyses. These phenomena were significantly inhibited following pre-treatment with pentoxifylline or flavopiridol.

Significance

We demonstrated that midbrain astrocytes play an important role in the development of analgesic tolerance to morphine.     

Further investigations on neurotensin as central modulator of intestinal motility in rats

Previous studies have shown that neurotensin (NT) administered intracerebroventricularly (i.c.v.) to rats provokes an inhibition of intestinal propulsion linearly related to the log of administered doses. In the present study it is demonstrated that, in contrast to morphine, repeated i.c.v. administrations of NT (2.5 nmol/rat/day) did not result in tolerance to the intestinal effect. Naloxone (Nx) administered i.c.v. fully antagonized the intestinal inhibition of i.c.v. morphine, but did not significantly alter the NT effect. However, centrally administered thyrotropin-releasing hormone (TRH) inhibited NT-induced (but not morphine-induced) intestinal inhibition. Direct microinjections of NT into the periaqueductal gray matter (PAG) produced complete inhibition of intestinal propulsion when the microinjections were localized in the dorsal portion. Finally, subdiaphragmatic vagotomy totally abolished the inhibition induced by NT into the PAG, while morphine was not affected. Some considerations are put forward concerning the existence in the central nervous system of a peptidergic pathway modulating intestinal function.     

Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model

Apelin, as the endogenous ligand of the APJ receptor, is a novel identified neuropeptide whose biological functions are not fully understood. APJ receptor mRNA was found in several brain regions related to descending control system of pain, such as amygdala, hypothalamus and dorsal raphe nucleus (DRN). The present study was designed to determine whether supraspinal apelin-13 may produce antinociceptive effect observed in the acetic acid-induced writhing test, a model of visceral pain. Apelin-13 not only significantly produced preemptive antinociception at the dose of 0.3, 0.5, 1 and 3μg/mouse when injected intracerebroventricularly (i.c.v.) before acetic acid, but also significantly induced antinociception at a dose of 0.5, 1 and 3μg/mouse when injected i.c.v. after acetic acid. And i.c.v. apelin-13 did not influence 30-min locomotor activity counts in mice. Intrathecal (i.t.) administration of apelin-13 (1 and 3μg/mouse) significantly decreased the number of writhes, however, intraperitoneal (i.p.) injection of apelin-13 (10-100μg/mouse) had no effect on the number of writhes in the writhing test. The specific APJ receptor antagonist apelin-13(F13A), no-specific opioid receptor antagonist naloxone and μ-opioid receptor antagonist β-funaltrexamine hydrochloride (β-FNA) could significantly antagonize the antinociceptive effect of i.c.v. apelin-13, suggesting APJ receptor and μ-opioid receptor are involved in this process. Central low dose of apelin-13 (0.3μg/mouse, i.c.v.) could significantly potentiate the analgesic potencies of modest and even relatively ineffective doses of morphine administrated at supraspinal level. This enhanced antinociceptive effect was reversed by naloxone, suggesting that the potentiated analgesic response is mediated by opioid-responsive neurons.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.