Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance

Compelling evidence has suggested that spinal glial cells were activated by chronic morphine treatment and involved in the development of morphine tolerance. However, the mechanisms of glial activation were still largely unknown in morphine tolerance. In present study, we investigated the role of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal cord in the development of chronic morphine antinociceptive tolerance. We found that intrathecal administration of morphine (15 microg) daily for 7 consecutive days significantly induced an increase in number of phospho-p38 (p-p38) immunoreactive cells in the spinal cord compared with chronic saline or acute morphine treated rats. Double immunofluorescence staining revealed that p-p38 immunoreactivity was exclusively restricted in the activated spinal microglia, not in astrocytes or neurons. Repeated intrathecal administration of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (10 microg or 2 microg), a specific p38 inhibitor, 30 min before each morphine injection for 7 consecutive days significantly attenuated tolerance to morphine analgesia assessed by tail flick test. However, a single intrathecal administration of SB203580 (10 microg) did not antagonize the established tolerance to morphine analgesia. Taken together, these findings suggested that p38 MAPK activation in the spinal microglia was involved in the development of morphine antinociceptive tolerance. Inhibition of p38 MAPK by SB203580 in the spinal cord attenuated but not reversed the tolerance to morphine analgesia. The present study provides the first evidence that p38 activation in spinal microglia played an important role in the development of tolerance to morphine analgesia.     

CCK(B) receptor antagonist L365,260 potentiates the efficacy to and reverses chronic tolerance to electroacupuncture-induced analgesia in mice

Cholecystokinin octapeptide (CCK-8) is a physiological antagonist of endogenous opioids in the central nervous system (CNS). Our previous work has shown that CCK-8 plays an important role in the development of tolerance to morphine analgesia and electroacupuncture (EA) analgesia in the rat. The present studies were designed to examine whether the CCK(B) receptor is involved in the modulation of EA analgesia and the development of EA tolerance in mice. The latency to flick the tail in the radiant heat was used as index to assess the efficacy of EA analgesia. Subcutaneous (s.c.) injection of the CCK(B) receptor antagonist L365,260 produced a dose-dependent (0.125-2.0 mg/kg) potentiation of the analgesia induced by 100 Hz EA, with a maximal effect occurred at 0.5 mg/kg. In addition, L365,260 (0.5 mg/kg) significantly reversed chronic tolerance to 100 Hz EA in mice. These results suggest that the CCK(B) receptor might play a role in the tonic inhibition of 100 Hz EA-induced analgesia and in the mediation of chronic tolerance to 100 Hz EA in mice. The results opened a way for further investigation of the function of CCK-8 in pain modulation using inbred strains of mice.     

A novel role of minocycline: attenuating morphine antinociceptive tolerance by inhibition of p38 MAPK in the activated spinal microglia

We have previously demonstrated that activation of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal microglia mediates morphine antinociceptive tolerance. Minocycline, a selective inhibitor of microglia activation, has been reported to attenuate peripheral inflammation-induced hyperalgesia by depressing p38 MAPK in the spinal microglia. The aim of the present study is to explore the effect of intrathecal minocycline on the development of morphine antinociceptive tolerance and p38 activation in the spinal microglia induced by chronic morphine treatment. Minocycline (20, 50 and 100 microg) was given intrathecally 30 min before each morphine (15 microg) administration for consecutive 7 days. It was shown that minocycline attenuated tolerance to morphine analgesia in a dose-dependent manner. Minocycline administration (50 microg) which was initiated on day 4 followed by another 4 days administration partially reversed the established morphine antinociceptive tolerance. However, minocycline treatment which was started on day 8 followed by its administration for 4 more days failed to reverse the established morphine tolerance. Immunohistochemical analysis showed that chronic intrathecal morphine-induced activation of p38 MAPK in the spinal microglia. Minocycline at a dose that was shown to antagonize tolerance to morphine analgesia significantly inhibited the increase in p38 MAPK activation in the spinal microglia. To our knowledge, this is the first study to demonstrate that minocycline antagonizes morphine antinociceptive tolerance, possibly due to the inhibition of p38 activation in the spinal microglia.     

Altered cholecystokinin binding site density in the supraoptic nucleus of morphine-tolerant and -dependent rats

The processes underlying the development of neuronal tolerance to and dependence upon opiates are not yet fully understood. To evaluate a possible role for cholecystokinin (CCK) in these processes, quantitative receptor autoradiography and in situ hybridisation histochemistry were used to study both the density and distribution of sulphated CCK octapeptide (CCK8S) binding sites and preproCCK peptide mRNA levels within the dorsal (oxytocin neurone-rich) supraoptic nuclei of rats given an intracerebroventricular (i.c.v.) infusion of morphine over 5 days, which is known to induce tolerance and dependence in mechanisms regulating oxytocin neurones. Specific CCK8S binding was significantly increased in the supraoptic nuclei of both morphine-dependent and salt-loaded (2% sodium chloride to drink for 48 h) rats compared to their respective controls (P<0.05). In situ hybridisation histochemistry revealed no difference in preproCCK mRNA levels within supraoptic neurones of (i.c.v.) morphine-treated compared with either i.c.v. vehicle-treated or untreated control animals. These results suggest that CCK receptor mechanisms involved in the control of magnocellular oxytocin neurone activation are upregulated during chronic morphine treatment, and this may favour increased sensitivity to CCK, thereby offsetting the inhibitory actions of morphine, contributing to tolerance and perhaps to the withdrawal excitation characteristic of dependence.     

Up-regulation of adenosine transporter-binding sites in striatum and hypothalamus of opiate tolerant mice

Opioid–adenosine interactions have been demonstrated at both cellular and behavioral levels. Short-term morphine treatment has been shown to enhance adenosine release in brain and spinal tissues. Since adenosine uptake and release is regulated by a nitrobenzylthioinosine-sensitive adenosine transporter, we examined the effects of morphine treatment on this transporter-binding site. Adenosine transporter-binding sites were examined using equilibrium binding studies with [³H]nitrobenzylthioinosine in brain regions of morphine-treated mice. A 72-hour morphine pellet implantation procedure, which previously produced up-regulation of central adenosine A₁ receptors and created a state of opiate dependence [G.B. Kaplan, K.A. Leite-Morris and M.T. Sears, Alterations in adenosine A₁ receptors in morphine dependence, Brain Res., 657 (1994) 347–350], was used in this current study. This chronic morphine treatment significantly increased adenosine transporter-binding site concentrations in striatum and hypothalamus by 12 and 37%, respectively, compared to vehicle pellet implantation. No effects of morphine treatment were demonstrated in cortex, hippocampus, brainstem or cerebellum. In behavioral studies, mice receiving this same chronic morphine or vehicle treatment were given saline or morphine injections (40 or 50 mg/kg i.p.) followed by ambulatory activity monitoring. In the chronic vehicle treatment group, morphine injections significantly stimulated ambulatory activity while in the chronic morphine treatment group there was no such stimulation by acute morphine, suggestive of opiate tolerance. Morphine-induced up-regulation of striatal and hypothalamic adenosine transporter sites could potentially alter extracellular adenosine release and adenosine receptor activation and mediate aspects of opiate tolerance and dependence.     

Morphine tolerance does not develop in mice treated with endothelin-A receptor antagonists

Long-term use of morphine leads to development of antinociceptive tolerance. We provide evidence that central endothelin (ET) mechanisms are involved in development of morphine tolerance. In the present study, we investigated the effect of ET(A) receptor antagonists, BQ123 and BMS182874, on morphine antinociception and tolerance in mice. Mechanism of interaction of ET(A) receptor antagonists with morphine was investigated. BQ123 (3 microg, i.c.v.) and BMS182874 (50 microg, i.c.v.) significantly enhanced antinociceptive effect of morphine (P < 0.05), through an opioid-mediated effect. Treatment with a single dose of BQ123 (3 microg, i.c.v.) reversed tolerance to morphine antinociception in morphine-tolerant mice. BQ123 or BMS182874 did not affect naloxone binding in the brain. Therefore, ET(A) receptor antagonists did not bind directly to opioid receptors. [35S]GTPgammaS binding was stimulated by morphine and ET-1 in non-tolerant mice. Morphine- and ET-1-induced GTP stimulation was significantly lower (P < 0.05) in morphine-tolerant group (33% and 42%, respectively) compared to control group. BQ123 and BMS182874 did not activate binding in non-tolerant mice. BQ123 and BMS182874 significantly increased G protein activation in morphine-tolerant mice (96% and 86%, respectively; P < 0.05). These results provide evidence that uncoupling of G protein occurs in morphine-tolerant mice, and ET(A) antagonists promote coupling of G protein to its receptors, thereby restoring antinociceptive effect. These findings indicate that ET(A) receptor antagonists potentiate morphine antinociception and reverse antinociceptive tolerance in mice, through their ability to couple G proteins to opioid receptors.     

Is cholecystokinin octapeptide (CCK-8) a candidate for endogenous antiopioid substrates?

Cholecystokinin octapeptide (CCK-8), given intracerebroventricularly (icv) or intrathecally (ith) at the dose range of 0.25-4.0 ng, dose-dependently antagonised the effect of morphine analgesia and electroacupuncture analgesia (EAA) in the rat. That CCK-8 antiserum was capable of reversing the tolerance to EAA and changing the non-responders of EAA into responders suggest CCK-8 to be the endogenous anti-opioid substrate and that blocking the effect of CCK-8 may prove to be a powerful way of augmenting the effect of morphine analgesia and EA analgesia.     

Toll-like receptor 4-mediated nuclear factor-κB activation in spinal cord contributes to chronic morphine-induced analgesic tolerance and hyperalgesia in rats

Nuclear factor kappa B（NF-κB） in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the underlying mechanisms remain unclear. In the present study, we found that the level of phosphorylated NF-κB p65（p-p65） was increased in the dorsal horn of the lumbar 4–6 segments after intrathecal administration of morphine for 7 consecutive days, and the p-p65 was co-localized with neurons and astrocytes. The expression of TNF-α and IL-1β was also increased in the same area. In addition, pretreatment with pyrrolidinedithiocarbamate（PDTC） or SN50, inhibitors of NF-κB, prevented the development of morphine analgesic tolerance and alleviated morphine withdrawal-induced allodynia and hyperalgesia. The increase in TNF-α and IL-1β expression induced by chronic morphine exposure was also partially blocked by PDTC pretreatment. In another experiment, rats receiving PDTC or SN50 beginning on day 7 of morphine injection showed partial recovery of the anti-nociceptive effects of morphine and attenuation of the withdrawal-induced abnormal pain. Meanwhile, intrathecal pretreatment with lipopolysaccharide from Rhodobacter sphae-roides, an antagonist of toll-like receptor 4（TLR4）, blocked the activation of NF-κB, and prevented the development of morphine tolerance and withdrawal-induced abnormal pain. These data indicated that TLR4-mediated NF-κB activation in the spinal cord is involved in the development and maintenance of morphine analgesic tolerance and withdrawalinduced pain hypersensitivity.     

Site and mechanism of morphine tolerance in the gastrointestinal tract

Opioid‐induced constipation is a major clinical problem. The effects of morphine, and other narcotics, on the gastrointestinal tract persist over long‐term use thus limiting the clinical benefit of these excellent pain relievers. The effects of opioids in the gut, including morphine, are largely mediated by the μ‐opioid receptors at the soma and nerve terminals of enteric neurons. Recent studies demonstrate that regional differences exist in both acute and chronic morphine along the gastrointestinal tract. While tolerance develops to the analgesic effects and upper gastrointestinal motility upon repeated morphine administration, tolerance does not develop in the colon with chronic opioids resulting in persistent constipation. Here, we review the mechanisms by which tolerance develops in the small but not the large intestine. The regional differences lie in the signaling and regulation of the μ‐opioid receptor in the various segments of the gastrointestinal tract. The differential role of β‐arrestin2 in tolerance development between central and enteric neurons defines the potential for therapeutic approaches in developing ligands with analgesic properties and minimal constipating effects.     

Nocistatin, a peptide reversing acute and chronic morphine tolerance.

It has been reported that intracerebroventricular (i.c.v.) injection of nociception/orphanin FQ (OFQ) can antagonize morphine analgesia, whereas i.c.v. OFQ antibody can reverse morphine tolerance. Nocistatin (NST) is a recently characterized neuropeptide possessing an antagonizing effect on OFQ. Here we examine whether i.c.v. NST would result in a reversal of morphine tolerance. The results showed that: (1) i.c.v. NST at doses of 0.005, 0.05, 0.5, 5 or 50 ng per rat produced a bell-shaped dose-dependent reversal of chronic morphine tolerance, with maximum response at 0.5 ng. (2) Acute morphine tolerance could also be reversed, albeit partially, by i.c.v. NST at 0.5 ng. (3) The reversal of acute and chronic morphine tolerance by NST was completely abolished when NST (0.5 ng) was co-injected with (8 microg) OFQ. Since OFQ and NST are derived from the same preprohormone, the profile of its splicing in the CNS may play an important role in determining the effectiveness of morphine analgesia.     

Excitatory amino acid antagonists (kynurenic acid and MK-801) attenuate the development of morphine tolerance in the rat

To investigate the possible role of excitatory amino acids (EAAs) in the mechanisms of morphine tolerance, rats were treated either with the wide-spectrum EAA antagonist, kynurenic acid (150 mg/kg), or the specific N-methyl-D-aspartic acid (NMDA) receptor antagonist. MK-801 (0.05 mg/kg), during a four-day induction period of morphine tolerance. Morphine was given once daily at a dose of 15 mg kg. On the fifth day rats were injected only with morphine (15 mg/kg), and analgesia was assessed using the hot-plate test. Morphine tolerance was significantly reduced by both EAA antagonists. Control experiments showed that at the same doses neither acute nor chronic administration of these antagonists affected morphine analgesia itself in a manner that can explain these findings. The possible involvement of EAAs in the mechanisms of morphine tolerance is discussed.     

Blockade of adrenomedullin receptors reverses morphine tolerance and its neurochemical mechanisms

Adrenomedullin (AM) has been demonstrated to be involved in the development of opioid tolerance. The present study further investigated the role of AM in the maintenance of morphine tolerance, morphine-associated hyperalgesia and its cellular mechanisms. Intrathecal (i.t.) injection of morphine for 6 days induced a decline of its analgesic effect and hyperalgesia. Acute administration of the AM receptor antagonist AM_22-52 resumed the potency of morphine in a dose-dependent manner (12, 35.8 and 71.5 μg, i.t.). The AM_22-52 treatment also suppressed morphine tolerance-associated hyperalgesia. Furthermore, i.t. administration of AM_22-52 at a dose of 35.8 μg reversed the morphine induced-enhancement of nNOS (neuronal nitric oxide synthase) and CGRP immunoreactivity in the spinal dorsal horn and/or dorsal root ganglia (DRG). Interestingly, chronic administration of morphine reduced the expression of the endogenous opioid peptide bovine adrenal medulla 22 (BAM22) in small- and medium-sized neurons in DRG and this reduction was partially reversed by the administration of AM_22-52 (35.8 μg). These results suggest that the activation of AM receptors was involved in the maintenance of morphine tolerance mediating by not only upregulation of the pronociceptive mediators, nNOS and CGRP but also the down-regulation of pain-inhibiting molecule BAM22. Our data support the hypothesis that the level of both pronociceptive mediators and endogenous pain-inhibiting molecules has an impact on the potency of morphine analgesia. Targeting AM receptors is a promising approach to maintain the potency of morphine analgesia during chronic use of this drug.     

Dexamethasone mimics the inhibitory effect of chronic pain on the development of tolerance to morphine analgesia and compensates for morphine induced changes in G proteins gene expression

It is previously reported that the HPA axis plays role in the inhibitory effect of pain on tolerance development to analgesic effect of opioids. The present study was designed to investigate whether the chronic co-administration of dexamethasone as a glucocorticoid is also able to prevent or reverse analgesic tolerance to morphine and to compare the expression of G(alphai/o) and G(beta) subunits of G proteins in the context of chronic dexamethasone, development of morphine tolerance and their combination. Analgesic tolerance to morphine was induced by chronic intraperitoneally (i.p.) administration of morphine 20 mg/kg to male Wistar rats weighing 200-240 g within 4 consecutive days and analgesia was assessed using tail-flick test. Chronic dexamethasone was applied using 4 daily i.p. injections. Lumbar spinal tissues were assayed for the expression of G(alphai/o) and G(beta) proteins using "semiquantitative PCR" normalized to beta-actin gene expression. Results showed that chronic administration of dexamethasone could reduce and reverse the development of tolerance in rats that received chronic i.p. injections of morphine. Chronic administration of dexamethasone significantly increased the expression of G(alphai/o), while chronic administration of morphine did not change its expression. The expression of G(beta), however, was increased after the chronic administration of morphine, but did not change after the administration of chronic dexamethasone. None of these increases were observed when morphine and dexamethasone were co-administered. We conclude that the development of tolerance to analgesic effect of morphine could be prevented and reversed by dexamethasone co-administration. The increase in G(alphai/o) genes expression produced by chronic dexamethasone may facilitate the opioid signaling pathway and compensate for morphine-induced tolerance.     

Attenuation of morphine tolerance by minocycline and pentoxifylline in naive and neuropathic mice

We have previously demonstrated that glial inhibitors reduce the development of allodynia and hyperalgesia, potentiating the effect of a single morphine dose in a neuropathic pain model. This study explores the effects of two glial activation inhibitors, minocycline and pentoxifylline, on the development of tolerance to morphine in naive and chronic constriction injury (CCI)-exposed mice. Administration of morphine to naive (20 mg/kg; i.p.) and CCI-exposed mice (40 mg/kg; i.p.) twice daily resulted in tolerance to its anti-nociceptive effect after 6 days. Injections of morphine were combined with minocycline (30 mg/kg, i.p.) or pentoxifylline (20 mg/kg, i.p.) administered as two preemptive doses before first morphine administration in naive or pre-injury in CCI-exposed mice, and repeated twice daily 30 min before each morphine administration. With treatment, development of morphine tolerance was delayed by 5 days (from 6 to 11 days), as measured by the tail-flick test in naive and by tail-flick, von Frey, and cold plate tests in CCI-exposed mice. Western blot analysis of CD11b/c and GFAP protein demonstrated that minocycline and pentoxifylline, at doses delaying development of tolerance to morphine analgesia, significantly diminished the morphine-induced increase in CD11b/c protein level. We found that repeated systemic administration of glial inhibitors significantly delays development of morphine tolerance by attenuating the level of this microglial marker under normal and neuropathic pain conditions. Our results support the idea that targeting microglial activation during morphine therapy/treatment is a novel and clinically promising method for enhancing morphine's analgesic effects, especially in neuropathic pain.     

Implication of delta opioid receptor subtype 2 but not delta opioid receptor subtype 1 in the development of morphine analgesic tolerance in a rat model of chronic inflammatory pain

Opioids are well known for their robust analgesic effects. Chronic activation of mu opioid receptors (MOPs) is, however, accompanied by various unwanted effects such as analgesic tolerance. Among other mechanisms, interactions between MOPs and delta opioid receptors (DOPs) are thought to play an important role in morphine‐induced behavioral adaptations. Interestingly, certain conditions such as inflammation enhance the function of the DOP through a MOP‐dependent mechanism. Here, we investigated the role of DOPs during the development of morphine tolerance in an animal model of chronic inflammatory pain. Using behavioral approaches, we first established that repeated systemic morphine treatment induced morphine analgesic tolerance in rats coping with chronic inflammatory pain. We then observed that blockade of DOPs with subcutaneous naltrindole (NTI), a selective DOP antagonist, significantly attenuated the development of morphine tolerance in a dose‐dependent manner. We confirmed that this effect was DOP mediated by showing that an acute injection of NTI had no effect on morphine‐induced analgesia in naive animals. Previous pharmacological characterizations revealed the existence of DOP subtype 1 and DOP subtype 2. As opposed to NTI, 7‐benzylidenenaltrexone and naltriben were reported to be selective DOP subtype 1 and DOP subtype 2 antagonists, respectively. Interestingly, naltriben but not 7‐benzylidenenaltrexone was able to attenuate the development of morphine analgesic tolerance in inflamed rats. Altogether, our results suggest that targeting of DOP subtype 2 with antagonists provides a valuable strategy to attenuate the analgesic tolerance that develops after repeated morphine administration in the setting of chronic inflammatory pain.     

Morphine and Fentanyl Differently Affect MOP and NOP Gene Expression in Human Neuroblastoma SH-SY5Y Cells

Morphine is widely used for the treatment of severe acute and chronic pain, but long-term therapy rapidly leads to tolerance. Morphine effects are mediated by μ opioid receptor (MOP) activation as well as for fentanyl that, in contrast to morphine, induces less tolerance to analgesia. The mechanisms underlying opioid tolerance involve complex processes, such as MOP desensitization, internalization, and/or changes of gene expression. The development of morphine tolerance also involves adaptive changes of the anti-opioid nociceptin/orphanin FQ–nociceptin receptor system, as suggested by the reduction of morphine tolerance in nociceptin opioid receptor (NOP) knockout mice. The aim of the present study was to investigate the MOP and NOP gene expression in the SH-SY5Y cells following morphine and fentanyl exposure. Results showed that cell exposure to 10 μM morphine for 5 h induced a significant decrease of MOP and NOP gene expression and that the MOP downregulation was reverted by the pretreatment with naloxone. Conversely, SH-SY5Y cells exposed to 0.1 and 1 μM fentanyl for 5 and 72 h showed a significant MOP upregulation, also reverted by naloxone pretreatment. Fentanyl induced no changes of NOP gene expression. The present findings showed a different effect by morphine and fentanyl on MOP mRNA levels that contributes to define the role of MOP gene expression changes in the mechanisms underlying the tolerance. Morphine also triggers an altered NOP-related signaling confirming that the nociceptin/orphanin FQ–nociceptin receptor system also plays a significant role in the development of morphine tolerance.     

Botulinum toxin A increases analgesic effects of morphine,counters development of morphine tolerance and modulates glia activation and μ opioid receptor expression in neuropathic mice

The use of botulinum neurotoxin type A (BoNT/A) against pain, with emphasis for its possible use in alleviating chronic pain, still represents an outstanding challenge for experimental research. In this study, we examined the effects of BoNT/A on morphine-induced tolerance during chronic morphine treatment in neuropathic CD1 mice subjected to sciatic nerve lesion according to the Chronic Constriction Injury (CCI) model of neuropathic pain. We measured the effects of BoNT/A on CCI-induced allodynia and hyperalgesia and on the expression of glial fibrillary acidic protein (GFAP, marker of astrocytes), complement receptor 3/cluster of differentiation 11b (CD11b, marker of microglia), and neuronal nuclei (NeuN) at the spinal cord level. We also analyzed the colocalized expression of GFAP, CD11b and NeuN with phosphorylated p-38 mitogen-activated protein kinase and with μ-opioid receptor (MOR). A single intraplantar injection of BoNT/A (15 pg/paw) into the injured hindpaw, the day before the beginning of chronic morphine treatment (9 days of twice daily injections of 40 mg/kg morphine), was able to counteract allodynia and enhancement of astrocytes expression/activation induced by CCI. In addition, BoNT/A increased the analgesic effect of morphine and countered morphine-induced tolerance during chronic morphine treatment. These effects were accompanied, in neurons, by re-expression of MORs that had been reduced by repeated morphine administration. The combinatory effects of BoNT/A and morphine could have relevant therapeutic implications for sufferers of chronic pain who could benefit of pain relief reducing tolerance due to repeated treatment with opiates.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKCγ) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of theN-methyl-d-aspartate receptor antagonist MK-801 on tolerance-associated PKCγ changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 μg morphine for eight days, PKCγ immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKCγ immunostained neurons in laminae I–II than in laminae III–IV and V–VI. Such PKCγ immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKCγ increases. Moreover, both the development of morphine tolerance and the increase in PKCγ immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKCγ immunoreactivity was not increased in rats receiving a single i.t. administration of 10 μg morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKCγ immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.     

Effects of Aquaporin 4 Deficiency on Morphine Analgesia and Chronic Tolerance: A Study at Spinal Level

Recent reports showed that aquaporin 4 (AQP4) deficiency potentiated morphine analgesia but attenuated chronic morphine-induced tolerance in hot-plate test, predominantly reflecting supraspinal pain response. The present study investigated the effects of AQP4 deficiency on morphine analgesia and tolerance using tail flick test, primarily reflecting spinal response. It was found that (1) chronic morphine treatment resulted in decreased expression of spinal AQP4 in mice detected by Western blotting analysis; (2) in tail flick test, AQP4 knockout mice displayed significant impaired morphine analgesia without influencing the progress of chronic tolerance; and (3) the expressions of mu-opioid receptor (MuOR) and glutamate transporter 1 (GLT-1) in AQP4 knockout mice spinal cord were lower than those in wild-type mice, whereas chronic morphine-induced alteration characteristics of spinal MuOR or GLT-1 expression were not affected by AQP4 deficiency. In conclusion, AQP4 deficiency attenuated morphine acute antinociception but did not affect chronic tolerance in tail flick test, implying a role for spinal AQP4 in morphine analgesia but not in chronic tolerance.     

A review of the role of anti-opioid peptides in morphine tolerance and dependence.

Studies on the mechanisms of tolerance and dependence have mostly focused on changes at the receptor level. These experiments, conducted with model systems ranging from clonal cell lines to whole animals, have identified a number of important adaptive mechanisms which occur at the receptor level. However, none of these adaptive mechanisms can completely account for the phenomena which serve to define the state of morphine tolerance and dependence, especially the observation that as an animal becomes more tolerant to morphine, less naloxone is required to trigger withdrawal. The data reviewed in this paper provide strong support for the hypothesis that the brain synthesizes and secretes neuropeptides which act as part of a homeostatic system to attenuate the effects of morphine and endogenous opioid peptides. According to this model, administration of morphine releases anti-opioid peptides (AOP), which then attenuate the effects of morphine. As more morphine is given, more AOP are released, thereby producing tolerance to the effects of morphine. Cessation of morphine administration, or administration of naloxone, produces a relative excess of anti-opioid, which is in part responsible for the withdrawal syndrome. Since endogenous and exogenous antagonists might together produce synergistic effects, less naloxone might be required to trigger withdrawal in the presence of higher levels of AOPs. Although the study of AOP is in its infancy, a deeper understanding of the central nervous system (CNS) anti-opioid systems may lead to new treatments for chronic pain, substance abuse, and psychiatric disorders.