Naloxone Binding Research Articles

Retraction: High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor-Gs Coupling Underlying Opioid Tolerance and Dependence.

Retraction: High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor-Gs Coupling Underlying Opioid Tolerance and Dependence.

Electrochemical Determination of Naloxone Using Molecularly Imprinted Poly(para-phenylenediamine) Sensor

A molecularly imprinted polymer (MIP)-based electrochemical sensor featuring an electrochemically grafted para-phenylenediamine functional monomer on a reduced graphene oxide-gold nanoparticles composite modified screen printed electrode is reported. The morphology and properties of the sensing material were characterized with microscopy, spectroscopy and electrochemical techniques. A number of factors affecting the performance of the MIP sensor were examined and optimized. Under an optimized condition, the imprinted electrochemical sensor yielded homogenous naloxone binding sites with a dissociation constant of 8.6 μM, and responded linearly up to 8 μM naloxone, with a limit of detection of 0.16 μM. The sensor showed good run-to-run repeatability and batch-to-batch performance reproducibility with relative standard deviation of 5.7%–9.6% (n = 4) and <9% (n = 3), respectively. The imprinted sensor retained 95% and 85% of its performance when stored at ambient conditions for one and two weeks, respectively, demonstrating the sensor’s good stability. Selectivity experiments showed that both the MIP sensor and non-imprinted polymer electrode had minimal response (<25%) to equal concentrations of structurally similar compounds such as morphine, naltrexone and noroxymorphone, indicating good selectivity of the MIP sensor towards naloxone. The MIP sensor was successfully used to quantify naloxone in artificial urine samples, yielding recoveries greater than 92%.

Naloxone Is a Potential Binding Ligand and Activator of the Capsaicin Receptor TRPV1.

The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a μ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by [3H]-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.

Modulation of μ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

Background and PurposeAdverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on μ‐opioid receptor binding and signalling induced by the μ‐opioid receptor ligands fentanyl, DAMGO, and naloxone.Experimental ApproachHEK 293 cells stably transfected with μ‐opioid receptors were used to study opioid ligand binding, [35S]‐GTPγS binding, and cAMP reduction at physiological and acidic pH. We used μ‐opioid receptors mutated at H2976.52 to A (MOR‐H2976.52A) to delineate ligand‐specific interactions with H2976.52.Key ResultsLow pH and the mutant receptor MOR‐H2976.52A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G‐protein activation were decreased under these conditions. Fentanyl‐induced signalling was not influenced by pH and largely independent of H2976.52.Conclusions and ImplicationsOur investigations indicate that low pH selectively impairs μ‐opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52. We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52.

Characterization of Union Sites for Opioids in Human Lymphocyte Membranes

The opioid peptides Methionine enkephalin and β-Endorphin are stimulators of Natural Killer cytolytic activity. To know the biochemical mechanisms that explain this action, the binding of Methionine enkephalin and Naloxone in presence or ausence of Na+ ion and GTP to human Lymphocyte membranes was characterized.

Biosensor-based kinetic and thermodynamic characterization of opioids interaction with human μ-opioid receptor

Development of opioid analgesics with minimal side effects requires substantial knowledge on structure–kinetic and –thermodynamic relationship of opioid-receptor interactions. Here, combined kinetics and thermodynamics of opioid agonist binding to human μ-opioid receptor (h-μOR) was investigated using real-time label-free surface plasmon resonance (SPR)–based method. The N-terminal end truncated and C-terminal 6His-tagged h-μOR was constructed and expressed in E. coli. Receptor was purified, detergent-solubilized and characterized by circular dichroism. The uniform immobilization of h-μOR on Ni-NTA chips was achieved using hybrid capture-coupling approach followed by reconstitution in lipid bilayer. Thermodynamic equilibrium affinities of opioids were in narrow nanomolar range and in near quantitative agreement with their Ki values. However, they did not correlate with their in vitro EC50 values, indicating that they might not have thermodynamic selectivity. Contrary, on and off rates exhibited much larger dispersion and well correlated with EC50 values, indicating that opioids might exhibit kinetic-selectivity towards their target. Temperature-dependent SPR assays provided access to rate and equilibrium thermodynamic data, which demonstrated binding of morphine and naloxone to μOR was exothermic and essentially enthalpy driven. This work suggests that kinetic-based structure-activity of opioids in drug design and incorporation into the pharmacokinetics-pharmacodynamics predictions may have more value than thermodynamic equilibrium constants alone.

Mapping the naloxone binding sites on the mu-opioid receptor using cell-based photocrosslinkers

Naloxone is an alkaloid antagonist that acts as an antidote to opioids through the mu-opioid receptor (MOR), a G protein-coupled receptor. However, its binding site on the MOR remains unknown. To investigate the binding interfaces necessary for naloxone and MOR, available structural information was combined with a cell-based photocrosslinking approach. Computer prediction revealed that four binding sites on MOR were required for naloxone binding. In addition, in the photocrosslinking approach, an amber stop codon was used to replace the sense codon of the MOR at 266 selected individual positions, in order to introduce the photoreactive amino acid p-benzoyl-l-phenylalanine (BzF) into MOR to evaluate the results of the computer analysis. The BzF-incorporated MOR mutant genes were expressed in CHO cells, in which MOR retained the ability to interact with its ligands, such as morphine, and exhibited MOR-dependent activation of ERK signaling following morphine stimulation. Notably, after treatment with tritium-labeled naloxone and exposure to UV light, we observed naloxone crosslinking with BzF replacement at hydrophobic residues and some polar/uncharged residues in the computer-predicted sites 1 and 3, indicating that these two sites in the MOR interact with naloxone. In conclusion, these results indicate that MOR has two naloxone binding sites and that the hydrophobic and polar/uncharged residues within these sites are important for naloxone binding.

A structural insight into the negative effects of opioids in analgesia by modulating the TLR4 signaling: An in silico approach.

Opioids are considered the gold standard therapy for pain. However, TLR-dependent negative effects in analgesia have highlighted the complexities in the pharmacodynamics of opioids. While successive studies have reported that morphine and Morphine-3-glucuronide (M3G) activate the TLR4 pathway, the structural details of this mechanism are lacking. Here, we have utilized various computational tools to reveal the structural dynamics of the opioid-bound TLR4/MD2 complex, and have proposed a potential TLR4 activation mechanism. Our results support previous findings, and include the novel insight that the stable binding of morphine and naloxone, but not M3G, in the MD2 cavity, is TLR4 dependent. Morphine interacts with MD2 near its Phe126 loop to induce the active conformation (MD2C); however, this binding is likely reversible, and the complex gains stability upon interaction with TLR4. M3G also induces the MD2C state, with both the Phe126 loop and the H1 loop being involved in MD2-M3G complex stability. Remarkably, naloxone, which requires TLR4 interaction for complex stability, switches the conformation of the gating loop to the inactive state (MD2°). Cumulatively, our findings suggest that ligand binding and receptor clustering occur successively in opioid-induced TLR4 signaling, and that MD2 plasticity and pocket hydrophobicity are crucial for the recognition and accommodation of ligands.

Decreased [³H]naloxone Binding in the Dentate Gyrus of Cloninger Type 1 Anxiety-Prone Alcoholics: A Postmortem Whole-Hemisphere Autoradiography Study

The opioid system of the central nervous system plays an essential role in the regulation of the rewarding effects of alcohol. Alcohol affects mu-opioid receptor (MOR) function. Enhanced MOR function inhibits the GABAergic inhibition of the nucleus accumbens (Nac), which leads to a release of dopamine in the Nac. Of the few pharmaceutical treatments for alcoholism, the MOR antagonists naltrexone and nalmefene benefit most a subset of alcoholics who are characterized with early onset and impulsivity. Our aim was to investigate possible differences in the binding density of [³H]naloxone, a MOR competitive antagonist, between Cloninger type 1 anxiety-prone and harm-avoidant alcoholics, Cloninger type 2 impulsive and antisocial alcoholics, and healthy controls in brain areas that are essential for reward, learning, impulse-control, and mood regulation. We used postmortem whole-hemisphere autoradiography with [³H]naloxone, as a binding ligand. A subsequent autoradiography was performed with [³H]DAMGO, a selective MOR agonist. Cloninger type 1 alcoholics displayed decreased [³H]naloxone binding density in all studied brain areas. This trend reached statistical significance in the dentate gyrus, where type 1 alcoholics' [³H]naloxone binding density was significantly decreased (p=0.019) when compared to controls. A similar trend of decreased binding in type 1 alcoholics was observed in the [³H]DAMGO study. Our finding suggest that Cloninger type 1 anxiety-prone alcoholics may have an altered [³H]naloxone binding in brain areas related to reward, impulse-control, mood, and learning. The finding lends support to the idea of Cloninger type 1 anxiety-prone alcoholics responding weaker to the opioidergic pharmaceuticals of the treatment of alcoholism than Cloninger type 2 impulsive alcoholics.

P.6.a.010 Decreased [H]naloxone binding density in the dentate gyrus of Cloninger type 1 alcoholics

P.6.a.010 Decreased [H]naloxone binding density in the dentate gyrus of Cloninger type 1 alcoholics

Chronic neuropathic pain in mice reduces μ-opioid receptor-mediated G-protein activity in the thalamus

Neuropathic pain is a debilitating condition that is often difficult to treat using conventional pharmacological interventions and the exact mechanisms involved in the establishment and maintenance of this type of chronic pain have yet to be fully elucidated. The present studies examined the effect of chronic nerve injury on μ-opioid receptors and receptor-mediated G-protein activity within the supraspinal brain regions involved in pain processing of mice. Chronic constriction injury (CCI) reduced paw withdrawal latency, which was maximal at 10 days post-injury. [d-Ala2,( N-Me)Phe4,Gly5-OH] enkephalin (DAMGO)-stimulated [ 35S]GTPγS binding was then conducted at this time point in membranes prepared from the rostral ACC (rACC), thalamus and periaqueductal grey (PAG) of CCI and sham-operated mice. Results showed reduced DAMGO-stimulated [ 35S]GTPγS binding in the thalamus and PAG of CCI mice, with no change in the rACC. In thalamus, this reduction was due to decreased maximal stimulation by DAMGO, with no difference in EC 50 values. In PAG, however, DAMGO E max values did not significantly differ between groups, possibly due to the small magnitude of the main effect. [ 3H]Naloxone binding in membranes of the thalamus showed no significant differences in B max values between CCI and sham-operated mice, indicating that the difference in G-protein activation did not result from differences in μ-opioid receptor levels. These results suggest that CCI induced a region-specific adaptation of μ-opioid receptor-mediated G-protein activity, with apparent desensitization of the μ-opioid receptor in the thalamus and PAG and could have implications for treatment of neuropathic pain.

Inhibition of [3H]naloxone binding in homogenates of rat brain by eseroline, a drug, with analgesic activity, related to physostigmine.

Inhibition of [3H]naloxone binding in homogenates of rat brain by eseroline, a drug, with analgesic activity, related to physostigmine.

Narcotic agonist and antagonist potencies of a homologous series of N-alkyl-norketobemidones measured by the guinea-pig ileum and mouse vas deferens methods

The narcotic agonist and antagonist potencies of the series of N-alkyl-norketobemidones from norketobemidone to decylnorketo-bemidone have been determined. The values obtained in the electrically stimulated preparations of the guinea-pig ileum and the mouse vas deferens are closely correlated. The agonist potencies observed in the guinea-pig ileum agree well with those found in the mouse hot-plate test (Oh-ishi & May) and those obtained by determining the inhibition of naloxone binding in brain homogenates (Wilson, Rogers, Pert & Snyder). The antagonist potencies in the guinea-pig ileum and, to a lesser extent, those in the mouse vas deferens agree with the values obtained in the morphine-dependent monkey.

Alteration of lymphocyte opioid receptors in methadone maintenance subjects

Methadone maintenance therapy is the most widely used treatment in patients with heroin addiction. Multiple studies have suggested that both current and former heroin addicts entering a methadone maintenance treatment program have altered immune function. Our previous study indicated that heroin addicts have depressed mitogen-stimulated lymphocyte proliferation and a decrease in the modulation of lymphocyte surface markers. This immunosuppression may be mediated via the direct interaction of opiates with lymphocyte opioid receptors. In order to test this hypothesis, the levels of opioid receptors on immune cells obtained from heroin users were determined using saturation binding, and it was found that former heroin addicts on methadone maintenance treatment had a significantly reduced maximum number ( B max) of [ 3H]naloxone binding. The B max values were 51.3 ± 7.6 fmol/mg protein for the non-addicted group and 25.3 ± 3.1 fmol/mg protein for the methadone maintenance group. Opioid receptor gene expression on the immune cell was determined using a semi-quantitative reverse-transcription polymerase chain reaction technique with specific pairs of primers to amplify mu- and delta-opioid receptor mRNAs. Both types of mRNAs were significantly decreased in lymphocytes obtained from the former heroin addicts on methadone maintenance subjects. Similarly, in an in vitro study, 100 μM methadone significantly down-regulated both mu- and delta-opioid receptor mRNA expressions in cultured lymphocytes obtained from naïve subjects. This effect was prevented by including 100 μM naloxone or pretreating with 50 ng/ml pertussis toxin. The data presented indicate that chronic opiate exposure was associated with down-regulation of G-protein-coupled opioid receptor gene expression in human lymphocytes.

High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.

Human Peripheral Blood Mononuclear Cells Express Nociceptin/Orphanin FQ, but Not μ, δ, or κ Opioid Receptors

In Brief BACKGROUND: Expression of opioid receptors on peripheral blood mononuclear cells (PBMC) is controversial. These receptors are currently classified as classical (MOP/mu/μ, DOP/delta/δ and KOP/kappa/κ) and nonclassical NOP (nociceptin/orphanin FQ; N/OFQ). METHODS: In this volunteer study we probed for the expression of both classical and nonclassical opioid receptors using 1) radioligand binding, 2) specific antibody binding, and 3) polymerase chain reaction-based experimental paradigms. RESULTS: Membranes prepared from PBMC from healthy volunteers did not bind either [3H]diprenorphine (a nonselective radioligand for classical opioid receptors) or [3H]N/OFQ. There was significant concentration-dependent binding of each radioligand to control tissues expressing recombinant MOP and NOP. In addition, using fluorescence-activated cell sorting paradigms, there was no binding of fluorescent naloxone or either of two MOP antibodies to whole PBMC, though fluorescent naloxone did bind to recombinant MOP (as a positive control). Using primers specific for classical and nonclassical opioid receptors, and RNA extracted from the PBMC of 10 healthy volunteers, we were also unable to detect MOP, DOP, and KOP transcripts. In contrast, NOP was detected in all samples. CONCLUSIONS: Despite using several complementary experimental strategies, we failed to demonstrate protein for classical or nonclassical opioid receptors on PBMC from healthy volunteers. We detected NOP mRNA, suggesting low-density NOP expression on these immunocytes. It is possible that N/OFQ, produced by the PBMC itself, may be involved in the control of immune function. IMPLICATIONS: Opioids depress the immune response, but the involvement of opioid receptors on immune cells is controversial. In the blood of volunteers we failed to detect MOP (μ), DOP (δ), KOP (κ) or Nociceptin receptor (NOP) protein, but we did find NOP mRNA. Direct immunomodulation by opioids in health is unlikely to involve classical opioid receptors, but the nociceptin system may represent a new clinical target.

Role of kappa and delta opioid receptors in mediating morphine-induced antinociception in morphine-tolerant infant rats

We have previously noted that the antinociceptive efficacy of morphine was significantly decreased in rat pups chronically infused with morphine from implanted osmotic minipumps. In this study, morphine was fully efficacious (i.e., 100% maximum possible effect, %MPE) in the 52 °C tail-immersion test after a 72-h infusion from implanted saline-filled osmotic minipumps. However, administration of up to 1000 mg/kg, s.c. morphine failed to elicit greater than a 27% MPE in rats infused with morphine at 2 mg/kg/h. Morphine was more efficacious when the water bath temperature was decreased to 49 °C. Experiments were conducted to determine the mechanisms whereby chronic morphine administration leads to a decrease in antinociceptive efficacy. The kappa-opioid antagonist nor-binalorphimine completely blocked the antinociceptive effects of morphine in morphine-infused rat pups. The kappa agonist U50,488 elicited antinociception; however, the requirement to use higher doses in morphine- than saline-infused rats indicates that kappa cross-tolerance was present. Thus, in tolerant rats the antinociceptive effects of high doses of morphine appear to be mediated through kappa-opioid receptors. The delta-opioid antagonist naltrindole was inactive in both treatment groups. DAMGO-stimulated [ 35S]GTPγS and [ 3H]naloxone binding reveals that the anatomical distribution of the mu-opioid receptor was consistent with that of the adult rat brain. In adult rats, the mu-opioid receptor is desensitized during morphine tolerance. However, desensitization was not evident in P17 rats based on the lack of significant decreases in [ 35S]GTPγS binding. Furthermore, [ 3H]naloxone binding indicated a lack of mu receptor downregulation in morphine-tolerant rat pups.

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 μg, i.c.v.) and BMS182874 (50 μg, 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 μg, 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]GTPγS 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.

A Novel Combination of Opiates and Endothelin Antagonists to Manage Pain Without Any Tolerance Development

Several neurotransmitter mechanisms have been proposed as playing a role in the development of morphine tolerance. We provide evidence for the first time that endothelin antagonists can restore morphine analgesia in morphine-tolerant rats and prevent the development of tolerance to morphine. Studies were carried out in rats and mice treated with implanted placebo or implanted morphine pellet. The maximal tail-flick latency in morphine pellet + vehicle-treated rats (7.54 seconds) was significantly lower when compared with placebo pellet + vehicle-treated rats (10 seconds), indicating that tolerance developed to the analgesic effect of morphine. BQ123 potentiated tail-flick latency by 30.0% in placebo-tolerant rats and 94.5% in morphine-tolerant rats compared with respective controls. BMS182874 potentiated tail-flick latency by 30.2% in placebo-tolerant rats and 66.7% in morphine-tolerant rats. The enhanced analgesic effect of morphine after treatment with endothelin antagonists could be blocked by naloxone, indicating an opiate-mediated effect; but naloxone binding to brain membranes was not affected by BQ123. Guanosine triphosphate binding was stimulated by morphine and endothelin-1 in non-tolerant mice and not in morphine-tolerant mice; however, guanosine triphosphate binding was stimulated by BQ123 in morphine-tolerant mice and was unaffected in non-tolerant mice. These results suggest that uncoupling of G-protein occurs in morphine tolerance and endothelin antagonist restores the coupling of G-protein to its receptors. A combination use of endothelin antagonist and opiates could provide a novel approach in improving analgesia and eliminating tolerance.

Role of Na(+), K(+)-ATPase in morphine-induced antinociception.

We evaluated the modulation by Na+,K+-ATPase inhibitors of morphine-induced antinociception in the tail-flick test and [3H]naloxone binding to forebrain membranes. The antinociception induced by morphine (1-32 mg/kg, s.c.) in mice was dose-dependently antagonized by ouabain (1-10 ng/mouse, i.c.v.), which produced a significant shift to the right of the morphine dose-response curve. The i.c.v. administration of three Na+,K+-ATPase inhibitors (ouabain at 0.1-100, digoxin at 1-1000, and digitoxin at 10-10000 ng/mouse) dose-dependently antagonized the antinociceptive effect of morphine (4 mg/kg, s.c.) in mice, with the following order of potency: ouabain > digoxin > digitoxin. This effect cannot be explained by any interaction at opioid receptors, since none of these Na+,K+-ATPase inhibitors displaced [3H]naloxone from its binding sites, whereas naloxone did so in a concentration-dependent manner. The antinociception induced by morphine (5 mg/kg, s.c.) in rats was antagonized by the i.c.v. administration of ouabain at 10 ng/rat, whereas it was not significantly modified by intrathecally administered ouabain (10 and 100 ng/rat). These results suggest that the activation of Na+,K+-ATPase plays a role in the supraspinal, but not spinal, antinociceptive effect of morphine.