guanosine-5--o-(3-thiotriphosphate) and Pain

Opioid δ receptor agonists are potent antihyperalgesics in chronic pain models, but tolerance develops after prolonged use. Previous evidence indicates that distinct forms of tolerance occur depending on the internalization properties of δ receptor agonists. As arrestins are important in receptor internalization, we investigated the role of arrestin 2 (β-arrestin 1) in mediating the development of tolerance induced by high- and low-internalizing δ receptor agonists.. We evaluated the effect of two δ receptor agonists with similar analgesic potencies, but either high-(SNC80) or low-(ARM390) internalization properties in wild-type (WT) and arrestin 2 knockout (KO) mice. We compared tolerance to the antihyperalgesic effects of these compounds in a model of inflammatory pain. We also examined tolerance to the convulsant effect of SNC80. Furthermore, effect of chronic treatment with SNC80 on δ agonist-stimulated [. Arrestin 2 KO resulted in increased drug potency, duration of action and decreased acute tolerance to the antihyperalgesic effects of SNC80. In contrast, ARM390 produced similar effects in both WT and KO animals. Following chronic treatment, we found a marked decrease in the extent of tolerance to SNC80-induced antihyperalgesia and convulsions in arrestin 2 KO mice. Accordingly, δ receptors remained functionally coupled to G proteins in arrestin 2 KO mice chronically treated with SNC80.. Overall, these results suggest that δ receptor agonists interact with arrestins in a ligand-specific manner, and tolerance to high- but not low-internalizing agonists are preferentially regulated by arrestin 2.

Topics: Analgesics; Animals; Benzamides; beta-Arrestin 1; Brain; Drug Tolerance; Female; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice, Knockout; Pain; Piperazines; Receptors, Opioid, delta; Seizures

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

Topics: Analgesia; Analgesics, Opioid; Animals; Constipation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Gene Deletion; Gene Expression; Gene Knockout Techniques; Gene Order; Gene Targeting; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Knockout; Morphine; NAV1.8 Voltage-Gated Sodium Channel; Nociception; Pain; Pain Measurement; Posterior Horn Cells; Protein Binding; Receptors, Opioid, mu; Sensory Thresholds

Bone cancer pain is chronic and often difficult to control with opioids. However, recent studies have shown that several opioids have distinct analgesic profiles in chronic pain.. To clarify the mechanisms underlying these distinct analgesic profiles, functional changes in the μ-opioid receptor were examined using a mouse femur bone cancer (FBC) model.. In the FBC model, the B(max) of [(3) H]-DAMGO binding was reduced by 15-45% in the periaqueductal grey matter (PAG), region ventral to the PAG (vPAG), mediodorsal thalamus (mTH), ventral thalamus and spinal cord. Oxycodone (10(-8) -10(-5)  M) and morphine (10(-8) -10(-5)  M) activated [(35) S]-GTPγS binding, but the activation was significantly attenuated in the PAG, vPAG, mTH and spinal cord in the FBC model. Interestingly, the attenuation of oxycodone-induced [(35) S]-GTPγS binding was quite limited (9-26%) in comparison with that of morphine (46-65%) in the PAG, vPAG and mTH, but not in the spinal cord. Furthermore, i.c.v. oxycodone at doses of 0.02-1.0 μg per mouse clearly inhibited pain-related behaviours, such as guarding, limb-use abnormalities and allodynia-like behaviour in the FBC model mice, while i.c.v. morphine (0.05-2.0 μg per mouse) had only partial or little analgesic effect on limb-use abnormalities and allodynia-like behaviour.. These results show that μ-opioid receptor functions are attenuated in several pain-related regions in bone cancer in an agonist-dependent manner, and suggest that modification of the μ-opioid receptor is responsible for the distinct analgesic effect of oxycodone and morphine.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Bone Neoplasms; Brain; Cell Line, Tumor; Disease Models, Animal; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Mice, Inbred C3H; Morphine; Oxycodone; Pain; Receptors, Opioid, mu; Spinal Cord

The perception of pain and its inhibition varies considerably between individuals, and this variability is still unexplained. The aim of the present study is to determine whether functional interactions between opioid receptors are involved in the inter-individual variability in the sensitivity to μ-opioid receptor agonists.. Anti-nociceptive tests, radioligand binding, stimulation of [(35) S]GTP-γ-S binding, inhibition of cAMP production and co-immunoprecipitation experiments were performed in two strains of rat (Sprague-Dawley bred at our university - SDU - and Wistar) that differ in their sensitivity to opioids.. The increased anti-nociceptive potency of µ-opioid receptor agonists in SDU rats was reversed by the δ-opioid receptor antagonist, naltrindole. Inhibition of the binding of [(3) H] naltrindole by µ-opioid receptor agonists was different in brain membranes from SDU and Wistar rats. Differences were also evident in the effect of δ-opioid receptor ligands on the binding of [(35) S]GTP-γ-S stimulated by µ-opioid receptors agonists. No strain-related differences were detected in spinal cord membranes. The potency of morphine to inhibit cAMP production in brain membranes varied between the strains, in the presence of deltorphin II and naltrindole. Co-immunoprecipitation experiments demonstrated that δ-opioid receptors were associated with μ-opioid receptors to a higher extent in brain synaptosomal fractions from SDU than in those from Wistar rats.. There was increased supraspinal cross-talk between μ and δ-opioid receptors in SDU, as compared with Wistar rats. This was related to an enhanced sensitivity to anti-nociception induced by µ-opioid receptor agonists.

Topics: Analgesics, Opioid; Animals; Brain; Cyclic AMP; Disease Models, Animal; Guanosine 5'-O-(3-Thiotriphosphate); Immunoprecipitation; Male; Naltrexone; Narcotic Antagonists; Pain; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptor Cross-Talk; Receptors, Opioid, delta; Receptors, Opioid, mu; Species Specificity; Spinal Cord

The synthesis and the effect of a combination of 6-glycine and 14-phenylpropoxy substitutions in N-methyl- and N-cycloproplymethylmorphinans on biological activities are described. Binding studies revealed that all new 14-phenylpropoxymorphinans (11-18) displayed high affinity to opioid receptors. Replacement of the 14-methoxy group with a phenylpropoxy group led to an enhancement in affinity to all three opioid receptor types, with most pronounced increases in δ and κ activities, hence resulting in a loss of μ receptor selectivity. All compounds (11-18) showed potent and long-lasting antinociceptive effects in the tail-flick test in rats after subcutaneous administration. For the N-methyl derivatives 13 and 14, analgesic potencies were in the range of their 14-methoxy analogues 9 and 10, respectively. Even derivatives 15-18 with an N-cyclopropylmethyl substituent acted as potent antinociceptive agents, being several fold more potent than morphine. Subcutaneous administration of compounds 13 and 14 produced significant and prolonged antinociceptive effects mediated through peripheral opioid mechanisms in carrageenan-induced inflammatory hyperalgesia in rats.

Topics: Analgesics; Animals; Binding, Competitive; Guanosine 5'-O-(3-Thiotriphosphate); Magnetic Resonance Spectroscopy; Morphinans; N-substituted Glycines; Pain; Rats; Receptors, Opioid; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship

Opioid receptors are major actors in pain control and are broadly distributed throughout the nervous system. A major challenge in pain research is the identification of key opioid receptor populations within nociceptive pathways, which control physiological and pathological pain. In particular, the respective contribution of peripheral vs. central receptors remains unclear, and it has not been addressed by genetic approaches. To investigate the contribution of peripheral delta opioid receptors in pain control, we created conditional knockout mice where delta receptors are deleted specifically in peripheral Na(V)1.8-positive primary nociceptive neurons. Mutant mice showed normal pain responses to acute heat and to mechanical and formalin stimuli. In contrast, mutant animals showed a remarkable increase of mechanical allodynia under both inflammatory pain induced by complete Freund adjuvant and neuropathic pain induced by partial sciatic nerve ligation. In these 2 models, heat hyperalgesia was virtually unchanged. SNC80, a delta agonist administered either systemically (complete Freund adjuvant and sciatic nerve ligation) or into a paw (sciatic nerve ligation), reduced thermal hyperalgesia and mechanical allodynia in control mice. However, these analgesic effects were absent in conditional mutant mice. In conclusion, this study reveals the existence of delta opioid receptor-mediated mechanisms, which operate at the level of Na(V)1.8-positive nociceptive neurons. Delta receptors in these neurons tonically inhibit mechanical hypersensitivity in both inflammatory and neuropathic pain, and they are essential to mediate delta opioid analgesia under conditions of persistent pain. This delta receptor population represents a feasible therapeutic target to alleviate chronic pain while avoiding adverse central effects. The conditional knockout of delta-opioid receptor in primary afferent Na(V)1.8 neurons augmented mechanical allodynia in persistent pain models and abolished delta opioid analgesia in these models.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Benzamides; Disease Models, Animal; Dose-Response Relationship, Drug; Freund's Adjuvant; Ganglia, Spinal; Guanosine 5'-O-(3-Thiotriphosphate); Inflammation; Mice; Mice, Inbred C57BL; Motor Activity; NAV1.8 Voltage-Gated Sodium Channel; Nociceptors; Pain; Pain Measurement; Piperazines; Protein Binding; Receptors, Opioid, delta; Sodium Channels; Sulfur Isotopes

We have previously developed quinolone-3-carboxamides with the aim of obtaining new ligands for both cannabinoid receptors, CB1 and CB2. Our preliminary screening led to the identification of cannabinoid receptor ligands characterized by high affinity and, in some cases, also selectivity for CB(2) receptors. Specifically, three compounds, 1, 2 and 3 showed high affinity for CB2 as well as high selectivity over CB1 receptors. In addition, the activity shown by 1 against the formalin-induced nocifensive response in mice, reported in our previous paper, suggests that quinolone-3-carboxamides possess anti-nociceptive properties. In the present work, we have performed functional in vitro bioassays with the aim of investigating the functional activity in the [35S]GTPgammaS binding assay of the other two compounds that, like 1, behave as CB2 selective ligands, and their potential analgesic actions in vivo. We found that both 2 and 3 behave in vitro as CB2 inverse agonists and are able to decrease nociceptive behaviour in the late phase of the formalin test only at the highest dose tested, although, at lower doses, they prevent the anti-nociceptive effects of a selective CB2 partial agonist in the formalin test. These results identify in 2 and 3 two novel, potent and selective CB2 antagonists/inverse agonists and confirm previous reports in the literature that, in addition to agonists at cannabinoid CB2 receptors, also inverse agonists/antagonists at these receptors show promise as anti-inflammatory agents.

Topics: Analgesics; Animals; CHO Cells; Cricetinae; Cricetulus; Cyclohexanols; Drug Inverse Agonism; Formaldehyde; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Molecular Structure; Pain; Quinolones; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Structure-Activity Relationship

The present studies examined the effect of chronic neuropathic pain on cannabinoid receptor density and receptor-mediated G-protein activity within supraspinal brain areas involved in pain processing and modulation in mice. Chronic constriction injury (CCI) produced a significant decrease in WIN 55,212-2-stimulated [(35)S]GTPgammaS binding in membranes prepared from the rostral anterior cingulate cortex (rACC) of CCI mice when compared to sham-operated controls. Saturation binding with [(3)H]SR 141716A in membranes of the rACC showed no significant differences in binding between CCI and sham mice. Analysis of levels of the endocannabinoids anandamide (AEA) or 2-arachidonoylglycerol (2-AG) in the rACC following CCI showed no significant differences between CCI and sham mice. These data suggest that CCI produced desensitization of the cannabinoid 1 receptor in the rACC in the absence of an overall decrease in cannabinoid 1 receptor density or change in levels of AEA or 2-AG. These data are the first to show alterations in cannabinoid receptor function in the rostral anterior cingulate cortex in response to a model of neuropathic pain.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cell Membrane; Constriction; Disease Models, Animal; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred Strains; Models, Neurological; Morpholines; Naphthalenes; Pain; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sulfur Radioisotopes; Tritium

Cannabinoid receptor 2 (CB2) agonists have recently gained attention as potential therapeutic targets in the management of neuropathic pain. In this study, we characterized the pharmacological profile of the novel compound N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide (MDA19), a CB2 agonist.. We used radioligand binding assays and multiple in vitro functional assays at human and rat CB(1) and CB(2) receptors. The effects of MDA19 in reversing neuropathic pain were assessed in various neuropathic pain models in rats and in CB2(+/+) and CB2(-/-) mice.. MDA19 displayed 4-fold-higher affinity at the human CB(2) than at the human CB1 receptor (K(i) = 43.3 +/- 10.3 vs 162.4 +/- 7.6 nM) and nearly 70-fold-higher affinity at the rat CB2 than at the rat CB1 receptor (K(i) = 16.3 +/- 2.1 vs 1130 +/- 574 nM). In guanosine triphosphate (GTP)gamma[(35)S] functional assays, MDA19 behaved as an agonist at the human CB1 and CB2 receptors and at the rat CB1 receptor but as an inverse agonist at the rat CB2 receptor. In 3',5'-cyclic adenosine monophosphate (cAMP) assays, MDA19 behaved as an agonist at the rat CB1 receptor and exhibited no functional activity at the rat CB(2) receptor. In extracellular signal-regulated kinases 1 and 2 activation assays, MDA19 behaved as an agonist at the rat CB2 receptor. MDA19 attenuated tactile allodynia produced by spinal nerve ligation or paclitaxel in a dose-related manner in rats and CB2(+/+) mice but not in CB2(-/-) mice, indicating that CB2 receptors mediated the effects of MDA19. MDA19 did not affect rat locomotor activity.. We found that MDA19 exhibited a distinctive in vitro functional profile at rat CB2 receptors and behaved as a CB1/CB2 agonist in vivo, characteristics of a protean agonist. MDA19 has potential for alleviating neuropathic pain without producing adverse effects in the central nervous system.

Topics: Animals; Behavior, Animal; Biotransformation; Cannabinoids; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrazines; Indoles; Male; Mice; Mice, Knockout; Paclitaxel; Pain; Pain Measurement; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Nerves

The phytocannabinoid, Delta(9)-tetrahydrocannabivarin (THCV), can block cannabinoid CB(1) receptors. This investigation explored its ability to activate CB(2) receptors, there being evidence that combined CB(2) activation/CB(1) blockade would ameliorate certain disorders.. We tested the ability of THCV to activate CB(2) receptors by determining whether: (i) it inhibited forskolin-stimulated cyclic AMP production by Chinese hamster ovary (CHO) cells transfected with human CB(2) (hCB(2)) receptors; (ii) it stimulated [(35)S]GTPgammaS binding to hCB(2) CHO cell and mouse spleen membranes; (iii) it attenuated signs of inflammation/hyperalgesia induced in mouse hind paws by intraplantar injection of carrageenan or formalin; and (iv) any such anti-inflammatory or anti-hyperalgesic effects were blocked by a CB(1) or CB(2) receptor antagonist.. THCV inhibited cyclic AMP production by hCB(2) CHO cells (EC(50)= 38 nM), but not by hCB(1) or untransfected CHO cells or by hCB(2) CHO cells pre-incubated with pertussis toxin (100 ng.mL(-1)) and stimulated [(35)S]GTPgammaS binding to hCB(2) CHO and mouse spleen membranes. THCV (0.3 or 1 mg.kg(-1) i.p.) decreased carrageenan-induced oedema in a manner that seemed to be CB(2) receptor-mediated and suppressed carrageenan-induced hyperalgesia. THCV (i.p.) also decreased pain behaviour in phase 2 of the formalin test at 1 mg.kg(-1), and in both phases of this test at 5 mg.kg(-1); these effects of THCV appeared to be CB(1) and CB(2) receptor mediated.. THCV can activate CB(2) receptors in vitro and decrease signs of inflammation and inflammatory pain in mice partly via CB(1) and/or CB(2) receptor activation.

Topics: Animals; Cell Line, Transformed; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Dronabinol; Edema; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Inflammation; Male; Membranes; Mice; Mice, Inbred C57BL; Pain; Pain Measurement; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spleen

Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity.. Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G-protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment.. We found that conventional subtypes of PKC (cPKC) were up-regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS-ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine-induced up-regulation of cPKC was completely reversed by treatment with AS-ODN targeting KOR. In addition, AS-ODN targeting KOR significantly reversed the chronic pain-induced down-regulation of PKC activity or up-regulation of MOR [(35)S]GTPgammaS binding activity after repeated morphine treatment.. These results indicate that KOR plays an important role in the inhibition of repeated morphine-induced cPKC up-regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.

Topics: Analgesics, Opioid; Animals; Brain; Chronic Disease; Disease Models, Animal; Down-Regulation; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Formaldehyde; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Morphine; Naphthalenes; Oligonucleotides, Antisense; Pain; Pain Measurement; Pain Threshold; Protein Kinase C; Protein Kinase Inhibitors; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Topics: Analgesics; Animals; Benzamides; Brain; Calcium; Cell Membrane; Disease Models, Animal; Drug Interactions; Drug Tolerance; Female; Freund's Adjuvant; Ganglia, Spinal; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Inflammation; Ligands; Locomotion; Male; Maze Learning; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pain; Pain Threshold; Patch-Clamp Techniques; Piperazines; Piperidines; Protein Binding; Protein Transport; Receptors, Opioid, delta; Sensory Receptor Cells; Spinal Cord; Statistics, Nonparametric; Sulfur Isotopes; Time Factors

Compounds that activate both NOP and mu-opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds.. Binding affinities were determined on membranes from cells transfected with NOP and opioid receptors. Functional activity was determined by [(35)S]GTPgammaS binding on cell membranes and using the mouse vas deferens preparation in vitro and the tail flick antinociception assay in vivo.. Compounds ranged in affinity from SR14150, 20-fold selective for NOP receptors, to buprenorphine, 50-fold selective for mu-opioid receptors. In the [(35)S]GTPgammaS assay, SR compounds ranged from full agonist to antagonist at NOP receptors and most were partial agonists at mu-opioid receptors. Buprenorphine was a low efficacy partial agonist at mu-opioid receptors, but did not stimulate [(35)S]GTPgammaS binding through NOP. In the mouse vas deferens, each compound, except for SR16430, inhibited electrically induced contractions. In each case, except for N/OFQ itself, the inhibition was due to mu-opioid receptor activation, as determined by equivalent results in NOP receptor knockout tissues. SR14150 showed antinociceptive activity in the tail flick test, which was reversed by the opioid antagonist naloxone.. Compounds that bind to both mu-opioid and NOP receptors have antinociceptive activity but the relative contribution of each receptor is unclear. These experiments help characterize compounds that bind to both receptors, to better understand the mechanism behind their biological activities, and identify new pharmacological tools to characterize NOP and opioid receptors.

Topics: Analgesics, Opioid; Animals; Buprenorphine; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Guanosine 5'-O-(3-Thiotriphosphate); Indoles; Ligands; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nociceptin Receptor; Pain; Pain Measurement; Protein Binding; Receptors, Opioid; Receptors, Opioid, mu; Vas Deferens

Although opioids are highly effective analgesics, they are also known to induce cellular adaptations resulting in tolerance. Experimental studies are often performed in the absence of painful tissue injury, which precludes extrapolation to the clinical situation. Here we show that rats with chronic morphine treatment do not develop signs of tolerance at peripheral mu-opioid receptors (micro-receptors) in the presence of painful CFA-induced paw inflammation. In sensory neurons of these animals, internalization of mu-receptors was significantly increased and G protein coupling of mu-receptors as well as inhibition of cAMP accumulation were preserved. Opioid receptor trafficking and signaling were reduced, and tolerance was restored when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing granulocytes, monocytes, and lymphocytes with cyclophosphamide (CTX). Our data indicate that the continuous availability of endogenous opioids in inflamed tissue increases recycling and preserves signaling of mu-receptors in sensory neurons, thereby counteracting the development of peripheral opioid tolerance. These findings infer that the use of peripherally acting opioids for the prolonged treatment of inflammatory pain associated with diseases such as chronic arthritis, inflammatory neuropathy, or cancer, is not necessarily accompanied by opioid tolerance.

Topics: Animals; beta-Endorphin; Cells, Cultured; Cyclophosphamide; Drug Tolerance; Fentanyl; Guanosine 5'-O-(3-Thiotriphosphate); Inflammation; Male; Morphine; Neurons, Afferent; Pain; Rats; Rats, Wistar; Receptors, Opioid, mu

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Topics: Acetic Acid; Algorithms; Analgesics; Animals; Benzeneacetamides; Binding, Competitive; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Injections, Intravenous; Male; Mice; Mice, Inbred ICR; Oligopeptides; Opioid Peptides; Pain; Pain Measurement; Peripheral Nervous System; Postural Balance; Pyrrolidines; Radioligand Assay; Reaction Time; Receptors, Opioid, kappa

Analgesics such as morphine cause many side effects including addiction, but kappa-opioid receptor agonist can produce antinociception without morphine-like side effects. With the aim of developing new and potent analgesics with lower abuse potential, we studied the antinociceptive and physical dependent properties of a derivate of ICI-199441, an analogue of (-)U50,488H, named (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (LPK-26). LPK-26 showed a high affinity to kappa-opioid receptor with the Ki value of 0.64 nM and the low affinities to micro-opioid receptor and delta-opioid receptor with the Ki values of 1170 nM and >10,000 nM, respectively. It stimulated [(35)S]GTPgammaS binding to G-proteins with an EC50 value of 0.0094 nM. In vivo, LPK-26 was more potent than (-)U50,488H and morphine in analgesia, with the ED50 values of 0.049 mg/kg and 0.0084 mg/kg in hot plat and acetic acid writhing tests, respectively. Moreover, LPK-26 failed to induce physical dependence, but it could suppress naloxone-precipitated jumping in mice when given simultaneously with morphine. Taken together, our results show that LPK-26 is a novel selective kappa-opioid receptor agonist with highly potent antinociception effects and low physical dependence potential. It may be valuable for the development of analgesic and drug that can be used to reduce morphine-induced physical dependence.

Topics: Acetic Acid; Analgesics, Opioid; Animals; Behavior, Animal; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Humans; Male; Mice; Motor Activity; Naloxone; Narcotic Antagonists; Opioid-Related Disorders; Pain; Pain Measurement; Pain Threshold; Protein Binding; Pyrroles; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors; Transfection

Endomorphin-1 and endomorphin-2 are endogenous peptides that are highly selective for mu-opioid receptors. However, studies of their functional efficacy and selectivity are controversial. In this study, we systematically compared the effects of intrathecal (i.t.) administration of endomorphin-1 and -2 on nociception assays and G protein activation with those of [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), a highly effective peptidic mu-opioid receptor agonist.. Male Sprague-Dawley rats were used. Acute and inflammatory pain models were used to compare the duration and magnitude of antinociception. Agonist-stimulated [(35)S]GTP gamma S binding was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. In addition, antagonists selective for each receptor type were used to verify the functional selectivity of endomorphins in the rat spinal cord.. After i.t. administration, endomorphin-1 and -2 produced less antinociceptive effects than DAMGO in the model of acute pain. Concentration-response curves for DAMGO-, endomorphin-1-, and endomorphin-2-stimulated [(35)S]GTP gamma S binding revealed that both endomorphin-1 and -2 produced less G protein activation (i.e., approximately 50%-60%) than DAMGO did in the membranes of spinal cord and thalamus. In addition, i.t. endomorphin-induced antinociception was blocked by mu-opioid receptor selective dose of naltrexone (P < 0.05), but not by delta- and kappa-opioid receptor antagonists, naltrindole and nor-binaltorphimine (P > 0.05).. Endomorphins are partial agonists for G protein activation at spinal and thalamic mu-opioid receptors. Both in vivo and in vitro measurements together suggest that DAMGO is more effective than endomorphins. Spinal endomorphins' antinociceptive efficacy may range between 53% and 84% depending on the intensity and modality of the nociceptive stimulus.

Topics: Analgesics; Analgesics, Opioid; Animals; Behavior, Animal; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Partial Agonism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Spinal; Male; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Spinal Cord; Sulfur Radioisotopes; Thalamus; Time Factors

Cholinesterase inhibitors which reach the central nervous system produce pain relief but are poorly tolerated because of gastrointestinal side effects. Here, the authors tested whether donepezil, a central nervous system penetrant cholinesterase inhibitor with a low incidence of gastrointestinal side effects, would relieve hypersensitivity in an animal model of neuropathic pain.. Male rats were anesthetized, and the L5 and L6 spinal nerves were ligated unilaterally. Hypersensitivity was measured by withdrawal threshold to von Frey filament application to the hind paw after oral donepezil, and antagonists administered centrally and peripherally. Efficacy of chronic oral donepezil to relieve hypersensitivity was tested, and activation of G proteins by M(2) muscarinic receptors was determined by carbachol-stimulated [(35)S]guanosine triphosphate (gamma)S autoradiography in brain and spinal cord.. Spinal nerve ligation resulted in hypersensitivity that was more severe ipsilateral than contralateral to surgery. Oral donepezil reduced hypersensitivity bilaterally in a dose-dependent manner for 2 h, and this effect was blocked by spinal but not supraspinal or peripheral muscarinic receptor antagonism. Oral donepezil maintained efficacy over 2 weeks of twice daily administration, and this treatment did not lead to desensitization of muscarinic receptor-coupled G proteins in brain or spinal cord.. Donepezil, a well-tolerated cholinesterase inhibitor used in the treatment of Alzheimer dementia, reduces hypersensitivity in this rat model of neuropathic pain by actions on muscarinic receptors in the spinal cord. Lack of tolerance to this effect, in contrast to rapid tolerance to direct receptor agonists, suggests that cholinesterase inhibition may be useful in the treatment of neuropathic pain.

Topics: Administration, Oral; Animals; Cholinesterase Inhibitors; Donepezil; Dose-Response Relationship, Drug; Drug Tolerance; Guanosine 5'-O-(3-Thiotriphosphate); Indans; Male; Pain; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Spinal Cord

This study investigated three possible mechanisms by which the antinociceptive effects of the mu-opioid receptor (MOR) agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and the delta-opioid receptor (DOR) agonist [d-Ala(2),Glu(4)]-deltorphin (deltorphin II) (DELT), microinjected into the rostral ventromedial medulla (RVM), are enhanced in rats with persistent inflammatory injury. Radioligand binding determined that neither the B(max) nor the K(d) values of [(3)H]DAMGO differed in RVM membranes from rats that received an intraplantar injection of saline or complete Freund's adjuvant (CFA) in one hindpaw 4 h, 4 days, or 2 weeks earlier. Likewise, neither the EC(50) nor the E(max) value for DAMGO-induced stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding differed in the RVM of saline- or CFA-treated rats at any time point. Microinjection of fixed dose combinations of DAMGO and DELT in the RVM of naive rats indicated that these agonists interact synergistically to produce antinociception when DAMGO is present in equal or greater amounts than DELT and, additively, when DELT is the predominant component. Thus, unlike the periphery or spinal cord, potentiation of MOR-mediated antinociception does not entail an increase in MOR number, affinity, or coupling. Rather, the data are concordant with our proposal that potentiation results from a synergistic interaction of exogenous MOR agonist with DOR-preferring enkephalins whose levels are increased in CFA-treated rats (J Neurosci 21:2536-2545, 2001). Virtually no specific [(3)H]DELT binding nor stimulation of [(35)S]GTPgammaS binding by DELT was obtained in RVM membranes from CFA- or saline-treated rats at any time point. The mechanisms responsible for the potentiation of DELT-mediated antinociception remain to be elucidated.

Topics: Analgesics; Animals; Cell Membrane; Dose-Response Relationship, Drug; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Freund's Adjuvant; Guanosine 5'-O-(3-Thiotriphosphate); Hindlimb; Hyperalgesia; Inflammation; Male; Medulla Oblongata; Oligopeptides; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid, delta; Receptors, Opioid, mu

Morphine and delta9-tetrahydrocannabinol (THC) produce antinociception via mu opioid and cannabinoid CB1 receptors, respectively, located in central nervous system (CNS) regions including periaqueductal gray and spinal cord. Chronic treatment with morphine or THC produces antinociceptive tolerance and cellular adaptations that include receptor desensitization. Previous studies have shown that administration of combined sub-analgesic doses of THC+morphine produced antinociception in the absence of tolerance. The present study assessed receptor-mediated G-protein activity in spinal cord and periaqueductal gray following chronic administration of THC, morphine or low dose combination. Rats received morphine (escalating doses from 1 to 6x75 mg s.c. pellets or s.c. injection of 100 to 200 mg/kg twice daily), THC (4 mg/kg i.p. twice daily) or low dose combination (0.75 mg/kg each morphine (s.c) and THC (i.p.) twice daily) for 6.5 days. Antinociception was measured in one cohort of rats using the paw pressure test, and a second cohort was assessed for agonist-stimulated [35S]GTPgammaS binding. Chronic administration of morphine or THC produced antinociceptive tolerance to the respective drugs, whereas combination treatment did not produce tolerance. Administration of THC attenuated cannabinoid CB1 receptor-stimulated G-protein activity in both periaqueductal gray and spinal cord, and administration of morphine decreased mu opioid receptor-stimulated [35S]GTPgammaS binding in spinal cord or periaqueductal gray, depending on route of administration. In contrast, combination treatment did not alter cannabinoid CB1 receptor- or mu opioid receptor-stimulated G-protein activity in either region. These results demonstrate that low dose THC-morphine combination treatment produces antinociception in the absence of tolerance or attenuation of receptor activity.

Topics: Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Benzoxazines; Dose-Response Relationship, Drug; Dronabinol; Drug Synergism; Drug Therapy, Combination; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraperitoneal; Injections, Subcutaneous; Male; Morphine; Morpholines; Naphthalenes; Pain; Pain Measurement; Pain Threshold; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Spinal Cord; Sulfur Radioisotopes; Time Factors

Improgan is a congener of the H(2) antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB(1) receptors, the effects of the CB(1) antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB(1) potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB(1) receptors, rimonabant (K(d) = 0.23 nM), and O-1691 (K(d) = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB(1) affinity (O-1876, K(d) = 139 nM and O-848, K(d) = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB(1) potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with (35)S-GTPgammaS on CB(1) receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB(1) receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB(1) receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB(1) may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB(1)-blocking properties, will help to identify these cannabinoid-like, non-CB(1) mechanisms.. This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB(1)) is not solely responsible for improgan analgesia.

Topics: Analgesics; Analysis of Variance; Animals; Cimetidine; Disease Models, Animal; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Pain; Pain Measurement; Pain Threshold; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Cannabinoid, CB1; Rimonabant; Time Factors

This study was conducted to assess the utility of unbound brain EC50 (EC50,u) as a measure of in vivo potency for centrally active drugs. Seven mu-opioid agonists (alfentanil, fentanyl, loperamide, methadone, meperidine, morphine, and sufentanil) were selected as model central nervous system drugs because they elicit a readily measurable central effect (antinociception) and their clinical pharmacokinetics/pharmacodynamics are well understood. Mice received an equipotent subcutaneous dose of one of the model opioids. The time course of antinociception and the serum and brain concentrations were determined. A pharmacokinetic/pharmacodynamic model was used to estimate relevant parameters. In vitro measures of opioid binding affinity (Ki) and functional activity [EC50 for agonist stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding] and relevant clinical parameters were obtained to construct in vitro-to-preclinical and preclinical-to-clinical correlations. The strongest in vitro-to-in vivo correlation was observed between Ki and unbound brain EC50,u (r2 approximately 0.8). A strong correlation between mouse serum and human plasma EC50 was observed (r2 = 0.949); the correlation was improved when corrected for protein binding (r2 = 0.995). Clinical equipotent i.v. dose was only moderately related to Ki. However, estimates of ED50 and EC50 (total serum, unbound serum, total brain, and unbound brain) were significant predictors of clinical equipotent i.v. dose; the best correlation was observed for brain EC50,u (r2 = 0.982). For each opioid, brain equilibration half-life in mice was almost identical to the plasma effect-site equilibration half-life measured clinically. These results indicate that the mouse is a good model for opioid human brain disposition and clinical pharmacology and that superior in vitro-to-preclinical and preclinical-to-clinical correlations can be achieved with relevant unbound concentrations.

Topics: Analgesics, Opioid; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; Blood-Brain Barrier; Brain; Drug Evaluation, Preclinical; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Mice; Models, Biological; Pain; Predictive Value of Tests; Protein Binding; Radioligand Assay; Receptors, Opioid, mu

Clinically, it has been reported that chronic pain induces depression, anxiety, and reduced quality of life. The endogenous opioid system has been implicated in nociception, anxiety, and stress. The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice. We found that either injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery. Under these conditions, the selective mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective delta-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPgammaS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation. CFA injection was associated with a significant increase in the kappa-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPgammaS binding in membranes of the amygdala. The intracerebroventricular administration and microinjection of a selective mu-opioid receptor antagonist, a selective delta-opioid receptor antagonist, and the endogenous kappa-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice. We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery. In the light-dark test, the time spent in the lit compartment was not changed at 8 weeks after surgery. Collectively, the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice. This phenomenon may be associated with changes in opioidergic function in the amygdala.

Topics: Amygdala; Analgesics, Opioid; Analysis of Variance; Animals; Anxiety; Behavior, Animal; Benzamides; Chronic Disease; Diazepam; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Freund's Adjuvant; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Maze Learning; Mice; Mice, Inbred C57BL; Naltrexone; Narcotic Antagonists; Narcotics; Pain; Pain Measurement; Piperazines; Protein Binding; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reaction Time; Sciatica; Somatostatin; Sulfur Isotopes; Time Factors; Tranquilizing Agents

Experiments were undertaken to examine whether once daily i.p. administration of either of two antidepressants used for the treatment of neuropathic pain, amitriptyline (10 mg/kg) and fluoxetine (5 mg/kg), to rats for 7 days modifies GABA(B) receptor function and subunit expression in the lumbar spinal cord. The results indicate that, as previously reported for desipramine, both amitriptyline and fluoxetine increase the pain threshold to a thermal stimulus, the expression of GABA(B(1)) subunits, and baclofen-stimulated [35S]GTPgammaS binding, a measure of GABA(B) receptor function. The effects of antidepressant administration on GABA(B(1b)) and GABA(B(2)) subunit expression in spinal cord are more variable than for GABA(B(1a)). It was also discovered that repeated daily exposure to a thermal stimulus or immobilization stress increases GABA(B(1a)) expression in the lumbar spinal cord, with no commensurate change in thermal pain threshold or GABA(B) receptor sensitivity. These results support a relationship between GABA(B) receptors and the action of antidepressants. The findings demonstrate that drug-induced increases in GABA(B) receptor function can occur independently of any change in GABA(B) receptor subunit expression and are consistent with the notion that GABA(B) receptor subunits have multiple functions, only one of which is dimerization to form GABA(B) receptors. The data also suggest that GABA(B) subunit gene expression may serve as a preclinical marker of antidepressant efficacy and of drug- or stress-induced modifications in central nervous system activity.

Topics: Amitriptyline; Analgesics; Animals; Antidepressive Agents; Antidepressive Agents, Second-Generation; Antidepressive Agents, Tricyclic; Biomarkers; Fluoxetine; Gene Expression; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Male; Pain; Pain Threshold; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Receptors, GABA-B; Restraint, Physical; Spinal Cord; Stress, Psychological

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.

Topics: Animals; Binding, Competitive; Central Nervous System; Cerebrospinal Fluid; Dynorphins; Endorphins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mice; Mice, Knockout; Neural Pathways; Neurons; Nociceptin Receptor; Pain; Radioimmunoassay; Radioligand Assay; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity.. The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined.. CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception.. CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

Topics: Action Potentials; Adenylyl Cyclases; Amides; Analgesics; Animals; Brain Stem; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Ligands; Male; Mice; Pain; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Resorcinols; Transfection

In the search for opioid agonists with delayed antagonist actions as potential treatments for substance abuse, the bridged morphinan BU74 (17-cyclopropylmethyl-3-hydroxy-[5beta,7beta,3',5']-pyrrolidino-2'[S]-phenyl-7alpha-methyl-6,14-endoetheno morphinan) (3f) was synthesized. In isolated tissue and [35S]GTPgammaS opioid receptor functional assays BU74 was shown to be a potent long-lasting kappa opioid receptor agonist, delta opioid receptor partial agonist and mu opioid receptor antagonist. In antinociceptive tests in the mouse, BU74 showed high efficacy and potent kappa opioid receptor agonism. When its agonist action had waned BU74 became an antagonist of kappa and mu opioid receptor agonists in the tail flick assay and of delta, kappa and mu opioid receptor agonists in the acetic acid writhing assay. The slow onset, long-duration kappa opioid receptor agonist effects of BU74 suggests that it could be a lead compound for the discovery of a treatment for cocaine abuse.

Topics: Acetic Acid; Analgesics; Animals; Benzomorphans; Binding, Competitive; Bridged-Ring Compounds; Cell Line, Tumor; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Hot Temperature; Ileum; In Vitro Techniques; Male; Mice; Mice, Inbred ICR; Morphinans; Narcotic Antagonists; Pain; Pain Measurement; Radioligand Assay; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sulfur Radioisotopes; Vas Deferens

CT-3 (ajulemic acid) is a synthetic analogue of a metabolite of Delta9-tetrahydrocannabinol that has reported analgesic efficacy in neuropathic pain states in man. Here we show that CT-3 binds to human cannabinoid receptors in vitro, with high affinity at hCB1 (Ki 6 nM) and hCB2 (Ki 56 nM) receptors. In a functional GTP-gamma-S assay CT-3 was an agonist at both hCB1 and hCB2 receptors (EC50 11 and 13.4 nM, respectively). In behavioural models of chronic neuropathic and inflammatory pain in the rat, oral administration of CT-3 (0.1-1 mg/kg) produced up to 60% reversal of mechanical hyperalgesia. In both models the antihyperalgesic activity was prevented by the CB1-antagonist SR141716A but not the CB2-antagonist SR144528. In the tetrad of tests for CNS activity, CT-3 (1-10 mg/kg, po) produced dose-related catalepsy, deficits in locomotor performance, hypothermia, and acute analgesia. Comparison of 50% maximal effects in the tetrad and chronic pain assays produced an approximate therapeutic index of 5-10. Pharmacokinetic analysis showed that CT-3 exhibits significant but limited brain penetration, with a brain/plasma ratio of 0.4 measured following oral administration, compared to ratios of 1.0-1.9 measured following subcutaneous administration of WIN55,212-2 or Delta9-THC. These data show that CT-3 is a cannabinoid receptor agonist and is efficacious in animal models of chronic pain by activation of the CB1 receptor. Whilst it shows significant cannabinoid-like CNS activity, it exhibits a superior therapeutic index compared to other cannabinoid compounds, which may reflect a relatively reduced CNS penetration.

Topics: Analgesics; Animals; Benzoxazines; Cannabinoids; Catalepsy; Cell Line; Chromatography; Cricetinae; Cricetulus; Cyclohexanols; Disease Models, Animal; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Freund's Adjuvant; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hypothermia; Inflammation; Ligation; Male; Morpholines; Motor Activity; Naphthalenes; Pain; Pain Measurement; Pain Threshold; Radioligand Assay; Rats; Rats, Wistar; Rotarod Performance Test; Sciatic Neuropathy; Sulfur Isotopes; Time Factors; Tritium

Peripheral analgesic effects of opioids are pronounced under inflammatory conditions, e.g., arthritis; however, little is known about adaptive changes of micro opioid receptor binding and G protein coupling in the peripheral versus central nervous system. The present study investigated the effects of inflammation on mu opioid receptor (MOP receptor) binding and G protein coupling of supraspinal, spinal, and peripheral MOP receptors. In addition, MOP receptors were identified in immunohistochemical experiments in dorsal root ganglia (DRG) of inflamed and noninflamed rats. The number of MOP receptor binding sites decreased from hypothalamus (HT) > spinal cord (SC) > DRG. Unilateral Freund's complete adjuvant inflammation of one hindpaw induced a significant up-regulation of MOP receptor sites only in DRG but not in HT or SC. This up-regulation was time-dependent, restricted to the inflamed side, and showed a peak at 24 h. The full-agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO) induced MOP receptor G protein coupling with decreasing efficacies (E(max)) from HT > SC > DRG. Inflammation resulted in significant increases in MOP receptor G protein coupling only in membranes of DRG, but not in HT, SC, or DRG on the contralateral side of inflammation. This suggests that changes in MOP receptor levels are not related to systemically released mediators. These findings show that inflammation causes changes in MOP receptor binding and G protein coupling after DAMGO stimulation selectively in primary afferent neurons but did not cause any adaptive changes of MOP receptor in HT or SC.

Topics: Animals; Binding, Competitive; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hypothalamus; Immunohistochemistry; Male; Neurons, Afferent; Pain; Rats; Rats, Wistar; Receptors, Opioid, mu; Spinal Cord; Sulfur Radioisotopes

The development of buprenorphine as a treatment for opiate abuse and dependence has drawn attention to opioid ligands that have agonist actions followed by long-lasting antagonist actions. In a search for alternatives to buprenorphine, we discovered a bridged pyrrolidinomorphinan (BU72). In vitro, BU72 displayed high affinity and efficacy for mu-opioid receptors, but was also a partial delta-opioid receptor agonist and a full kappa-opioid receptor agonist. BU72 was a highly potent and long-lasting antinociceptive agent against both thermal and chemical nociception in the mouse and against thermal nociception in the monkey. These effects were prevented by mu-, but not kappa- or delta-, opioid receptor antagonists. Once the agonist effects of BU72 had subsided, the compound acted to attenuate the antinociceptive action of morphine. BU72 is too efficacious for human use but manipulation to reduce efficacy could provide a lead to the development of a treatment for opioid dependence.

Topics: Analgesics; Animals; Binding, Competitive; Buprenorphine; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Female; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Ileum; In Vitro Techniques; Macaca mulatta; Male; Mice; Mice, Inbred ICR; Morphinans; Morphine; Muscle Contraction; Pain; Pain Measurement; Pyrroles; Radioligand Assay; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sulfur Radioisotopes; Time Factors; Tritium; Vas Deferens

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.

Topics: Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dansyl Compounds; Disease Models, Animal; Drug Therapy, Combination; Drug Tolerance; Endothelin A Receptor Antagonists; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Mice; Morphine; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Pain Threshold; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptors, Opioid, mu; Sulfur Radioisotopes; Time Factors

The present study was designed to investigate whether a state of neuropathic pain induced by sciatic nerve ligation could alter the rewarding effect, antinociception, and G-protein activation induced by a prototype of mu-opioid receptor agonist morphine in the mouse. The sciatic nerve ligation caused a long-lasting and profound thermal hyperalgesia. Under this neuropathic pain-like state, an i.c.v. morphine-induced place preference was observed in sham-operated mice but not in sciatic nerve-ligated mice. However, no differences in the antinociceptive effect of i.c.v.-administered morphine were noted between the groups. The increases in the binding of guanosine-5'-o-(3-[(35)S]thio)triphosphate induced by morphine in lower midbrain membranes including the ventral tegmental area, which contributes to the expression of the rewarding effect of opioid, were significantly attenuated in sciatic nerve-ligated mice. On the other hand, there were no differences in the stimulation of guanosine-5'-o-(3-[(35)S]thio)triphosphate binding to pons/medulla membranes, which plays an important role in the antinociception of mu-opioid receptor agonists, between the groups. In addition, no changes in levels of guanosine-5'-o-(3-[(35)S]thio)triphosphate binding by either the selective delta- or kappa-opioid receptor agonists were noted in membrane of the lower midbrain and limbic forebrain membranes obtained from sciatic nerve-ligated mice. Reverse transcription-polymerase chain reaction analysis showed that sciatic nerve ligation did not alter the mRNA product of mu-opioid receptors in the lower midbrain, indicating that a decrease in some mu-opioid receptor functions may result from the uncoupling of mu-opioid receptors from G-proteins. We found a significant increase in protein levels of G-protein-coupled receptor kinase 2, which causes receptor phosphorylation in membranes of the lower midbrain but not in the pons/medulla, obtained from mice with nerve injury, whereas there were no changes in the protein level of phosphorylated-protein kinase C in the lower midbrain. These results suggest that the uncoupling of mu-opioid receptors from G-proteins by G-protein-coupled receptor kinase 2 in the lower midbrain may, at least in part, contribute to the suppression of the rewarding effect of morphine under neuropathic pain.

Topics: Analgesics, Opioid; Animals; beta-Adrenergic Receptor Kinases; Blotting, Western; Cyclic AMP-Dependent Protein Kinases; G-Protein-Coupled Receptor Kinase 3; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Male; Medulla Oblongata; Mesencephalon; Mice; Mice, Inbred ICR; Morphine; Pain; Pain Measurement; Phosphorylation; Pons; Prosencephalon; Protein Kinase C; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; Reward; RNA, Messenger; Sciatic Nerve; Sulfur Radioisotopes

Murine models are increasingly used for investigations of sleep, yet no previous studies have characterized cholinergic activation of guanine nucleotide binding proteins (G proteins) in mouse brainstem nuclei known to regulate sleep. This study used in vitro [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiography to test the hypothesis that muscarinic cholinergic receptors activate G proteins in C57BL/6J (B6) mouse brainstem. The nuclei studied are homologous to those known in rat and cat to modulate sleep and nociception. In B6 mouse, carbachol significantly increased specific binding of [(35)S]GTPgammaS in the pontine reticular nucleus, caudal part (79%); pontine reticular nucleus, oral part (131%); laterodorsal tegmental nucleus (56%); pedunculopontine tegmental nucleus (86%); dorsal raphe nucleus (53%); dorsal medial periaqueductal gray (54%); and ventrolateral periaqueductal gray (52%) when compared with basal binding. Carbachol-induced G protein activation was concentration-dependent and blocked by atropine, demonstrating mediation by muscarinic receptors. G protein activation by carbachol was heterogeneous across B6 mouse brainstem nuclei. Comparison of [(35)S]GTPgammaS binding between mouse and rat revealed different magnitudes of G protein activation in the pontine reticular formation. In the same pontine reticular formation area of B6 mouse where in vitro treatment with carbachol activates G proteins, in vivo microinjection of cholinomimetics causes a rapid eye movement sleep-like state. These data provide the first direct measurement of muscarinic receptor-activated G proteins in B6 mouse brainstem nuclei known in other species to regulate sleep.

Topics: Animals; Autoradiography; Brain Stem; Carbachol; Cats; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Models, Animal; Nociceptors; Pain; Rats; Reticular Formation; Sleep; Species Specificity; Sulfur Radioisotopes; Wakefulness

Opioids mediate their analgesic effects by activating mu-opioid receptors (MOR) not only within the central nervous system but also on peripheral sensory neurons. The peripheral analgesic effects of opioids are best described under inflammatory conditions (e.g., arthritis). The present study investigated the effects of inflammation on MOR binding and G-protein coupling of full versus partial MOR agonists in dorsal root ganglia (DRG) of primary afferent neurons. Our results show that Freund's complete adjuvant (FCA) unilateral hindpaw inflammation induces a significant up-regulation of MOR binding sites (25 to 47 fmol/mg of protein) on DRG membranes without affecting the affinity of either full or partial MOR agonists. In our immunohistochemical studies, the number of MOR-immunoreactive neurons consistently increased. This increase was mostly caused by small-diameter nociceptive DRG neurons. The full agonist DAMGO induced MOR G-protein coupling in DRG of animals without FCA inflammation (EC50 = 56 nM; relative Emax = 100%). FCA inflammation resulted in significant increases in DAMGO-induced MOR G-protein coupling (EC50 = 29 nM; relative Emax = 145%). The partial agonist buprenorphine hydrochloride (BUP) showed no detectable G-protein coupling in DRG of animals without FCA inflammation; however, partial agonist activity of BUP-induced MOR G-protein coupling was detectable in animals with FCA inflammation (EC50 = 1.6 nM; relative Emax = 82%). In behavioral studies, administration of BUP produced significant antinociception only in inflamed but not in noninflamed paws. These findings show that inflammation causes changes in MOR binding and G-protein coupling in primary afferent neurons. They further underscore the important differences in clinical studies testing peripherally active opioids in inflammatory painful conditions.

Topics: Animals; Behavior, Animal; Binding Sites; Buprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Immunohistochemistry; Inflammation; Male; Neurons, Afferent; Pain; Rats; Rats, Wistar; Receptors, Opioid, mu; Sciatic Nerve; Sulfur Radioisotopes

Although chronic inflammatory pain is known to be associated with hypersensitivity to mu opioid receptor agonists, no evidence for changes in the expression and/or characteristics of central mu opioid receptors has yet been reported in relevant models of this type of pain. In the present study, both immunohistochemical and autoradiographic approaches were used to address this question in polyarthritic rats, on the 4th week after intradermal injection of complete Freund's adjuvant, when inflammatory pain was at its maximum. Immunohistochemical labeling with specific anti-mu opioid receptor antibodies and autoradiographic labeling with [3H]DAMGO showed an upregulation of mu opioid receptors in the dorsal root ganglia but no changes in the density of these receptors in the dorsal horn at the level of L4-L6 segments in polyarthritic compared to age-paired control rats. On the other hand, autoradiographic quantification of the concentration-dependent increase in [35S]GTP-gamma-S binding by the mu-opioid receptor agonist DAMGO did not show any significant differences within the lumbar dorsal horn between polyarthritic and control rats. These data indicate that chronic inflammatory pain caused by polyarthritis was associated with an increased expression of mu-opioid receptors in dorsal root ganglion sensory neurones that did not result in an increased spinal density of these receptors, in spite of their well established axonal transport in the central portion of primary afferent fibres to the dorsal horn. In contrast, axonal transport of mu-opioid receptors in the peripheral portion of these fibres probably accounts for the increased receptor density in inflamed tissues already reported in the literature.

Topics: Analgesics, Opioid; Animals; Arthritis, Experimental; Autoradiography; Chronic Disease; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Lumbar Vertebrae; Male; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Spinal Cord; Sulfur Radioisotopes; Tritium

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors produce effective pain relief. Intrathecal injection of a small dose of neostigmine produces a profound antiallodynic effect in rats with diabetic neuropathy. However, the mechanisms of increased antinociceptive effect of cholinergic agents on diabetic neuropathic pain are not clear. In the present study, we tested the hypothesis that spinal muscarinic receptors are up-regulated in diabetes. The withdrawal threshold of the hindpaw in response to noxious heat and pressure stimuli was determined in streptozotocin-induced diabetic and age-matched normal rats. Muscarine-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding was used to assess the change of functional muscarinic receptors in the spinal cord in diabetes. The [3H]AF-DX 384 membrane binding was performed to determine the number and affinity of spinal cord M2 muscarinic receptors in normal and diabetic rats. We found that the antinociceptive effect of intrathecal 2 to 12 mug muscarine in diabetic animals was potentiated significantly compared with that in normal animals. The maximal muscarine-stimulated [35S]GTPgammaS binding was 112.5 +/- 8.3% in normal rats and 168.8 +/- 12.1% (P < 0.05) in diabetic rats. Although the KD value (2.9 nM) was similar in both groups, the Bmax of [3H]AF-DX 384 membrane binding was significantly higher in diabetic than in normal rats (255.2 +/- 5.9 versus 165.9 +/- 3.5 fmol/mg protein, P < 0.05). Collectively, these data strongly suggest that the muscarinic receptor is up-regulated in the dorsal spinal cord in diabetic rats. This finding probably accounts for the increased efficacy of the antinociceptive effect of intrathecal muscarinic agonists in diabetic neuropathic pain.

Topics: Analgesics; Animals; Binding Sites; Diabetic Neuropathies; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Spinal; Male; Muscarine; Muscarinic Agonists; Pain; Pain Measurement; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Spinal Cord; Sulfur Radioisotopes; Tritium; Up-Regulation

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G protein-coupled receptor (opioid receptor like 1, ORL1), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiological functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited high affinity ligands to ORL1, and particularly the lack of availability of useful specific antagonists. Herein we describe the pharmacological activity of a series of N-terminally modified hexapeptides with high affinity for ORL1. These compounds were tested for binding affinity using [3H]N/OFQ binding to human ORL1 in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS binding in CHO cell membranes. The N-terminal modifications have produced compounds that maintained very high receptor affinity, but led to significant changes in intrinsic activity. One compound, pentanoyl-RYYRWR-NH2, with barely measurable agonist activity was tested in vivo. It was found to possess modest analgesic activity, but it was unable to block the morphine modulatory activity of N/OFQ.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; CHO Cells; Colforsin; Cricetinae; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Mice; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Pain; Receptors, Opioid; Sulfur Radioisotopes

The present study was designed to investigate the possible change in spinal micro -opioid receptor function after repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] in the ICR mouse. A single s.c. or i.t. injection of (-)U-50,488H produced a dose-dependent antinociception. Repeated s.c. or i.t. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect induced by the i.t. administration of a selective micro -opioid receptor agonist [d-Ala2,N-Me-Phe4,Gly5-ol] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTPgammaS) binding assay, we found that (-)U-50,488H was able to produce a dose-dependent increase in [35S]GTPgammaS binding to membranes of the mouse spinal cord. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTPgammaS binding in this region, whereas repeated treatment with (-)U-50,488H exhibited an increase in the DAMGO-stimulated [35S]GTPgammaS binding in membranes of the spinal cord. Using a receptor binding assay, repeated treatment with (-)U-50,488H significantly increased the density of [3H]DAMGO binding sites in membranes of the mouse spinal cord. In contrast, the expression of micro -opioid receptor was not affected after repeated treatment with (-)U-50,488H. These results suggest that repeated stimulation of kappa-opioid receptors leads to the up-regulation of micro -opioid receptor functions in the spinal cord, which may be associated with an increase in the number of functional micro -opioid receptors in the mouse spinal cord.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred ICR; Pain; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Up-Regulation

A novel mu-opioid receptor (MOR) subtype, named MOR-1B, derived from alternatively spliced variants of MOR gene, has been isolated from the rat brain. Here we found for the first time that CXBK recombinant-inbred mice display a significant reduction in the expression of MOR-1B mRNA in the brain as compared to that in their progenitor C57BL/6 mice. In contrast, the expression level of MOR-1 mRNA in the brain of CXBK mice was similar to that found in C57BL/6 mice. Furthermore, relatively lower levels of MOR-1B immunoreactivity were detected in the periaqueductal grey matter (PAG) of CXBK mice than that observed in C57BL/6 mice. To investigate further the possible changes in MOR function to activate G-proteins under the condition of a reduced MOR-1B expression, the guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assay was performed. We found that the increased level of [35S]GTPgammaS bindings to whole brain membranes induced by a selective MOR agonist endomorphin-1 was significantly decreased in CXBK mice, indicating that CXBK strain can be classified as MOR-1B-knockdown mice. We next investigated whether intracerebroventricular (i.c.v.) pretreatment with an antisence oligodeoxynucleotide against exon 5 of MOR gene (MOR-1B) could affect the endomorphin-1-induced supraspinal antinociception. The i.c.v. pretreatment with antisence oligodeoxynucleotide against MOR-1B produced a significant reduction in the i.c.v.-administered endomorphin-1-induced antinociceptive effect. The present data provide first evidence that a lack of MOR-1B expression may, at least in part, contribute to the reduced sensitivity to MOR agonists in CXBK mice, and MOR-1B may play a potential role in the MOR-mediated supraspinal antinociception.

Topics: Alternative Splicing; Animals; Brain; Brain Chemistry; Cell Membrane; Down-Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Oligonucleotides, Antisense; Oligopeptides; Pain; Protein Isoforms; Radioligand Assay; Receptors, Opioid, mu; Sulfur Radioisotopes

The present study was designed to investigate the rewarding effect, G-protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham-operated and sciatic nerve-ligated rats in the increases in guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N-propylnoraporphine or the D3 receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH DPAT). In contrast, the increases in [35S]GTPgammaS binding to membranes of the VTA induced by either morphine or a selective micro -opioid receptor agonist [d-Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve-ligated rats as compared with sham- operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with reduction in the mu-opioid receptor-mediated G-protein activation in the VTA.

Topics: Animals; Behavior, Animal; Binding, Competitive; Disease Models, Animal; Dopamine; Extracellular Space; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Ligation; Male; Microdialysis; Morphine; Nucleus Accumbens; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Reward; Sciatic Neuropathy; Spatial Behavior; Ventral Tegmental Area

We report the discovery of F 13640 and evidence suggesting this agent to produce powerful, broad-spectrum analgesia by novel molecular and neuroadaptative mechanisms. F 13640 stimulates G(alphaomicron) protein coupling to 5-HT(1A) receptors to an extent unprecedented by selective, non-native 5-HT(1A) ligands. Fifteen minutes after its injection in normal rats, F 13640 (0.01-2.5 mg/kg) decreases the vocalization threshold to paw pressure; 15 min upon injection in rats that are exposed to formalin-induced tonic nociception, F 13640 inhibits pain behavior. The initial hyperalgesia induced by 0.63 mg/kg F 13640 was followed, 8 hrs later, by paradoxical hypo-algesia; 5 mg/kg of morphine produces the opposite effects (i.e., hypo-algesia followed by hyper-algesia). Repeated F 13640 injections cause an increase in the basal vocalization threshold and a reduction of F 13640-produced hyperalgesia; in these conditions, morphine causes basal hyperalgesia and antinociceptive tolerance. Continuous two-week infusion of F 13640 (0.63 mg/day) exerts little effect on the threshold in normal rats, but markedly reduces analgesic self-administration in arthritic rats. F 13640 infusion also decreases allodynic responses to tactile and thermal stimulations in rats sustaining spinal cord or sciatic nerve injury. In these models of chronic nociceptive and neuropathic pain, the analgesia afforded by F 13640 consistently surpasses that of morphine (5 mg/day), imipramine (2.5 mg/day), ketamine (20 mg/day) and gabapentin (10 mg/day). Very-high-efficacy 5-HT(1A) receptor activation constitutes a novel mechanism of central analgesia that grows rather than decays with chronicity, that is amplified by nociceptive stimulation, and that may uniquely relieve persistent nociceptive and neuropathic pains.

Topics: Acetates; Adrenergic Uptake Inhibitors; Amines; Aminopyridines; Analgesia; Analgesics; Animals; Cells, Cultured; CHO Cells; Cricetinae; Cyclohexanecarboxylic Acids; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Synergism; Female; Fentanyl; Gabapentin; gamma-Aminobutyric Acid; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Imipramine; Ketamine; Male; Morphine; Pain; Pain Measurement; Pain Threshold; Piperidines; Pyridines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Serotonin Agents; Time Factors; Transfection

Many clinical and experimental studies have suggested that diabetes or hyperglycemia alter pain sensitivity, and sensitivity to several drugs. It has been reported that the antinociceptive potency of morphine is decreased in several rodent models of hyperglycemia, including streptozotocin-induced diabetes, an animal models of type I diabetes. The present study was designed to investigate in streptozotocin-induced diabetic mice the effect of the selective micro-opioid agonist [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO) on G-protein activation by monitoring guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to pons/medulla membranes, which contain the key areas for opioid antinociception. In the tail-flick test, DAMGO (1-10 ng, intracerebroventricularly) produced a marked dose-dependent antinociception in non-diabetic mice. In streptozotocin-induced diabetic mice, the effect of DAMGO was significantly attenuated as compared to that in non-diabetic mice. In the [35S]GTPgammaS binding assay, DAMGO (0.1-10 microM) increased the binding of [35S]GTPgammaS to pons/medulla membranes from non-diabetic mice in a concentration-dependent manner, affording approximately 100% maximal stimulation at 10 microM. The maximal stimulation of [35S]GTPgammaS binding by DAMGO (10 microM) in streptozotocin-induced diabetic mice (100.55+/-3.12%), was similar to non-diabetic mice. The present results indicated that the antinociceptive effect of DAMGO given supraspinally was less potent in streptozotocin-induced diabetic mice than that in non-diabetic mice, whereas the mu-opioid receptor-mediated G-protein activation in pons/medulla was unaltered in streptozotocin-induced diabetic mice. Thus, the attenuation of DAMGO-induced antinociception in streptozotocin-induced diabetic mice is probably caused by dysfunction in cellular pathways after the activation of G-proteins.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Medulla Oblongata; Membranes; Mice; Mice, Inbred ICR; Pain; Pain Measurement; Pons; Receptors, Opioid, mu; Sulfur Radioisotopes

Previous evaluation of antinociceptive action in experimental diabetes has been conducted almost exclusively in chemically induced diabetes mellitus. The purpose of the present study was to evaluate antinociceptive response and G-protein activation by mu-opioid receptor and delta-opioid receptor agonists in the genetic non-obese diabetic (NOD) mouse, a model of type I insulin-dependent diabetes mellitus (IDDM). Tail-flick latency before and after hyperglycemia was unaltered. Hyperglycemic NOD mice were hyporesponsive to intracerebroventricular (i.c.v.) injections of [D-Ala(2)]deltorphin II but not to [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO); however, G-protein activation in pons/medulla assessed by [35S]GTPgammaS binding was not diminished. This suggests that a G-protein defect in signaling cannot account for the hyporesponsiveness of antinociception in this genetic model of IDDM.

Topics: Analgesics, Opioid; Animals; Benzamides; Binding, Competitive; Diabetes Mellitus, Type 1; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Medulla Oblongata; Membranes; Mice; Mice, Inbred NOD; Nociceptors; Oligopeptides; Pain; Piperazines; Pons; Receptors, Opioid; Sulfur Radioisotopes

Previous studies in rats have shown that spinal morphine loses potency and efficacy to suppress an acute nociceptive stimulus applied to the tail or the paw following injury to peripheral nerves by tight ligation of the L5/L6 spinal nerves. Additionally, intrathecal (i.th.) morphine is ineffective in suppressing tactile allodynia at fully antinociceptive doses in these animals. The molecular basis for this loss of morphine potency and efficacy in nerve injury states is not known. One possible explanation for this phenomenon is a generalized, multi-segmental loss of opioid mu (mu) receptors in the dorsal horn of the spinal cord after nerve injury. This hypothesis was tested here by determining whether nerve injury produces (a) a decrease in mu receptors in the lumbar spinal cord; (b) a decrease in the affinity of ligand-receptor interaction, (c) a decrease in the fraction of high-affinity state of the mu receptors and (d) a reduced ability of morphine to activate G-proteins via mu receptors. Lumbar spinal cord tissues were examined 7 days after the nerve injury, a time when stable allodynia was observed. At this point, no differences were observed in the receptor density or affinity of [3H]DAMGO (mu selective agonist) or [3H]CTAP (mu selective antagonist) in the dorsal quadrant of lumbar spinal cord ipsilateral to nerve injury. Additionally, no change in morphine's potency and efficacy in activating G-proteins was observed. In contrast, staining for mu opioid receptors using mu-selective antibodies revealed a discrete loss of mu opioid receptors localized ipsilateral to the nerve injury and specific for sections taken at the L6 level. At these spinal segments, mu opioid receptors were decreased in laminae I and II. The data indicate that the loss of mu opioid receptors are highly localized and may contribute to the loss of morphine activity involving input at these spinal segments (e.g., foot-flick response). On the other hand, the lack of a generalized loss of opioid mu receptors across spinal segments makes it unlikely that this is the primary cause for the loss of potency and efficacy of mu opioids to suppress multi-segmental reflexes, such as the tail-flick response.

Topics: Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Morphine; Narcotic Antagonists; Narcotics; Nociceptors; Pain; Peptide Fragments; Peptides; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord; Tritium

In this study we have evaluated the second messenger system that might couple 5-HT1A receptor activation to produce peripheral hyperalgesia. The intradermal injection of the serotonin (5-hydroxytryptamine; 5-HT) receptor agonist for the 1A receptor subset (5-HT1A), (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH DPAT) produces a dose-dependent hyperalgesia which was attenuated by a cAMP kinase inhibitor (the R-isomer of cyclic adenosine-3'-5'-monophosphate), but prolonged by the inhibition of endogenous phosphodiesterase by rolipram, supporting a role for the cAMP second messenger system. The 5-HT1A receptor agonist, 8-OH-DPAT, and the adenyl cyclase activator, forskolin administered together, produced an additive hyperalgesia, suggesting that the 5-HT1A receptor in peripheral terminals of the primary afferent neurons is positively coupled to the cAMP second messenger system in producing hyperalgesia. The inability of pertussis toxin to inhibit 8-OH DPAT-induced hyperalgesia further supports this hypothesis. The coupling of the 5-HT1A receptor to the cAMP second messenger system appears to be through guanine regulatory proteins since guanosine 5'-O-(3-thiotriphosphate) and cholera toxin both markedly enhanced 8-OH DPAT hyperalgesia. In further support of the role of guanine nucleotide regulatory proteins, guanosine 5'-O-(2-thiodiphosphate), as well as activators of inhibitory guanine regulatory proteins (the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, and the adenosine A1 agonist, N6-cyclopentyladenosine, significantly attenuated 8-OH DPAT hyperalgesia.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; 8-Hydroxy-2-(di-n-propylamino)tetralin; Adenosine; Analgesics; Animals; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Isomerism; Male; Nociceptors; Pain; Protein Kinase Inhibitors; Pyrrolidines; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptors, Serotonin; Rolipram; Second Messenger Systems; Serotonin; Tetrahydronaphthalenes

Bradykinin is one of several pro-inflammatory, pain-inducing substances produced during inflammation--the body's response to injury. In previous work we have shown that bradykinin and guanosine-5'-O-3-thiotriphosphate increase excitability in a subpopulation of cultured neonatal rat dorsal root ganglion neurons. We now describe experiments in which the mechanism underlying the stimulatory action of these two substances has been examined in more detail. Using the whole-cell voltage-clamp technique, bradykinin-sensitive cells were distinguished by their response to a 1-s depolarizing voltage-pulse which evoked more than one inward current during the step command. The secondary inward currents are likely to represent action potentials generated at the poorly clamped neurites of these cells. Bradykinin- and guanosine-5'-O-3-thiotriphosphate-induced changes in excitability were measured indirectly by a change in the number of inward currents recorded during the 1-s depolarizing voltage-step. The effect of activators and inhibitors of protein kinase C, arachidonic acid metabolism, G-protein activation and release of intracellular Ca2+ were examined on this response. In the presence of extracellular staurosporine (1.0 microM) or nordihydroguaiaretic acid (10 microM), these excitatory effects were reduced but not abolished, whilst indomethacin (20 microM) had no effect. Intracellular application of guanosine-5'-O-2-thiodiphosphate (10 mM) or ryanodine (100 microM) substantially reduced the effect of bradykinin. The excitatory effect of internal guanosine-5'-O-3-thiotriphosphate (500 microM) occurred gradually over time, and this was mimicked by internal application of myo-inositol 1,4,5-trisphosphorothioate (1.0 microM). From the results, it is proposed that G-protein activation is an essential component of the bradykinin response, which may also require a Ca(2+)-activated conductance modulated by protein kinase C and lipoxygenase metabolites of arachidonic acid.

Topics: Alkaloids; Animals; Animals, Newborn; Bradykinin; Cells, Cultured; Evoked Potentials; Ganglia, Spinal; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Indomethacin; Kinetics; Masoprocol; Neurons, Afferent; Pain; Protein Kinase C; Rats; Rats, Wistar; Ryanodine; Signal Transduction; Staurosporine