enkephalin--ala(2)-mephe(4)-gly(5)- and Pain

Opioids are broad-spectrum analgesics with potent pain-relieving qualities but also with potential adverse effects related to both short-term and long-term therapy. Researchers have attempted to alter existing opioid analgesics, utilize different routes/formulations, or combine opioid analgesics with other compounds in efforts to improve analgesia while minimizing adverse effects. Exogenous opioids, administered in efforts to achieve analgesia, work by mimicking the actions of endogenous opioids. Endogenous opioids and their receptors are located in the brain (supraspinal areas), spinal cord, and periphery. Although opioids and opioid receptors in the brain and spinal cord have received much attention over many years, peripheral endogenous opioid analgesic systems have only been extensively studied during the past decade. It has been known since 1990 that following injection into the rodent hindpaw, D-Ala(2),N-Me-Phe(4), Gly(5)-ol-enkephalin (DAMGO) [a muopioid receptor agonist] probably exerts its antinociceptive effects locally, since the doses administered are too low to have an effect in the central nervous system (CNS). This notion has been supported by the observation that the quaternary compound morphine methyliodide, which does not as readily cross the bloodbrain barrier and enter the CNS, produced antinociception following intradermal administration into the hindpaw, but not when the same dose was administered systemically (subcutaneously at a distant site). With a growing appreciation of peripheral endogenous opioids, peripheral endogenous opioid receptors, and peripheral endogenous opioid analgesic systems, investigators began growing hopeful that it may be possible to achieve adequate analgesics while avoiding unwanted central untoward adverse effects (e.g. respiratory depression, somnolence, addiction). Peripherally-acting opioids, which capitalize on peripheral endogenous opioid analgesic systems, may be one potential future strategy which may be utilized in efforts to achieve potent analgesia with minimal side effects.

Topics: Analgesics, Opioid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Inflammation; Leukocytes; Pain; Peripheral Nervous System

Topics: Analgesia, Epidural; Analgesics, Opioid; Animals; Benzeneacetamides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Humans; Nerve Fibers; Pain; Posterior Horn Cells; Pyrrolidines; Receptors, Opioid; Synapses; Synaptic Transmission

This review summarizes our studies using pharmacological, neurochemical and molecular biological methods on the nociception in the CNS and opioid receptors (OPRs). We designed an in vitro fluorometric on-line monitoring system including an immobilized glutamate dehydrogenase column, and for the first time actually demonstrated that capsaicin induced the release of glutamate from rat dorsal horn slices containing the terminal area of primary afferents, in concentration-dependent, extracellular Ca(2+)-dependent and tetrodotoxin-resistant manners. Further, such a release was shown to be inhibited through mu- and delta-opioid receptors and alpha 2-adrenoceptors. On the other hand, we found that intracerebroventricular injections of interleukin (IL)-1 beta in rats produced biphasic effects on the mechanical nociception in rats (hyperalgesia in lower concentrations but analgesia in higher ones) and that similar injections of cytokine-induced neutrophil chemoattractant-1 (CINC-1) facilitated mechanical nociception in rats. The above described facts suggest that glutamate and some sorts of cytokines (IL-1 beta and CINC-1) contribute to nociception at least from the primary afferents to the spinal dorsal horn neurons and in higher brain, respectively. We have cloned rat kappa- and mu-opioid receptors. Using cloned cDNA for OPRs, we demonstrated (1) the distribution of mRNAs for OPRs in the rat central nervous system, (2) coexistence of each type of mRNA for mu-, delta- and kappa-OPRs and pre-protachykinin A mRNA in the dorsal root ganglion neurons, (3) an increased expression of mu- and kappa-OPR mRNAs in the I-II layers of rat lumbar dorsal horn with an adjuvant arthritis in the hind limb, (4) the inhibitions of N- and Q-types of Ca2+ channels by mu- and kappa-OPR agonists and (5) cross-desensitization of the inhibition through a common intracellular phosphorylation-independent mechanism, (6) pharmacological characterization of "antagonist analgesics" as partial agonists at every type of OPRs, and (7) the key-structure(s) of OPRs for discriminative binding of DAMGO to mu-OPR.

Topics: Analgesics, Opioid; Animals; Calcium Channels; Central Nervous System; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glutamates; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1; Nociceptors; Pain; Rats; Receptors, Opioid; RNA, Messenger

The mechanisms underlying trigeminal pain conditions are incompletely understood. In vitro animal studies have elucidated various targets for pharmacological intervention; however, a lack of clinical models that allow evaluation of viable innervated human tissue has impeded successful translation of many preclinical findings into clinical therapeutics. Therefore, we developed and characterized an in vitro method that evaluates the responsiveness of isolated human nociceptors by measuring basal and stimulated release of neuropeptides from collected dental pulp biopsies. Informed consent was obtained from patients presenting for extraction of normal wisdom teeth. Patients were anesthetized using nerve block injection, teeth were extracted and bisected, and pulp was removed and superfused in vitro. Basal and capsaicin-evoked peripheral release of immunoreactive calcitonin gene-related peptide (iCGRP) was analyzed by enzyme immunoassay. The presence of nociceptive markers within neurons of the dental pulp was characterized using confocal microscopy. Capsaicin increased the release of iCGRP from dental pulp biopsies in a concentration-dependent manner. Stimulated release was dependent on extracellular calcium, reversed by a TRPV1 receptor antagonist, and desensitized acutely (tachyphylaxis) and pharmacologically by pretreatment with capsaicin. Superfusion with phorbol 12-myristate 13-acetate (PMA) increased basal and stimulated release, whereas PGE2 augmented only basal release. Compared with vehicle treatment, pretreatment with PGE2 induced competence for DAMGO to inhibit capsaicin-stimulated iCGRP release, similar to observations in animal models where inflammatory mediators induce competence for opioid inhibition. These results indicate that the release of iCGRP from human dental pulp provides a novel tool to determine the effects of pharmacological compounds on human nociceptor sensitivity.

Topics: Adolescent; Adult; Biological Assay; Biopsy; Calcitonin Gene-Related Peptide; Capsaicin; Dental Pulp; Dinoprostone; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Models, Biological; Models, Neurological; Nociceptors; Pain; Receptors, Opioid, mu; Sensory Receptor Cells; Sensory System Agents; Tetradecanoylphorbol Acetate; TRPV Cation Channels; Young Adult

The most commonly used opioid analgesics are limited by their severe side-effects in the clinical treatment of pain. Preliminary reports indicate that the combination of classical opioids and N/OFQ receptor (NOP) ligands may be an effective strategy to reduce unwanted side-effects and improve antinociception. But the interaction of these two receptor ligands in pain regulation at the peripheral level remains unclear. In this study, the antinociception of a designed amide analogue of the mu opioid receptor (MOP) peptide agonist DAMGO, DAMGO-NH

Topics: Analgesics; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ligands; Mice; Nociceptin Receptor; Pain; Peptides; Receptors, Opioid; Receptors, Opioid, mu

Opioids are powerful analgesics commonly used in pain management. However, opioids can induce complex neuroadaptations, including synaptic plasticity, that ultimately drive severe side effects, such as pain hypersensitivity and strong aversion during prolonged administration or upon drug withdrawal, even following a single, brief administration. The lateral parabrachial nucleus (LPBN) in the brainstem plays a key role in pain and emotional processing; yet, the effects of opioids on synaptic plasticity in this area remain unexplored. Using patch-clamp recordings in acute brainstem slices from male and female Sprague Dawley rats, we demonstrate a concentration-dependent, bimodal effect of opioids on excitatory synaptic transmission in the LPBN. While a lower concentration of DAMGO (0.5 µM) induced a long-term depression of synaptic strength (low-DAMGO LTD), abrupt termination of a higher concentration (10 µM) induced a long-term potentiation (high-DAMGO LTP) in a subpopulation of cells. LTD involved a metabotropic glutamate receptor (mGluR)-dependent mechanism; in contrast, LTP required astrocytes and N-methyl-D-aspartate receptor (NMDAR) activation. Selective optogenetic activation of spinal and periaqueductal gray matter (PAG) inputs to the LPBN revealed that, while LTD was expressed at all parabrachial synapses tested, LTP was restricted to spino-parabrachial synapses. Thus, we uncovered previously unknown forms of opioid-induced long-term plasticity in the parabrachial nucleus that potentially modulate some adverse effects of opioids. PERSPECTIVE: We found a previously unrecognized site of opioid-induced plasticity in the lateral parabrachial nucleus, a key region for pain and emotional processing. Unraveling opioid-induced adaptations in parabrachial function might facilitate the identification of new therapeutic measures for addressing adverse effects of opioid discontinuation such as hyperalgesia and aversion.

Topics: Analgesics, Opioid; Animals; Brain Stem; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Neuronal Plasticity; Pain; Pain Clinics; Rats; Rats, Sprague-Dawley

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the

Topics: Analgesics, Opioid; Animals; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ligands; Male; Mice; Pain; Rats; Receptors, Opioid, mu

The development of σ

Topics: Animals; Guinea Pigs; Hydrogen; Hydrogen Sulfide; Ligands; Male; Morpholines; Pain; Piperazines; Rats, Sprague-Dawley; Receptors, sigma; Sigma-1 Receptor

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E

Topics: Aged; Analgesics, Opioid; Animals; Capsaicin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Hyperalgesia; Nociceptors; Pain; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Sensory Receptor Cells

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Hyperalgesia; Male; Mice; Morphine; Neurons; Neurons, Afferent; Pain; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spinal Cord

Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for μ-, δ-, and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands,

Topics: Administration, Intravenous; Analgesics, Opioid; Animals; CHO Cells; Cricetulus; Female; Humans; Infusions, Subcutaneous; Male; Mice; Models, Molecular; Opioid Peptides; Pain; Peptides, Cyclic; Receptors, Opioid

Topics: Analgesics; Animals; Female; Ligands; Mice; Models, Molecular; Pain; Piperidines; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Structure-Activity Relationship

Evidence indicates that periaqueductal gray matter (PAG) plays an important role in defensive responses and pain control. The activation of cannabinoid type-1 (CB1) or mu-opioid (MOR) receptors in the dorsal region of this structure (dPAG) inhibits fear and facilitates antinociception induced by different aversive stimuli. However, it is still unknown whether these two receptors work cooperatively in order to achieve these inhibitory actions. This study investigated the involvement and a likely interplay between CB1 and MOR receptors localized into the dPAG on the regulation of fear-like defensive responses and antinociception (evaluated in tail-flick test) evoked by dPAG chemical stimulation with N-methyl-d-aspartate (NMDA). Before the administration of NMDA, animals were first intra-dPAG injected with the CB1 agonist ACEA (0.5 pmol), or with the MOR agonist DAMGO (0.5 pmol) in combination with the respective antagonists AM251 (CB1 antagonist, 100 pmol) or CTOP (MOR antagonist, 1 nmol). To investigate the interplay between these receptors, microinjection of CTOP was combined with ACEA, or microinjection of AM251 was combined with DAMGO. Our results showed that both the intra-PAG treatments with ACEA or DAMGO inhibited NMDA-induced freezing expression, whereas only the treatment with DAMGO increased antinociception induced with NMDA, which are completely blocked by its respective antagonists. Interestingly, the inhibitory effects of ACEA or DAMGO on freezing was blocked by CTOP and AM251, respectively, indicating a functional interaction between these two receptors in the mediation of defensive behaviors. However, this cooperative interaction was not observed during the NMDA-induced antinociception. Our findings indicate that there is a cooperative action between the MOR and CB1 receptors within the dPAG and it is involved in the mediation of NMDA-induced defensive responses. Additionally, the MORs into the dPAG are involved in the modulation of the antinociceptive effects that follow a fear-like defense-reaction induced by dPAG chemical stimulation with NMDA.

Topics: Analgesics, Opioid; Animals; Arachidonic Acids; Behavior, Animal; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Agonists; Fear; Freezing Reaction, Cataleptic; Male; Microinjections; N-Methylaspartate; Nociception; Pain; Pain Measurement; Periaqueductal Gray; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Somatostatin

Chronic pain is a worldwide major health problem and many pain-suffering patients are under opioid based therapy. Epidemiological data show that pain intensity correlates with the risk of misuse of prescription opioids, and other drugs of abuse including alcohol. This increased vulnerability to suffer Substance Use Disorders could be, in part, caused by functional changes that occur over the mesocorticolimbic system, a brain pathway involved in reward processing and addiction. Previous data in rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA). As a consequence, pain alters dopamine release in the nucleus accumbens (NAc) derived from MOR activation in the VTA and also increases intake of high doses of heroine. Given that the VTA neurons target different brain regions, in the present study we first analyzed changes induced by inflammatory pain in the MOR dependent activation pattern of the main VTA projecting areas. To do that, we administered two doses (7 or 14 ng) of DAMGO (MORs agonist) or artificial cerebrospinal fluid (aCSF) focally into the VTA of rats and measured the activation in projection areas by cFos immunohistochemistry. Our results show that focal injections of DAMGO in the VTA increases cFos expression in the majority of its projecting areas, namely NAc, basolateral amygdala (BLA), cingulate cortex (ACC) and bed nucleus of the stria terminalis (BNST), as compared to aCSF. Second, we analyzed whether inflammatory pain would affect to cFos expression using a group of rats injected with CFA in the hind paw. In this case, we found that cFos expression was not significantly different between DAMGO and aCSF administered rats in BLA, ACC and BNST. Our results confirm that inflammatory pain induces desensitization of VTA MORs in a region dependent manner which can be very relevant for addictive behaviours.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Freund's Adjuvant; Gene Expression Regulation; Genes, fos; Immunohistochemistry; Inflammation; Male; Microinjections; Neural Pathways; Pain; Rats; Receptors, Opioid, mu; Ventral Tegmental Area

The mechanisms underlying the transition from acute nociceptive pain to centrally maintained chronic pain are not clear. We have studied the contributions of the peripheral and central nervous systems during the development of osteoarthritis (OA) pain. Male Sprague-Dawley rats received unilateral intra-articular injections of monosodium iodoacetate (MIA 1 mg) or saline, and weight-bearing (WB) asymmetry and distal allodynia measured. Subgroups of rats received intra-articular injections of, QX-314 (membrane impermeable local anaesthetic) + capsaicin, QX-314, capsaicin or vehicle on days 7, 14 or 28 post-MIA and WB and PWT remeasured. On days 7&14 post-MIA, but not day 28, QX-314 + capsaicin signficantly attenuated changes in WB induced by MIA, illustrating a crucial role for TRPV1 expressing nociceptors in early OA pain. The role of top-down control of spinal excitability was investigated. The mu-opioid receptor agonist DAMGO was microinjected into the rostroventral medulla, to activate endogenous pain modulatory systems, in MIA and control rats and reflex excitability measured using electromyography. DAMGO (3 ng) had a significantly larger inhibitory effect in MIA treated rats than in controls. These data show distinct temporal contribtuions of TRPV1 expressing nociceptors and opioidergic pain control systems at later timepoints.

Topics: Anesthetics, Local; Animals; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation; Humans; Hyperalgesia; Injections, Intra-Articular; Iodoacetic Acid; Nociceptors; Osteoarthritis; Pain; Pain Measurement; Rats; Receptors, Opioid, mu; TRPV Cation Channels

Placental Opioid Enhancing Factor (POEF) is found in amniotic fluid (AF) and placenta. When ingested, it enhances opioid-mediated pain relief. Our laboratory has shown that ingestion of AF specifically enhances the hypoalgesia associated with δ-opioid receptor activation in the brain. The specific biochemical compound in AF responsible for the enhancement of δ-opioid activity is of great interest as an analgesic adjunct for pain but is unknown at this time. Research efforts to isolate and characterize this biochemical compound are hampered by the lack of an algesiometric assay that allows repeated measurement of pain threshold and repeated exposure to δ-opioid receptor activation. The cold water tail-flick assay (CWTF) may be a sensitive and reliable pain threshold test of (a) all species of opioids that is (b) not subject to repeated-testing effects. Therefore the CWTF test is potentially ideal for the study of δ opioid systems in a repeated measures design. Here, we confirm these attributes of the CWTF test, and determined that (a) there are no repeated-exposure effects associated with the CWTF assay; (b) there are no repeated-exposure effects associated with repeated central injections of DPDPE ([D-Pen2,D-Pen5]-Enkephalin, a selective δ-opioid agonist) as measured by the CWTF assay; and (c) ingestion of AF in conjunction with a central injection of DPDPE produced the same hypoalgesic enhancement as previously found using another assay.

Topics: Amniotic Fluid; Analgesics; Analgesics, Opioid; Animals; Cold Temperature; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Female; Pain; Pain Measurement; Pain Threshold; Pregnancy; Rats; Rats, Long-Evans; Receptors, Opioid, delta; Water

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.

Topics: Analgesics; Animals; Benzylidene Compounds; Ligands; Mice; Oxymorphone; Pain; Receptors, Opioid, delta; Receptors, Opioid, mu

Topics: Analgesics, Opioid; Animals; Cells, Cultured; CHO Cells; Cricetulus; Diterpenes; Diterpenes, Clerodane; Dose-Response Relationship, Drug; Male; Molecular Structure; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Salvia; Structure-Activity Relationship

Buprenorphine is a potent analgesic with high affinity at μ, δ and κ and moderate affinity at nociceptin opioid (NOP) receptors. Nevertheless, NOP receptor activation modulates the in vivo activity of buprenorphine. Structure activity studies were conducted to design buprenorphine analogues with high affinity at each of these receptors and to characterize them in in vitro and in vivo assays.. Compounds were tested for binding affinity and functional activity using [(35) S]GTPγS binding at each receptor and a whole-cell fluorescent assay at μ receptors. BU08073 was evaluated for antinociceptive agonist and antagonist activity and for its effects on anxiety in mice.. BU08073 bound with high affinity to all opioid receptors. It had virtually no efficacy at δ, κ and NOP receptors, whereas at μ receptors, BU08073 has similar efficacy as buprenorphine in both functional assays. Alone, BU08073 has anxiogenic activity and produces very little antinociception. However, BU08073 blocks morphine and U50,488-mediated antinociception. This blockade was not evident at 1 h post-treatment, but is present at 6 h and remains for up to 3-6 days.. These studies provide structural requirements for synthesis of 'universal' opioid ligands. BU08073 had high affinity for all the opioid receptors, with moderate efficacy at μ receptors and reduced efficacy at NOP receptors, a profile suggesting potential analgesic activity. However, in vivo, BU08073 had long-lasting antagonist activity, indicating that its pharmacokinetics determined both the time course of its effects and what receptor-mediated effects were observed.. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Topics: Analgesics, Opioid; Animals; Anxiety; Behavior, Animal; Buprenorphine; CHO Cells; Cricetulus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Humans; Male; Membrane Potentials; Mice, Inbred ICR; Narcotic Antagonists; Pain; Receptors, Opioid, mu

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the μ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-β-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.

Topics: Animals; Baclofen; Calcium Channels, L-Type; Calcium Channels, N-Type; Calcium Channels, T-Type; Dithiothreitol; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABA-B Receptor Agonists; gamma-Aminobutyric Acid; Ganglia, Spinal; Guanosine Diphosphate; HEK293 Cells; Humans; Nociception; Pain; Patch-Clamp Techniques; Pertussis Toxin; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, GABA-B; Sensory Receptor Cells; Signal Transduction; Thionucleotides

Pain management in opioid abusers engenders ethical and practical difficulties for clinicians, often resulting in pain mismanagement. Although chronic opioid administration may alter pain states, the presence of pain itself may alter the propensity to self-administer opioids, and previous history of drug abuse comorbid with chronic pain promotes higher rates of opioid misuse. Here, we tested the hypothesis that inflammatory pain leads to increased heroin self-administration resulting from altered mu opioid receptor (MOR) regulation of mesolimbic dopamine (DA) transmission. To this end, the complete Freund's adjuvant (CFA) model of inflammation was used to assess the neurochemical and functional changes induced by inflammatory pain on MOR-mediated mesolimbic DA transmission and on rat intravenous heroin self-administration under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement. In the presence of inflammatory pain, heroin intake under an FR schedule was increased for high, but attenuated for low, heroin doses with concomitant alterations in mesolimbic MOR function suggested by DA microdialysis. Consistent with the reduction in low dose FR heroin self-administration, inflammatory pain reduced motivation for a low dose of heroin, as measured by responding under a PR schedule of reinforcement, an effect dissociable from high heroin dose PR responding. Together, these results identify a connection between inflammatory pain and loss of MOR function in the mesolimbic dopaminergic pathway that increases intake of high doses of heroin. These findings suggest that pain-induced loss of MOR function in the mesolimbic pathway may promote opioid dose escalation and contribute to opioid abuse-associated phenotypes.. This study provides critical new insights that show that inflammatory pain alters heroin intake through a desensitization of MORs located within the VTA. These findings expand our knowledge of the interactions between inflammatory pain and opioid abuse liability, and should help to facilitate the development of novel and safer opioid-based strategies for treating chronic pain.

Topics: Action Potentials; Analgesics, Opioid; Animals; Conditioning, Operant; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; Glycine Agents; Heroin; Hyperalgesia; Inflammation; Inhibitory Postsynaptic Potentials; Male; Neurons; Pain; Pain Threshold; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Strychnine; Sucrose; Ventral Tegmental Area

The nociceptin opioid receptor (NOP) and its endogenous peptide ligand nociceptin/orphanin FQ have been shown to modulate the pharmacological effects of the classical opioid receptor system. Suppression of opioid-induced reward associated with mu-opioid receptor (MOP)-mediated analgesia, without decreasing anti-nociceptive efficacy, can potentially be achieved with NOP agonists having bifunctional agonist activity at MOP, to afford 'non-addicting' analgesics. In Part II of this series, we describe a continuing structure-activity relationship (SAR) study of the NOP-selective piperidin-4-yl-1,3-dihydroindol-2-one scaffold, to obtain bifunctional activity at MOP, and a suitable ratio of NOP/MOP agonist activity that produces a non-addicting analgesic profile. The SAR reported here is focused on the influence of various piperidine nitrogen aromatic substituents on the ratio of binding affinity and intrinsic activity at both the NOP and MOP receptors.

Topics: Analgesics; Animals; Ligands; Mice; Models, Animal; Nociceptin Receptor; Pain; Piperidines; Protein Binding; Receptors, Opioid; Receptors, Opioid, mu; Structure-Activity Relationship

The μ-opioid receptor (MOR) is the major opioid receptor targeted by most analgesics in clinical use. However, the use of all known MOR agonists is associated with severe adverse effects. We reported that the 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-ones are novel opioid receptor agonists. Subsequent structural modification resulted in the potent MOR/KOR (κ-opioid receptor) agonists 19, 20, and 21. Testing the analgesic effect of these in WT B6 mice (tail-flick test) gave ED50 values of 8.4, 10.9, and 26.6mg/kg, respectively. The 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one core could be addressed in 1 or 2 synthetic steps with moderate to high percent of yield. In the adenylyl cyclase assay, compound 19 displayed a MOR/KOR agonist profile, with IC50 values of 0.73 and 0.41μM, respectively. Current results suggest that compound 19 is a promising lead to go further development and in vitro/in vivo adverse effects studies.

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Drug Discovery; HEK293 Cells; Humans; Indazoles; Mice; Mice, Congenic; Molecular Structure; Pain; Pain Measurement; Receptors, Opioid, kappa; Receptors, Opioid, mu; Structure-Activity Relationship; Tail

Extracellular acidosis is a common feature in pain-generating pathological conditions. Acid-sensing ion channels (ASICs), pH sensors, are distributed in peripheral sensory neurons and participate in nociception. Morphine exerts potent analgesic effects through the activation of opioid receptors for various pain conditions. A cross-talk between ASICs and opioid receptors in peripheral sensory neurons has not been shown so far. Here, we have found that morphine inhibits the activity of native ASICs in rat dorsal root ganglion (DRG) neurons. Morphine dose-dependently inhibited proton-gated currents mediated by ASICs in the presence of the TRPV1 inhibitor capsazepine. Morphine shifted the proton concentration-response curve downwards, with a decrease of 51.4±3.8% in the maximum current response but with no significant change in the pH0.5 value. Another μ-opioid receptor agonist DAMGO induced a similar decrease in ASIC currents compared with morphine. The morphine inhibition of ASIC currents was blocked by naloxone, a specific opioid receptor antagonist. Pretreatment of forskolin, an adenylyl cyclase activator, or the addition of cAMP reversed the inhibitory effect of morphine. Moreover, morphine altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, peripheral applied morphine relieved pain evoked by intraplantar of acetic acid in rats. Our results indicate that morphine can inhibit the activity of ASICs via μ-opioid receptor and cAMP dependent signal pathway. These observations demonstrate a cross-talk between ASICs and opioid receptors in peripheral sensory neurons, which was a novel analgesic mechanism of morphine.

Topics: Acetic Acid; Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Action Potentials; Analgesics, Opioid; Animals; Capsaicin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; In Vitro Techniques; Male; Morphine; Naloxone; Narcotic Antagonists; Neurons; Nociception; Pain; Protons; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; TRPV Cation Channels

Opiate analgesia in the spinal cord is impaired during neuropathic pain. We hypothesized that this is caused by a decrease in μ-opioid receptor inhibition of neurotransmitter release from primary afferents. To investigate this possibility, we measured substance P release in the spinal dorsal horn as neurokinin 1 receptor (NK1R) internalization in rats with chronic constriction injury (CCI) of the sciatic nerve. Noxious stimulation of the paw with CCI produced inconsistent NK1R internalization, suggesting that transmission of nociceptive signals by the injured nerve was variably impaired after CCI. This idea was supported by the fact that CCI produced only small changes in the ability of exogenous substance P to induce NK1R internalization or in the release of substance P evoked centrally from site of nerve injury. In subsequent experiments, NK1R internalization was induced in spinal cord slices by stimulating the dorsal root ipsilateral to CCI. We observed a complete loss of the inhibition of substance P release by the μ-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in CCI rats but not in sham-operated rats. In contrast, DAMGO still inhibited substance P release after inflammation of the hind paw with complete Freund's adjuvant and in naïve rats. This loss of inhibition was not due to μ-opioid receptor downregulation in primary afferents, because their colocalization with substance P was unchanged, both in dorsal root ganglion neurons and primary afferent fibers in the dorsal horn. In conclusion, nerve injury eliminates the inhibition of substance P release by μ-opioid receptors, probably by hindering their signaling mechanisms.

Topics: Analgesics, Opioid; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; Hyperalgesia; Inflammation; Male; Neurons; Pain; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Opioid, mu; Sciatic Nerve; Sciatica; Spinal Cord; Substance P

Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR-GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50=1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment.

Topics: Analgesics; Animals; Arrestins; Behavior, Animal; beta-Arrestins; Cell Line, Tumor; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Isoquinolines; Ligands; Male; Mice, Inbred ICR; Morphinans; Morphine; Motor Activity; Naloxone; Narcotic Antagonists; Opioid-Related Disorders; Pain; Receptors, Opioid, mu

Pain has sensory-discriminative and emotional-affective dimensions. Recent studies show that the affective component can be assessed with a conditioned place avoidance (CPA) test. We hypothesized that systemic morphine before a post-conditioning test would more potently attenuate the affective aspect compared to the sensory component and that [d-Ala2-N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a μ-selective opioid receptor agonist, injected into the central nucleus of the amygdala (CeA) would reduce established CPA. A rat model of inflammatory pain, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a CPA test. Three experiments were performed on adult male Sprague-Dawley rats. Systemic morphine (0.5 or 1.0mg/kg) in Experiment 1, intrathecal (i.t.) morphine (2.5 μg/rat) in Experiment 2, and intra-CeA DAMGO (7.7-15.4 ng/0.4 μl) in Experiment 3 were given to CFA-injected rats (n=6-8/group) prior to a post-conditioning test. Saline-injected rats were used as control. Time spent in a pain-paired compartment was recorded twice, before conditioning and after a post-conditioning test. Paw withdrawal latency (PWL) to a noxious thermal stimulus was measured before experiment at day-1 and after the post-conditioning test; hyperalgesia was defined as a decrease in PWL. The data showed that CFA-injected rats had significantly negative CPA compared to those of saline-injected rats (P<0.05). Low-dosage systemic morphine significantly (P<0.05) reduced CFA-induced CPA but had no effect on PWL. I.t. morphine did not inhibit the display of CPA but significantly increased PWL, suppressing hyperalgesia (P<0.05). Intra-CeA DAMGO significantly inhibited the display of CPA compared to saline (P<0.05) but had no effect on PWL. The data demonstrate that morphine attenuates the affective component more powerfully than it does the sensory and suggests that the sensory and the emotional-affective dimensions are underpinned by different mechanisms.

Topics: Amygdala; Analgesics, Opioid; Animals; Behavior, Animal; Conditioning, Classical; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hyperalgesia; Inflammation; Male; Morphine; Pain; Rats; Rats, Sprague-Dawley

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

A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural α-methylene-β-amino acids (Map) at position 3 or/and position 4. Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the μ-opioid receptor and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation and ERK1/2 phosphorylation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH(2) (analogue 12) exhibited the highest binding potency (K(i)(μ) = 0.221 nM) and efficacy (EC(50) = 0.0334 nM, E(max) = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the μ-opioid receptor.

Topics: Amino Acid Sequence; Amino Acids; Aminoisobutyric Acids; Analgesics; Animals; Cyclic AMP; Guinea Pigs; HEK293 Cells; Humans; Ileum; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Molecular; Molecular Dynamics Simulation; Muscle Contraction; Oligopeptides; Pain; Protein Binding; Receptors, Opioid, mu; Structure-Activity Relationship; Vas Deferens

We have previously shown that the balance of electrically evoked descending brainstem control of spinal nociceptive reflexes undergoes a switch from excitation to inhibition in preadolescent rats. Here we show that the same developmental switch occurs when μ-opioid receptor agonists are microinjected into the rostroventral medulla (RVM). Microinjections of the μ-opioid receptor agonist [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) into the RVM of lightly anaesthetised adult rats produced a dose-dependent decrease in mechanical nociceptive hindlimb reflex electromyographic activity. However, in preadolescent (postnatal day 21 [P21]) rats, the same doses of DAMGO produced reflex facilitation. RVM microinjection of δ-opioid receptor or GABA(A) receptor agonists, on the other hand, caused reflex depression at both ages. The μ-opioid receptor-mediated descending facilitation is tonically active in naive preadolescent rats, as microinjection of the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) into the RVM at this age decreases spinal nociceptive reflexes while having no effect in adults. To test whether tonic opioid central activity is required for the preadolescent switch in RVM descending control, naloxone hydrochloride was delivered continuously from subcutaneous osmotic mini-pumps for 7-day periods, at various postnatal stages. Blockade of tonic opioidergic activity from P21 to P28, but not at earlier or later ages, prevented the normal development of descending RVM inhibitory control of spinal nociceptive reflexes. Enhancing opioidergic activity with chronic morphine over P7 to P14 accelerated this development. These results show that descending facilitation of spinal nociception in young animals is mediated by μ-opioid receptor pathways in the RVM. Furthermore, the developmental transition from RVM descending facilitation to inhibition of pain is determined by activity in central opioid networks at a critical period of periadolescence.

Topics: Age Factors; Analgesics, Opioid; Animals; Brain Stem; Critical Period, Psychological; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Medulla Oblongata; Microinjections; Pain; Rats; Rats, Sprague-Dawley; Spinal Cord

Opioid sensitivity varies among individuals. Although opioids can act partly in the rostral ventromedial medulla (RVM), which has a major role in pain perception, individual differences in the functions of the RVM in response to opioids have not been elucidated. Pain-related behavior among inbred mouse strains may reflect individual differences in sensitivity to pain. We therefore investigated the changes in action potentials of RVM neurons in response to opioid in different mouse strains.. Two inbred strains of mice (A/J and CBA/J) were used. Their behavior to noxious stimuli was measured after intracerebroventricular injection of the μ-opioid receptor agonist, DAMGO. Using an in vivo extracellular recording technique, action potentials from single RVM neurons and their functional type (ON-like, OFF-like, or NEUTRAL-like cell) were identified. Evoked responses of the RVM neurons to noxious stimuli were recorded before and after DAMGO administration.. The behavioral study showed that the dose-dependent antinociceptive effect in the A/J strain was significantly stronger than in the CBA/J strain. The electrophysiological study showed that the number of inhibitory OFF-like cells in A/J mice was significantly larger than in CBA/J mice (P<0.01), and that the evoked responses of neurons of A/J mice were inhibited significantly more than in CBA/J mice both for ON-like and OFF-like cells (P<0.01).. The strain differences in the physiological properties of RVM neurons corresponded to the behavioral strain differences. Genetic differences may contribute to the interindividual variation seen in opioid-induced analgesia.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Data Interpretation, Statistical; Electrophysiological Phenomena; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; Hot Temperature; Injections, Intraventricular; Male; Medulla Oblongata; Mice; Mice, Inbred A; Mice, Inbred CBA; Neurons; Pain; Pain Measurement; Physical Stimulation; Receptors, Opioid, mu; Species Specificity

We have previously demonstrated an opioid link in nucleus accumbens (NAc) that mediates antinociception produced by a novel ascending pain modulation pathway. For example, noxious stimulation induces heterosegmental antinociception that is mediated by both mu- and delta-opioid receptors in NAc. However, spinal intrathecal administration of the mu-receptor agonist [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) also induces heterosegmental antinociception. The aim of the present study in the rat was to identify the intra-NAc opioid receptors that mediate the antinociceptive effects of spinally administered DAMGO and also to determine the effect of NAc efferent activity on nociception. Intra-NAc administration of either the mu-opioid receptor antagonist Cys(2),Tyr(3), Orn(5),Pen(7)amide (CTOP) or the delta-opioid receptor antagonist naltrindole blocked the antinociceptive effect of spinally administered DAMGO on the jaw-opening reflex (JOR). Injection of quaternary lidocaine (QX-314) attenuated the JOR, suggesting that the output of NAc is pronociceptive. In support of this, intra-NAc injection of the excitatory amino acid agonist kainate enhanced the JOR. Thus, it is possible to modulate activity in NAc to bidirectionally attenuate or enhance nociception, suggesting a potential role for NAc in setting nociceptive sensitivity.

Topics: Analysis of Variance; Animals; Electrodes, Implanted; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Spinal; Lidocaine; Male; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Pain; Pain Measurement; Pain Perception; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Spinal Cord

The regulator of G protein signaling 9-2 (RGS9-2) is a constituent of G protein-coupled receptor (GPCR) macromolecular complexes with a major role in regulation of GPCR activity in the central nervous system. Previous in situ hybridization and Western blot studies revealed that RGS9-2 is expressed in the superficial dorsal horn of the spinal cord. In the present study, we monitored tail withdrawal latencies to noxious thermal stimuli and performed in vitro whole-cell patch clamp electrophysiological recordings from neurons in lamina II of the spinal dorsal horn to examine the role of RGS9-2 in the dorsal horn of the spinal cord in nociceptive behaviours and opiate mediated modulation of synaptic transmission. Our findings obtained from RGS9 knockout mice indicate that the lack of RGS9-2 protein decreases sensitivity to thermal stimuli and to the analgesic actions of morphine in the tail immersion paradigm. This modulatory role of RGS9-2 on opiate-mediated responses was further supported by electrophysiological studies showing that hyperpolarization of neurons in lamina II of the spinal dorsal horn evoked by application of DAMGO ([d-Ala2, N-MePhe4, Gly-ol]-enkephalin, a mu opioid receptor agonist) was diminished in RGS9 knockout mice. The results indicate that RGS9-2 enhances the effect of morphine and may play a crucial role in opiate-mediated analgesic mechanisms at the level of the spinal cord.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Mice; Mice, Knockout; Morphine; Pain; Posterior Horn Cells; Receptors, Opioid, mu; RGS Proteins; Spinal Cord; Synaptic Transmission

Over the past few years, δ-opioid receptors (DOPRs) and μ-opioid receptors (MOPRs) have been shown to interact with each other. We have previously seen that expression of MOPR is essential for morphine and inflammation to potentiate the analgesic properties of selective DOPR agonists. In vivo, it is not clear whether MOPRs and DOPRs are expressed in the same neurons. Indeed, it was recently proposed that these receptors are segregated in different populations of nociceptors, with MOPRs and DOPRs expressed by peptidergic and nonpeptidergic fibers, respectively. In the present study, the role and the effects of DOPR- and MOPR-selective agonists in two different pain models were compared. Using preprotachykinin A knock-out mice, we first confirmed that substance P partly mediates intraplantar formalin- and capsaicin-induced pain behaviors. These mice had a significant reduction in pain behavior compared with wild-type mice. We then measured the effects of intrathecal deltorphin II (DOPR agonist) and DAMGO (MOPR agonist) on pain-like behavior, neuronal activation, and substance P release following formalin and capsaicin injection. We found that both agonists were able to decrease formalin- and capsaicin-induced pain, an effect that was correlated with a reduction in the number of c-fos-positive neurons in the superficial laminae of the lumbar spinal cord. Finally, visualization of NK(1) (neurokinin 1) receptor internalization revealed that DOPR and MOPR activation strongly reduced formalin- and capsaicin-induced substance P release via direct action on primary afferent fibers. Together, our results indicate that functional MOPRs and DOPRs are both expressed by peptidergic nociceptors.

Topics: Animals; Capsaicin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Formaldehyde; Injections, Spinal; Male; Mice; Mice, Knockout; Neurons; Neurons, Afferent; Oligopeptides; Pain; Pain Measurement; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord; Substance P; Tachykinins

14-O-Methyloxymorphone and 14-methoxymetopon were reported as highly selective and potent micro opioid receptor agonists. The aim of this study was to demonstrate the opioid activity of these compounds in vitro and in vivo in comparison to oxymorphone, morphine and DAMGO. The micro opioid receptor efficacy, full or partial agonist nature of opioids was analyzed in the rat vas deferens (RVD) bioassay. Compared to oxymorphone, 14-O-methyloxymorphone and 14-methoxymetopon showed greater affinities to the rodent brain micro opioid receptors in receptor binding assays. In isolated organs 14-O-methyloxymorphone and 14-methoxymetopon were 3-10-fold more potent than the micro agonist opioid peptide, DAMGO. All tested compounds reached at least 70% maximum inhibition in mouse vas deferens (MVD) except morphine and oxymorphone. In the RVD, morphine could not exceed 50% inhibition of the twitches while 14-O-methyloxymorphone and 14-methoxymetopon showed inhibitory effects more than 70%. Oxymorphone reached only 4% maximal agonist effect and antagonized the inhibitory effect of DAMGO. The investigated morphinans produced dose-dependent antinociceptive activities in mice and rats. Both, 14-O-methyloxymorphone and 14-methoxymetopon are highly efficacious micro opioid receptor agonists in the RVD exhibiting full micro agonist properties. The RVD tissue contains mu receptors indicated by the comparable K(e) values of the micro antagonist naltrexone against DAMGO in the MVD. RVD may be a good alternative to assess the mu receptor efficacy of opioid agonists providing a more physiological environment for the ligand-receptor interaction than other efficacy measuring methods such as the [(35)S]GTPgammaS binding assay.

Topics: Analgesics, Opioid; Animals; Brain; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Mice; Mice, Inbred Strains; Models, Biological; Morphine; Morphine Derivatives; Naltrexone; Oxymorphone; Pain; Rats; Rats, Wistar; Receptors, Opioid, mu; Vas Deferens

A previous study from our laboratories showed that a significant reduction in spinal N-methyl-D-aspartate (NMDA) receptor NR1 subunit phosphorylation (pNR1) is associated with the antiallodynic effect produced by intrathecal (IT) injection of the alpha-2 adrenoceptor agonist, clonidine, in neuropathic rats. In this study, we determined whether the spontaneous pain and increased pNR1 expression induced by NMDA injection are reduced by IT injection of either clonidine or the mu-opioid receptor agonist, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO).. We examined the effect of clonidine (20 microg/rat) or DAMGO (1 microg/rat) injection on IT NMDA-induced spontaneous nociceptive behavior and pNR1 expression in the spinal dorsal horn. We also determined whether the effect of clonidine is mediated by alpha-2A or alpha-2C adrenoceptors. Finally, rat spinal cords were immunohistochemically processed for double staining of pNR1 and alpha-2A or alpha-2C adrenoceptors or mu-opioid receptors.. The NMDA-induced increase in both pNR1 expression and nociceptive behavior was significantly reduced by IT clonidine but not DAMGO. This analgesic effect of clonidine was blocked by administration of either an alpha-2A (BRL44408, 30 microg/rat) or an alpha-2C (JP-1302, 50 microg/rat) adrenoceptor antagonist. In addition, immunocytochemistry revealed that spinal pNR1 immunoreactive cells co-contain alpha-2A and alpha-2C adrenoceptors.. These results demonstrate that the IT NMDA-induced increase in pNR1 expression and nociceptive behavior is significantly reduced by activation of alpha-2 adrenoceptors, but not mu-opioid receptors, in the spinal cord dorsal horn. Furthermore, these findings suggest that the modulation of spinal NR1 phosphorylation is linked to the effect of IT clonidine on postsynaptic neuronal activity.

Topics: Adrenergic alpha-2 Receptor Antagonists; Adrenergic alpha-Agonists; Analgesics; Animals; Behavior, Animal; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Immunohistochemistry; Injections, Spinal; Male; N-Methylaspartate; Pain; Phosphorylation; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu

The role of mu-opioid receptors (MORs) in the inflammatory pain processing mechanisms within the ventrobasal complex of the thalamus (VB) is not well understood. This study investigated the effect of modulating MOR activity upon nociception, by stereotaxically injecting specific ligands in the VB. Nociceptive behaviour was evaluated in two established animal models of inflammatory pain, by using the formalin (acute and tonic pain) and the ankle-bend (chronic monoarthritic pain) tests. Control (saline intra-VB injection) formalin-injected rats showed acute and tonic pain-related behaviours. In contrast, intrathalamic administration of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate (DAMGO), a MOR-specific agonist, induced a statistically significant decrease of all tonic phase pain-related behaviours assessed until 30-35min after formalin hind paw injection. In the acute phase only the number of paw-jerks was affected. In monoarthritic rats, there was a noticeable antinociceptive effect with approximately 40min of duration, as denoted by the reduced ankle-bend scores observed after DAMGO injection. Intra-VB injection of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), a specific MOR antagonist, or of CTOP followed, 10min after, by DAMGO had no effects in either formalin or ankle-bend tests. Data show that DAMGO-induced MOR activation in the VB has an antinociceptive effect in the formalin test as well as in chronic pain observed in MA rats, suggesting an important and specific role for MORs in the VB processing of inflammatory pain.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Ankle; Behavior, Animal; Disease Models, Animal; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Inflammation; Male; Narcotic Antagonists; Pain; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Reaction Time; Receptors, Opioid, mu; Somatostatin; Time Factors; Ventral Thalamic Nuclei

Clinical pain conditions may remain responsive to opiate analgesics for extended periods, but such persistent acute pain can undergo a transition to an opiate-resistant chronic pain state that becomes a much more serious clinical problem. To test the hypothesis that cellular mechanisms of chronic pain in the primary afferent also contribute to the development of opiate resistance, we used a recently developed model of the transition of from acute to chronic pain, hyperalgesic priming. Repeated intradermal administration of the potent and highly selective mu-opioid agonist, [d-Ala(2),N-MePhe(4),gly-ol]-enkephalin (DAMGO), to produce tolerance for its inhibition of prostaglandin E(2) hyperalgesia, simultaneously produced hyperalgesic priming. Conversely, injection of an inflammogen, carrageenan, used to produce priming produced DAMGO tolerance. Both effects were prevented by inhibition of protein kinase Cepsilon (PKCepsilon). Carrageenan also induced opioid dependence, manifest as mu-opioid receptor antagonist (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2))-induced hyperalgesia that, like priming, was PKCepsilon and G(i) dependent. These findings suggest that the transition from acute to chronic pain, and development of mu-opioid receptor tolerance and dependence may be linked by common cellular mechanisms in the primary afferent.

Topics: Acute Disease; Analgesics, Opioid; Animals; Carrageenan; Chronic Disease; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Hyperalgesia; Inflammation; Male; Nociceptors; Opioid-Related Disorders; Pain; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome

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

Nociceptin/Orphanin FQ (N/OFQ) counteracts supraspinal opioid effects and plays a role in antinociceptive morphine tolerance. Therefore, in the present study, the selective mu-opioid agonist [D-Ala(2)-NMe-Phe(4)-Gly-ol(5)]-enkephalin (DAMGO) was used. Repeated injection of DAMGO (1 microg/ 1 microl) into the ventrolateral periaqueductal gray (vlPAG), a key site for the development of opioid tolerance, induced analgesia that lasted up to 3 days. In DAMGO-tolerant rats, injection of the N/OFQ antagonist (+/-)-J 113397 (4 microg/1 microl), into the same site, restored the antinociceptive effect of DAMGO. If (+/-)-J 113397 treatment preceded each DAMGO injection, tolerance did not develop. Inhibition of N/OFQ signaling can reverse and prevent the development of DAMGO tolerance in the vlPAG. The results confirm that N/OFQ acts as a functional opioid antagonist.

Topics: Analgesia; Analgesics, Opioid; Animals; Benzimidazoles; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Morphine; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Opioid Peptides; Pain; Periaqueductal Gray; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Vasodilator Agents

Data available on the role of the opioid systems of the naked mole-rat in nociception is scanty and unique compared to that of other rodents. In the current study, the effect of DAMGO, DPDPE and U-50488 and U-69593 on formalin-induced (20 microl, 10%) nociception were investigated. Nociceptive-like behaviors were quantified by scoring in blocks of 5 min the total amount of time (s) the animal spent scratching/biting the injected paw in the early (0-5 min) and in the late (25-60 min) phase of the test. In both the early and late phases, administration of 1 or 5 mg/kg of DAMGO or DPDPE caused a naloxone-attenuated decrease in the mean scratching/biting time. U-50488 and U-69593 at all the doses tested did not significantly change the mean scratching/biting time in the early phase. However, in the late phase U-50488 or U-69593 at the highest doses tested (1 or 5 mg/kg or 0.025 or 0.05 mg/kg, respectively) caused a statistically significant and naloxone-attenuated decrease in the mean scratching/biting time. The data showed that mu, delta or kappa-selective opioids causes antinociception in the formalin test in this rodent, adding novel information on the role of opioid systems of the animal on pain regulation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Benzeneacetamides; Data Interpretation, Statistical; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Formaldehyde; Injections, Intraperitoneal; Male; Mole Rats; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Pyrrolidines; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

The single nucleotide polymorphism 118A>G of the human micro-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the mu-opioid receptor-specific agonist DAMGO, we analyzed the micro-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D micro-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n=18), DAMGO was only 62% as efficient (p=0.002) as in homozygous carriers of the wild-type 118A allele (n=15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the micro-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p<0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of mu-opioid receptor variant N40D.

Topics: Alleles; Amino Acid Substitution; Analgesics, Opioid; Asparagine; Aspartic Acid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation; Homozygote; Humans; Pain; Polymorphism, Single Nucleotide; Protein Structure, Tertiary; Receptors, Opioid, mu; Somatosensory Cortex; Thalamus

Women are more sensitive to most noxious visceral stimuli, both in terms of intensity and frequency. The spinoparabrachial (spino-PBn) pathway is an essential neural circuit for the central relay of viscerosensitive information, but studies characterizing the anatomical and physiological characteristics of this pathway have only been conducted in males. Sex differences in the anatomical and/or physiological organization of the spino-PBn may contribute to the sexually dimorphic incidence rate for visceral pain syndromes. Retrograde labeling and colorectal distention (CRD) induced Fos expression was used to delineate the spino-PBn circuit in male and cycling female Sprague-Dawley rats. The ability of morphine to suppress CRD evoked responses was also examined. Neurons retrogradely labeled from the PBn were localized primarily within the superficial dorsal horn and sacral parasympathetic nucleus of the L5-S1 spinal cord. While no sex differences were noted in either the distribution of spino-PBn neurons or in CRD-induced Fos expression, significantly greater Fos expression was noted specifically in spino-PBn neurons in males compared to females. Morphine selectively attenuated Fos expression in spino-PBn neurons in males, but not females. Subsequent anatomical studies showed significantly reduced mu opioid receptor protein levels and radioligand binding within the PBn of females in comparison to males. Together, these data indicate that there are profound sex differences in how a noxious visceral stimulus and opiates engage the spino-PBn pathway, which may account for the observed clinical differences in visceral pain sensitivity and morphine antinociception.

Topics: Analgesics, Opioid; Animals; Autoradiography; Brain Mapping; Colon; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Medulla Oblongata; Morphine; Neural Pathways; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sex Characteristics; Spinal Cord; Stilbamidines; Stress, Mechanical; Tritium

A single nucleotide polymorphism (SNP) in the human mu-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in a variety of drug addiction and pain sensitivity phenotypes; however, the extent of these adaptations and the mechanisms underlying these associations remain elusive. To clarify the functional mechanisms linking the OPRM1 A118G SNP to addiction and analgesia phenotypes, we derived a mouse model possessing the equivalent nucleotide/amino acid substitution in the Oprm1 gene. Mice harboring this SNP (A112G) demonstrated several phenotypic similarities to humans carrying the A118G SNP, including reduced mRNA expression and morphine-mediated antinociception. We found additional phenotypes associated with this SNP including significant reductions of receptor protein levels, morphine-mediated hyperactivity, and the development of locomotor sensitization in mice harboring the G112 allele. In addition, we found sex-specific reductions in the rewarding properties of morphine and the aversive components of naloxone-precipitated morphine withdrawal. Further cross-species analysis will allow us to investigate mechanisms and adaptations present in humans carrying this SNP.

Topics: Analgesics, Opioid; Animals; Base Sequence; Binding, Competitive; Conditioning, Operant; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Gene Expression; Gene Frequency; Genotype; Humans; Male; Mice; Mice, Inbred C57BL; Models, Animal; Morphine; Motor Activity; Pain; Pain Measurement; Polymorphism, Single Nucleotide; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sex Factors; Substance Withdrawal Syndrome

cis-4-(Piperazin-1-yl)-5,6,7a,8,9,10,11,11a-octahydrobenzofuro[2,3-h]quinazolin-2-amine, 4 (A-987306) is a new histamine H(4) antagonist. The compound is potent in H(4) receptor binding assays (rat H(4), K(i) = 3.4 nM, human H(4) K(i) = 5.8 nM) and demonstrated potent functional antagonism in vitro at human, rat, and mouse H(4) receptors in cell-based FLIPR assays. Compound 4 also demonstrated H(4) antagonism in vivo in mice, blocking H(4)-agonist induced scratch responses, and showed anti-inflammatory activity in mice in a peritonitis model. Most interesting was the high potency and efficacy of this compound in blocking pain responses, where it showed an ED(50) of 42 mumol/kg (ip) in a rat post-carrageenan thermal hyperalgesia model of inflammatory pain.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzofurans; Carrageenan; Disease Models, Animal; Drug Design; Drug Evaluation, Preclinical; Humans; Hyperalgesia; Ligands; Mice; Molecular Structure; Pain; Peritonitis; Quinazolines; Rats; Receptors, G-Protein-Coupled; Receptors, Histamine; Receptors, Histamine H4; Stereoisomerism; Structure-Activity Relationship

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.

Topics: Animals; Binding, Competitive; Brain Stem; Camphanes; Cannabinoid Receptor Modulators; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glycerides; Mice; Mice, Inbred C57BL; Mice, Knockout; Nociceptors; Pain; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Opioid, mu; RNA, Messenger

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

The antagonism by Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) analog, of the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [D-Ala2,NMePhe4,Gly(ol)5]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) was studied with the mouse tail-flick test. D-Pro2-Tyr-W-MIF-1 (0.5-3 nmol) given intracerebroventricularly (i.c.v.) had no effect on the thermal nociceptive threshold. High doses of D-Pro2-Tyr-W-MIF-1 (4-16 nmol) administered i.c.v. produced antinociception with a low intrinsic activity of about 30% of the maximal possible effect. D-Pro2-Tyr-W-MIF-1 (0.25-2 nmol) co-administered i.c.v. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 or DAMGO without affecting endomorphin-2-induced antinociception. A 0.5 nmol dose of D-Pro2-Tyr-W-MIF-1 significantly attenuated Tyr-W-MIF-1-induced antinociception but not DAMGO- or endomorphin-1-induced antinociception. The highest dose (2 nmol) of D-Pro2-Tyr-W-MIF-1 almost completely attenuated Tyr-W-MIF-1-induced antinociception. However, that dose of D-Pro2-Tyr-W-MIF-1 significantly but not completely attenuated endomorphin-1 or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by D-Pro2-Tyr-W-MIF-1. Pretreatment i.c.v. with various doses of naloxonazine, a mu1-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID50 values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than that of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that D-Pro2-Tyr-W-MIF-1 is a selective antagonist for the mu2-opioid receptor in the mouse brain. D-Pro2-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, which both show a preference for the mu2-opioid receptor in the brain.

Topics: Analgesics, Opioid; Animals; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Injections, Intraventricular; Male; Mice; MSH Release-Inhibiting Hormone; Naloxone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Reaction Time; Receptors, Opioid, mu; Somatostatin; Time Factors

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

We determined responses to noxious thermal stimuli, before and after morphine, and mu-opioid receptor binding in brain regions involved in nociception in maternally separated (MS), neonatally handled (H) and nonhandled (NH) female rats. Long-Evans dams were randomly assigned to either 180-minute (MS) or 15-minute (H) minute daily separations from their litters or left undisturbed (NH). At 120 days of age, paw lick latency (50 degrees C hot plate) was determined in offspring during diestrous. Rats were then given 1, 2, 5, or 10 mg/kg morphine and paw lick latency was measured. Rats were killed during diestrous and mu-opioid receptor binding was determined in discrete brain regions, using [(3)H]DAMGO autoradiography. MS rats had significantly longer (P < .05) paw lick latencies compared with H rats. The percent maximal possible effect of morphine was significantly (P < .05) lower in MS compared with H rats for the 5 mg/kg dose. Mu-Opioid receptor binding capacity was significantly greater (P < .05) in MS rats compared with H rats in the medial preoptic nucleus. In conclusion, MS and H treatments led to antipodal differences in pain sensitivity in female rats and differential mu-opioid receptor binding in the medial preoptic nucleus.. This article describes the persistent impact of early life adversity on pain sensitivity and the analgesic potency of morphine. Clinically, early life history may play an important role in pain symptoms and responses to opioid analgesics.

Topics: Analysis of Variance; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Estrous Cycle; Female; Maternal Deprivation; Morphine; Narcotics; Pain; Pain Measurement; Pain Threshold; Pregnancy; Protein Binding; Radiography; Random Allocation; Rats; Rats, Long-Evans; Reaction Time; Time Factors; Tritium

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [D-Ala(2), N-MePhe(4),Gly-ol(5)] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for beta-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Tolerance; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; G-Protein-Coupled Receptor Kinases; Gene Silencing; Hot Temperature; Injections, Intraventricular; Male; Mice; Molecular Sequence Data; Morphine; Neurons; Oligonucleotides, Antisense; Pain; Pain Measurement; Pain Threshold; Periaqueductal Gray; Phosphorylation; Receptors, Opioid, mu; RGS Proteins; Serine; Synaptosomes; Time Factors

Peripheral micro-opioid receptors (MOR) have emerged as important components of inhibitory nociceptive pathways. Here, the antinociceptive effects of MOR agonists, the 6beta-glycine derivative of 14-O-methyloxymorphone (HS-731), DAMGO and morphine were evaluated in a mouse model of visceral pain. The abdominal acetic acid-induced writhing test was used to examine the peripheral, preemptive antinociceptive opioid action on visceral nociception. HS-731 administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) dose-dependently and completely inhibited writhing, being 24-598-fold more potent, depending on the administration route, than two selective MOR agonists, the enkephalin analogue [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO) and morphine. A longer duration of action (2-3 h) was induced by HS-731 given before acetic acid, while shorter effect was produced by morphine (30-60 min) and DAMGO (30-45 min). The antinociceptive effects of systemic opioids were reversed by the s.c. opioid antagonist, naloxone. Blocking of central MOR by the selective MOR antagonist D-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, i.c.v.) resulted in a significant reduction of antinociception of s.c. morphine, whereas it completely failed to antagonize the effects of systemic HS-731 or DAMGO. In in vitro studies, HS-731 and DAMGO, but not morphine showed high intrinsic efficacy, naltrexone-sensitive agonist effect at MOR of the rat vas deferens. These data demonstrate that selective activation of peripheral MOR by systemic s.c. HS-731 or DAMGO produces potent peripheral, preemptive visceral antinociception, while morphine's effects are mediated primarily through central mechanisms. Our findings support the role of peripheral MOR in the pathology of pain states involving sensitization of peripheral nociceptors.

Topics: Acetic Acid; Analgesics, Opioid; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Epoxy Compounds; In Vitro Techniques; Male; Mice; Morphinans; Morphine; Pain; Pain Measurement; Peptide Fragments; Peptides; Rats; Rats, Wistar; Somatostatin; Time Factors; Vas Deferens

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

A growing body of evidences suggests that receptor desensitization is implicated in the development of tolerance to opioids, which is generally regulated by protein kinases and receptor trafficking proteins. In the present study, we demonstrated that repeated s.c. treatment with etorphine, but not morphine, produced a significant increase in protein levels of G protein-coupled receptor kinase 2, dynamin II, beta-arrestin 2 and phosphorylated-conventional protein kinase C in membranes of the mouse spinal cord, suggesting that the etorphine-induced mu-opioid receptor desensitization may result from G protein-coupled receptor kinase 2/dynaminII/beta-arrestin2-dependent phosphorylation of mu-opioid receptors. Unlike etorphine, morphine failed to change the levels of these trafficking proteins. Furthermore, we found that the level of glial fibrillary acidic protein in the mouse spinal cord was clearly increased by chronic in vivo and in vitro treatment with morphine, whereas no such effect was noted by etorphine. In the behavioral study, intraperitoneal pretreatment with the glial-modulating agent propentofylline suppressed the development of tolerance to morphine-induced antinociception. In addition, intrathecal injection of astrocytes and astrocyte-conditioned medium mixture, which were obtained from cultured astrocytes of the newborn mouse spinal cord, aggravated the development of tolerance to morphine. In contrast, these agents failed to affect the development of tolerance induced by etorphine. These findings provide direct evidence for the distinct mechanisms between etorphine and morphine on the development of tolerance to spinal antinociception. These findings raise the possibility that the increased astroglia response produced by chronic morphine could be associated with the lack of mu-opioid receptor internalization.

Topics: Animals; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Etorphine; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Morphine; Neuroglia; Neurons; Pain; Phosphorylation; Protein Transport; Receptors, Opioid, mu; Spinal Cord; Xanthines

Recent evidence suggests that neurons in the medullary raphe are critical to the activation of brown adipose tissue (BAT), the major source of nonshivering heat production in the rat. Yet it is unclear which medullary raphe cells participate in cold defense and how participating cells contribute to BAT activation. Therefore, we recorded extracellularly from raphe cells during three thermoregulatory challenges that evoked an increase in BAT temperature in anesthetized rats: central cold, ambient cold, or intracerebroventricular prostaglandin E2 (PGE2) injection. Physiologically identified serotonergic (p5HT) cell discharge increased in response to cold or PGE2 administration and was positively correlated with BAT temperature. However, none of the 147 physiologically identified non-serotonergic (non-p5HT) cells recorded responded to thermoregulatory challenges that evoked an increase in BAT temperature. To test for modulation of BAT activation by non-p5HT cells that are either excited (ON cells) or inhibited (OFF cells) by noxious cutaneous stimulation, noxious stimuli were applied during evoked BAT temperature increases. Noxious stimulation suppressed BAT activation, suggesting that cells inhibited by noxious stimulation facilitate spinal circuits controlling BAT. To test whether medullary OFF cells modulate BAT activity, the mu-opiate receptor agonist (d-Ala2, N-Me-Phe4, Gly-ol5)-enkephalin (DAMGO) was microinjected into the raphe magnus, a manipulation that selectively activates OFF cells. DAMGO microinjection blocked noxious stimulation-evoked suppression of PGE2-induced BAT temperature increases. Thus, both p5HT and non-p5HT OFF cells in the medullary raphe facilitate BAT activation in response to cold challenge or pyrogen.

Topics: Adipose Tissue, Brown; Animals; Body Temperature; Cold Temperature; Dinoprostone; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Hot Temperature; Injections, Intraventricular; Male; Microinjections; Neurons; Nociceptors; Pain; Preoptic Area; Pressure; Pyrogens; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Serotonin; Thermogenesis

Suppression of reactions to one noxious stimulus by a spatially distant noxious stimulus is termed heterotopic antinociception. In lightly anesthetized rats, a noxious visceral stimulus, colorectal distension (CRD), suppressed motor withdrawals but not blood pressure or heart rate changes evoked by noxious hindpaw heat. Microinjection of muscimol, a GABA(A) receptor agonist, into raphe magnus (RM) reduced CRD-evoked suppression of withdrawals, evidence that RM neurons contribute to this heterotopic antinociception. To understand how brain stem neurons contribute to heterotopic antinociception, RM neurons were recorded during CRD-elicited suppression of hindpaw withdrawals. Although subsets of RM neurons that were excited (on cells) or inhibited (off cells) by noxious cutaneous stimulation were either excited or inhibited by CRD, on cells were inhibited and off cells excited by an intracerebroventricularly administered opioid, evidence that the nociception-facilitating and -inhibiting functions of on and off cells, respectively, are predicted by the cellular response to noxious cutaneous stimulation alone and not by the response to CRD. When recorded during CRD-elicited antinociception, RM cell discharge resembled the pattern observed in response to CRD stimulation alone. However, when hindpaw withdrawal suppression was incomplete, RM cell discharge resembled the pattern observed in response to heat alone. We propose that on cells excited by CRD facilitate responses to CRD itself, which in turn augments excitation of off cells that then act to suppress cutaneous nociception. RM cells may thereby contribute to the dominance of quiet recuperative reactions evoked by potentially life-threatening visceral stimuli over transient somatomotor activity elicited by less-injurious noxious cutaneous stimuli.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Catheterization; Electric Stimulation; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABA Agonists; Hot Temperature; Male; Microelectrodes; Muscimol; Neurons; Pain; Physical Stimulation; Posterior Horn Cells; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Rectum; Serotonin; Skin

Nociceptin activation of ORL1 (opioid receptor-like 1 receptor) has been shown to antagonize mu receptor-mediated analgesia at the supraspinal level. ORL1 and mu-opioid receptor (muR) are co-expressed in several subpopulations of CNS neurons involved in regulating pain transmission. The amino acid sequence of ORL1 also shares a high degree of homology with that of mu receptor. Thus, it is hypothesized that ORL1 and muR interact to form the heterodimer and that ORL1/muR heterodimerization may be one molecular basis for ORL1-mediated antiopioid effects in the brain. To test this hypothesis, myc-tagged ORL1 and HA-tagged muR are co-expressed in human embryonic kidney (HEK) 293 cells. Co-immunoprecipitation experiments demonstrate that ORL1 dimerizes with muR and that intracellular C-terminal tails of ORL1 and muR are required for the formation of ORL1/muR heterodimer. Second messenger assays further indicate that formation of ORL1/muR heterodimer selectively induces cross-desensitization of muR and impairs the potency by which [D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin (DAMGO) inhibits adenylate cyclase and stimulates p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. These results provide the evidence that ORL1/muR heterodimerization and the resulting impairment of mu receptor-activated signaling pathways may contribute to ORL1-mediated antiopioid effects in the brain.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cell Line; Central Nervous System; Dimerization; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Mitogen-Activated Protein Kinase 1; Neurons; Nociceptin Receptor; Pain; Protein Structure, Tertiary; Rats; Receptors, Opioid; Receptors, Opioid, mu; Signal Transduction

Male rats are more sensitive than female rats to the antinociceptive action of morphine. The present study used age-matched (9-10 weeks old) male and female Sprague-Dawley rats to investigate whether this difference is due to variation in micro-opioid receptor binding and G protein activation. In the warm-water tail-withdrawal assay at both 50 degrees C and 55 degrees C, morphine was 2-3 times more potent in males than females. In contrast, micro-opioid receptor number and the binding affinity of the micro-opioid agonists morphine and DAMGO in membranes from whole brain, cortex, thalamus, and spinal cord were not different between males and females. Similarly, morphine and DAMGO stimulation of G protein, determined using GTPase and [(35)S]GTPgammaS binding assays, did not show a difference between the sexes. The long-lasting micro-opioid receptor antagonist methocinnamox (0.32 mg/kg), given 24 h prior to morphine, reduced micro-opioid receptor number by approximately 50% in thalamic and spinal cord tissue from female and male rats and reduced the antinociceptive potency of morphine. Pretreatment of male rats with 0.32 mg/kg methocinnamox reduced the antinociceptive potency of morphine to that observed in female rats expressing a full complement of micro-opioid receptors. However, with increasing pretreatment doses of methocinnamox, the maximal antinociceptive effect of morphine was decreased in females but not males. The results suggest that pathways downstream of the micro-opioid receptor and G protein are more efficient in male rats than in female rats such that there is a larger receptor reserve for morphine-mediated antinociception.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Central Nervous System; Cinnamates; Down-Regulation; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Morphine; Morphine Derivatives; Nociceptors; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Sex Characteristics

Previous studies from our laboratory have shown that application of the mu opioid agonist DAMGO into the basolateral region of the amygdala (BLA) suppresses the radiant heat tail flick (TF) reflex in anesthetized rats. This antinociceptive effect can be blocked by lesions of brainstem regions such as the periaqueductal gray (PAG) or the rostral ventromedial medulla (RVM) or by functional inactivation of neurons in these regions, suggesting the activation of brainstem-descending antinociceptive systems from the amygdala. However, little is known about the direct interaction of DAMGO with mu receptors in the amygdala. In the present series of experiments, the BLA was pretreated with opioid receptor antagonists and a G protein inhibitor prior to TF testing with application of DAMGO into the same site. Rats pretreated with the non-selective opioid antagonist naltrexone (1.25-3.75 microg/0.25 microl per side) or the G protein inhibitor pertussis toxin (0.25 microg) failed to show inhibition of TF reflexes following infusion of DAMGO (0.168-0.50 microg), indicating that DAMGO works through G-protein-coupled opioid receptors in the BLA. Furthermore, pretreatment with the mu antagonist beta-FNA (1.00-2.00 microg) attenuated antinociception induced by DAMGO injection, suggesting DAMGO's action on mu receptors in the BLA. Accordingly, we confirm a direct interaction of DAMGO with G-protein-coupled mu receptors in the BLA contributing to induction of opioid antinociception in the amygdala.

Topics: Amygdala; Analgesics, Opioid; Anesthetics, Local; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Microinjections; Neurons; Pain; Pain Threshold; Rats; Rats, Long-Evans; Receptors, Opioid, mu

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

To test our hypothesis that the abnormally small efficacy of mu-opioid agonists in diabetic rats may be due to functional changes in the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway, we evaluated the effects of N-iminoethyl-L-ornithine, methylene blue, and 3-morpholino-sydnonimine on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced antinociception in both streptozotocin (STZ)-diabetic and nondiabetic rats. Animals were rendered diabetic by an injection of STZ (60 mg/kg intraperitoneally). Antinociception was evaluated by the formalin test. The mu-opioid receptor agonist DAMGO (1 microg per paw) suppressed the agitation response in the second phase. The antinociceptive effect of DAMGO in STZ-diabetic rats was significantly less than in nondiabetic rats. N-Iminoethyl-L-ornithine (100 microg per paw), an NO synthase inhibitor, or methylene blue (500 microg per paw), a guanylyl cyclase inhibitor, significantly decreased DAMGO-induced antinociception in both diabetic and nondiabetic rats. Furthermore, 3-morpholino-sydnonimine (200 microg per paw), an NO donor, enhanced the antinociceptive effect of DAMGO in nondiabetic rats but did not change in diabetic rats. These results suggest that the peripheral antinociceptive effect of DAMGO may result from activation of the L-arginine/NO/cGMP pathway and dysfunction of this pathway; also, events that are followed by cGMP activation may have contributed to the demonstrated poor antinociceptive response of diabetic rats to mu-opioid agonists.. This is the first study on the role of the nitric oxide (NO)/cyclic guanosine monophosphate pathway on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced peripheral antinociception and the effect of diabetes on this pathway. The study suggests a possible role of DAMGO as a peripherally-acting analgesic drug.

Topics: Analgesics, Opioid; Animals; Arginine; Blood Glucose; Cyclic GMP; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Formaldehyde; Guanylate Cyclase; Male; Methylene Blue; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Pain; Pain Measurement; Rats; Rats, Wistar

We cloned kappa and mu opioid receptor cDNAs. Using these cDNAs, first, we examined the molecular mechanism for the subtype selectivity of opioid ligands, especially a mu-selective ligand DAMGO. Binding experiments using various chimera and mutated receptors revealed that DAMGO discriminates between mu and delta receptors by recognizing the difference in only one amino acid residue, that is, N(127) in mu and K(108) in delta, at the first extracellular loop, and that it distinguishes between mu and kappa receptors by the difference in four amino acid residues at the third extracellular loop. Second, we established the cell lines expressing the cloned mu, delta, or kappa receptor and elucidated the pharmacological properties, that is, binding affinity and agonistic activity of several opioid agonists. Third, distribution of the mRNAs for mu, delta, and kappa receptors in the brain, spinal cord, and DRG was examined by in situ hybridization histochemistry (ISHH). Double ISHH demonstrated that most of the substance P-producing DRG neurons express the micro receptor. Recently, we are interested in the emotional aspect of pain and its regulation by opioids. Behavioral and microdialysis studies showed that sustained pain evoked by the intraplanter injection of formalin induced conditioned place aversion through the increment of glutamate release followed by the activation of NMDA receptors in the basolateral nucleus of amygdala (BLA). Intra-BLA injection of morphine suppressed the place aversion by inhibiting the glutamate release.

Topics: Analgesics, Opioid; Animals; Brain; Cloning, Molecular; DNA, Complementary; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Pain; Receptors, Opioid; Receptors, Opioid, mu

The dermorphin-derived tetrapeptide Tyr-D-Arg(2)-Phe-Sar(4) (TAPS) was tested for its ability to induce tolerance, cross-tolerance, withdrawal and its substitution properties in rats subjected to chronic intracerebroventricular (i.c.v.) infusions of mu-opiate receptor agonists. Tolerance and cross-tolerance were assessed by quantification of the thermally induced tail-flick response. Chronic intracerebroventricular infusion of TAPS resulted in antinociception at almost 1000-fold lower doses compared to morphine sulphate and [D-Ala(2), MePhe(4)Gly(ol)(5)]enkephalin (DAMGO). Tolerance to the antinociceptive effect of TAPS developed similar to DAMGO and morphine sulphate. Cross-tolerance to intracerebroventricular bolus injections of DAMGO, but not of TAPS, was evident in rats rendered tolerant to morphine sulphate and TAPS. Naloxone-induced withdrawal was equally pronounced in animals treated with morphine sulphate, DAMGO or TAPS. TAPS substituted for morphine sulphate and vice versa regarding the withdrawal syndrome in a cross-over experimental design. In contrast to DAMGO, TAPS retains its antinociceptive effect following bolus administration in rats rendered tolerant to mu-opioid receptor agonists.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; In Vitro Techniques; Infusion Pumps; Injections, Intraventricular; Male; Morphine; Naloxone; Oligopeptides; Pain; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Substance-Related Disorders; Time Factors

The ventral or inner region of spinal substantia gelatinosa (SG; lamina II(i)) is a heterogeneous sublamina important for the generation and maintenance of hyperalgesia and neuropathic pain. To test whether II(i) neurons can be hyperpolarized by the mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of mu-opioid-evoked inhibition of II(i) neurons, we combined in vitro whole-cell, tight-seal recording methods with fluorescent labeling of the intracellular tracer biocytin and confocal microscopy. Twenty-one of 23 neurons studied had identifiable axons. Nine possessed axons that projected ventrally into laminae III-V; six of these were hyperpolarized by DAMGO. Three of four neurons with identifiable axons that projected to lamina I were hyperpolarized by DAMGO. Most neurons could be classified as either islet cells or stalked cells. Five of nine labeled islet cells and only two of seven stalked cells were hyperpolarized by DAMGO. Three were stellate cells: one resembled a spiny cell and three could not be classified. DAMGO hyperpolarized each of the stellate cells, the spiny cell, and 1 of the unclassified cells. Our data support the hypothesis that part of the action of mu-opioid agonists involves the inhibition of interneurons that are part of a polysynaptic excitatory pathway from primary afferents to neurons in the deep and/or superficial dorsal horn.

Topics: Afferent Pathways; Analgesics, Opioid; Animals; Dendrites; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Interneurons; Membrane Potentials; Microscopy, Confocal; Neural Inhibition; Pain; Posterior Horn Cells; Presynaptic Terminals; Rats; Rats, Long-Evans; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Synaptic Transmission

Antagonistic properties of buprenorphine for epsilon- and micro -opioid receptors were characterized in beta-endorphin- and [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO)-induced antinociception, respectively, with the tail-flick test in male ICR mice. epsilon-Opioid receptor agonist beta-endorphin (0.1-1 micro g), micro -opioid receptor agonist DAMGO (0.5-20 ng), or buprenorphine (0.1-20 micro g) administered i.c.v. dose dependently produced antinociception. The antinociception induced by 10 micro g of buprenorphine given i.c.v. was completely blocked by the pretreatment with beta-funaltrexamine (beta-FNA) (0.3 micro g i.c.v.), indicating that the buprenophine-induced antinociception is mediated by the stimulation of the micro -opioid receptor. The antinociceptive effects induced by beta-endorphin (1 micro g i.c.v.) and DAMGO (16 ng i.c.v.) were dose dependently blocked by pretreatment with smaller doses of buprenorphine (0.001-1 micro g i.c.v.), but not by a higher dose of buprenorphine (10 micro g i.c.v.). beta-FNA at a dose (0.3 micro g i.c.v.) that strongly attenuated DAMGO-induced antinociception had no effect on the antinociception produced by beta-endorphin (1 micro g i.c.v.). However, pretreatment with buprenorphine (0.1-10 micro g) in mice pretreated with this same dose of beta-FNA was effective in blocking beta-endorphin-induced antinociception. beta-FNA was 226-fold more effective at antagonizing the antinociception induced by DAMGO (16 ng i.c.v.) than by beta-endorphin (1 micro g i.c.v.). The antinociception induced by delta-opioid receptor agonist [d-Ala2]deltorphin II (10 micro g i.c.v.) or kappa1-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamine methanesulfonate salt [(-)-U50,488H] (75 micro g i.c.v.) was not affected by pretreatment with buprenorphine (0.1-1.0 micro g i.c.v.). It is concluded that buprenorphine, at small doses, blocks epsilon-opioid receptor-mediated beta-endorphin-induced antinociception and micro -opioid receptor-mediated DAMGO-induced antinociception, and at high doses produces a micro -opioid receptor-mediated antinociception.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; beta-Endorphin; Buprenorphine; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors

We evaluated the ability of the specific micro opioid receptor agonist DAMGO, the kappa-specific agonist U-50488 in vitro, and exogenous opioid morphine administered in vivo both acutely and chronically to modulate IL-12 and IL-10 production by murine peritoneal macrophages. Damgo and U-50488 at the concentrations of 10(-6) and 10(-8) M decreased the production of IL-12 without affecting IL-10. One hour after the acute administration of 5, 10, and 20 mg/kg of morphine a dose-related decrease of both IL-10 and IL-12 levels was present. The pretreatment with naltrexone at doses up to 20 mg/kg did not prevent the decrease of IL-10 and IL-12 induced by morphine. When the drug was administered chronically, a differential development of tolerance to the immune effects was observed. After 3 days of treatment the effect of the acute challenge with 20 mg/kg of morphine on IL-12 was lost. In contrast, morphine-induced inhibition of IL-10 disappeared between 10 and 12 days of treatment, in parallel with tolerance to the antinociceptive effect. These results suggest that opioids modulate macrophage cytokine production. However, the effects exerted by the in vivo administration of morphine are not superimposable onto those induced by the in vitro administration of specific opioid agonists.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analysis of Variance; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme-Linked Immunosorbent Assay; Interleukin-10; Interleukin-12; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Morphine; Naltrexone; Pain

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

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

The role of N-methyl-D-aspartate (NMDA) receptors in supraspinal and spinal sites on the reduction of supraspinal micro-opioid receptor-induced antinociception in diabetic mice was examined. The antinociceptive effect of i.c.v. [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO, 20 pmol) in diabetic mice was significantly less than that in non-diabetic mice. The antinociceptive effect of i.c.v. DAMGO (20 pmol) was significantly and dose dependently reduced by i.c.v. (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) in both non-diabetic (0.03-0.3 nmol) and diabetic mice (0.1-3.0 nmol). While the antinociceptive effect of i.c.v. DAMGO (10 pmol) was significantly enhanced by i.c.v NMDA (0.01-0.1 nmol) in non-diabetic mice, the same doses of i.c.v. NMDA had no significant effect on the antinociceptive effect of i.c.v. DAMGO (20 pmol) in diabetic mice. In non-diabetic mice, the antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose dependently reduced by intrathecal administration of MK-801 (0.1-1.0 nmol). The antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose-dependently enhanced by MK-801 (0.1-1.0 nmol, i.t.) in diabetic mice. Furthermore, NMDA (0.1 nmol, i.t.) significantly enhanced the antinociceptive effect of DAMGO (10 pmol, i.c.v.) in non-diabetic mice. However, in non-diabetic mice, the antinociceptive effect of DAMGO (40 pmol, i.c.v.) was dose dependently reduced by NMDA (0.03-0.3 nmol, i.t.). These results suggest that NMDA receptor function in supraspinal and spinal sites appear to be modulated differently by the diabetic state, and this functional modulation may play an important role in the reduction of supraspinal micro-opioid receptor-induced antinociception in diabetic animals.

Topics: Animals; Diabetes Mellitus, Experimental; Dizocilpine Maleate; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Pain; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu

Prolonged exposure to opioid agonists can induce adaptive changes resulting in tolerance and dependence. Here, rats were rendered tolerant by subcutaneous injections of increasing doses of morphine from 10 to 60 mg/kg for 3, 5, or 10 consecutive days. Binding parameters of the mu-opioid receptor in subcellular fractions were measured with [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin). Although the density of surface mu-sites did not change after the 5-day morphine treatment, up-regulation of synaptic plasma membrane binding was detected after the 10-day drug administration. In contrast, the number of mu-binding sites in a light vesicle or microsomal fraction (MI) was elevated by 68 and 30% after 5 and 10 days of morphine exposure, respectively. The up-regulated MI mu-sites displayed enhanced coupling to G proteins compared with those detected in saline-treated controls. Pertussis toxin catalyzed ADP ribosylation, and Western blotting with specific antisera was used to quantitate chronic morphine-induced changes in levels of various G protein alpha-subunits. Morphine treatment of 5 days and longer induced significant increases in levels of Galpha(o), Galpha(i1), and Galpha(i2) in MI fractions that are part of an adaptation process. Up-regulation of intracellular mu-sites may be the result of post-translational changes and in part de novo synthesis. The results provide the first evidence that distinct regulation of intracellular mu-opioid receptor G protein coupling and G protein levels may accompany the development of morphine tolerance.

Topics: Analgesics, Opioid; Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Morphine; Morphine Dependence; Organ Specificity; Pain; Rats; Receptors, Opioid, mu; Subcellular Fractions

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that activates signalling pathways. The present study was designed to investigate whether PI3K could be involved in supraspinal antinociception induced by intracerebroventricular (i.c.v.) administration of micro- and delta-opioid receptor agonists in the mouse. We demonstrated using the mouse warm-plate assay that the prototype of micro-opioid receptor agonist morphine, selective mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) and delta-opioid receptor agonists [D-Ala(2)]deltorphin II and [D-Pen(2,5)]enkephalin (DPDPE) when given i.c.v. produced profound antinociceptive responses. Under these conditions, i.c.v. pretreatment with cell-permeable and specific PI3K inhibitors wortmannin (0.7-2.3 nmol) and LY294002 (3-33 nmol), which alone had no effects on the basal warm-plate latencies, caused a dose-dependent inhibition of either morphine-, DAMGO-, DPDPE- or [D-Ala(2)]deltorphin II-induced antinociception. Furthermore, LY294002 at 33 nmol significantly shifted the dose-response curves for DAMGO-, DPDPE- and [D-Ala(2)]deltorphin II-induced antinociception to the right. In the immunoblotting assay, we found that PI3K gamma is dense in the periaqueductal gray and lower medulla regions that include several key sites for the production of opioid-induced antinociception. Our findings provide evidence that central PI3K pathways may, at least in part, contribute to the expression of supraspinal antinociception induced by both mu- and delta-opioid receptor agonists in the mouse.

Topics: Analgesics, Opioid; Animals; Blotting, Western; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Injections, Intraventricular; Male; Mice; Pain; Phosphatidylinositol 3-Kinases; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Time Factors

The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor ORL-1, also designated opioid receptor 4 (OP(4)) are involved in the modulation of nociception. Using OP(4)-knockout mice, we have studied their response following opioid receptor stimulation and under neuropathic conditions.In vas deferens from wild-type and OP(4)-knockout mice, DAMGO (mu/OP(3) agonist), deltorphine II (delta/OP(1) agonist) and (-)-U-50488 (kappa/OP(2) agonist) induced similar concentration-dependent inhibition of electrically-evoked contractions. Naloxone and naltrindole (delta/OP(1) antagonists) shifted the curves of DAMGO (pA(2)=8.6) and deltorphine II (pA(2)=10.2) to the right, in each group. In the hot-plate assay, N/OFQ (10 nmol per mouse, i.t.) increased baseline latencies two-fold in wild-type mice while morphine (10mg/kg, s.c.), deltorphine II (10 nmol per mouse, i.c.v.) and dynorphin A (20 nmol per mouse, i.c.v.) increased hot-plate latencies by about four- to five-fold with no difference observed between wild-type and knockout mice. Furthermore, no change was evident in the development of the neuropathic condition due to chronic constriction injury (CCI) of the sciatic nerve, after both thermal and mechanical stimulation. Altogether these results suggest that the presence of OP(4) receptor is not crucial for (1) the development of either acute or neuropathic nociceptive responses, and for (2) the regulation of full receptor-mediated responses to opioid agonists, even though compensatory mechanisms could not be excluded.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Mice; Mice, Knockout; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Pain; Receptors, Opioid; Time Factors; Vas Deferens

We previously demonstrated that noxious peripheral stimulation (e.g. subdermal capsaicin injection in the hind paw) produces antinociception that is mediated by opioid receptors in nucleus accumbens. The current study used the trigeminal jaw-opening nociceptive reflex responses in the rat to assess the role of intra-accumbens mu-, delta- and kappa-opioid receptors in the antinociceptive effect of noxious stimulation and intra-accumbens opioid agonism. Whilst intra-accumbens injection of either the mu-receptor-selective antagonist Cys2,Tyr3,Orn5,Pen7amide (CTOP) or the delta-receptor-selective antagonist naltrindole blocked capsaicin-induced antinociception, neither the selective mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO; 150 or 300 ng) nor the selective delta-agonist D-Pen2,5-enkephalin (DPDPE; 150 or 300 ng) alone induced antinociception. Simultaneous injection of DAMGO and DPDPE (150 ng each), however, produced significant antinociception. Capsaicin-induced antinociception was not blocked by the selective kappa-receptor antagonist nor-binaltorphimine, but was blocked by the kappa-agonist U69,593. U69,593 also antagonized the antinociceptive effect of the DAMGO/DPDPE combination. Thus, in nucleus accumbens, mu- and delta- but not kappa-opioid receptors contributed to capsaicin-induced antinociception; selective activation of individual receptor subtypes was insufficient, but coactivation of mu- and delta-opioid receptors induced antinociception, and kappa-receptors appeared to play an antianalgesic role in nucleus accumbens.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; Capsaicin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Male; Narcotic Antagonists; Neurons; Nociceptors; Nucleus Accumbens; Pain; Pyrrolidines; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reflex

The actions of the endogenous ORL1 receptor (opioid receptor-like1) ligand nociceptin on the membrane properties of rat trigeminal nucleus caudalis neurons were examined by use of whole cell and perforated patch clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested (EC50 112 nM). The outward current produced by nociceptin was completely reversed with the addition of the non-peptide ORL1 antagonist J-113397. Outward currents reversed polarity at -99+/-2 mV, close to the potential for K+ of -102 mV, suggesting that they were mediated by an increased K+ conductance. These results suggest that the analgesic action of nociceptin might be mediated by direct postsynaptic inhibition within the dorsal horn.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Postsynaptic Potentials; Nociceptin; Nociceptin Receptor; Opioid Peptides; Pain; Patch-Clamp Techniques; Posterior Horn Cells; Potassium; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Substantia Gelatinosa; Trigeminal Caudal Nucleus; Vasodilator Agents

This study examined a mechanism responsible for the enhanced antihyperalgesic and antinociceptive effects of the mu opioid receptor agonist (ORA) [D-Ala(2), NMePhe(4), Gly(5)-ol]enkephalin (DAMGO) microinjected in the rostroventromedial medulla (RVM) of rats with inflammatory injury induced by injection of complete Freund's adjuvant (CFA) in one hindpaw. In rats injected with CFA 4 hr earlier, microinjection of the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) in the RVM antagonized both the marginal enhancement of the potency of DAMGO and its antinociceptive effect. The delta opioid receptor antagonist naltriben (NTB) was without effect. In rats injected with CFA 2 weeks earlier, CTAP antagonized the effects of DAMGO to a lesser extent. However, NTB completely prevented the enhancement of the potency of DAMGO, whereas it did not antagonize DAMGO's antinociceptive effects. Microinjection of NTB alone, but not CTAP in the RVM of CFA-treated rats, enhanced the hyperalgesia present in the ipsilateral hindpaw and induced hyperalgesia in the contralateral, uninjured hindpaw. These results suggest that persistent inflammatory injury increased the release in the RVM of opioid peptides with preferential affinity for the delta opioid receptor, which can interact in a synergistic or additive manner with an exogenously administered mu opioid receptor agonist. Indeed, the levels of [Met(5)]enkephalin and [Leu(5)]enkephalin were increased in the RVM and in other brainstem nuclei in CFA-treated rats. This increase most likely presents a compensatory neuronal response of the CNS of the injured animal to mitigate the full expression of inflammatory pain and to enhance the antinociceptive and antihyperalgesic effects of exogenously administered mu opioid receptor analgesics.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Inflammation; Male; Microinjections; Naltrexone; Pain; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin

Some opioid-resistant pain conditions can be alleviated by voltage-dependent Na(+) channel blockers such as lamotrigine. The mu-opioid-receptor agonist morphine can modulate cation entry into cells to affect overall cellular excitability, an effect which can in turn be endogenously antagonised by the neuropeptide cholecystokinin (CCK). However, lamotrigine may also modulate cellular excitability by non-specifically blocking voltage-dependent ion channels. We have looked for interactions of lamotrigine with the opioid/CCK pathway within the spinal dorsal horn, to rule out the possibility that lamotrigine may attenuate nociceptive responses via actions on this pathway. Both lamotrigine and the mu-opioid agonist DAMGO inhibited mustard oil-evoked cell firing by approximately 50% compared with control levels. Co-application of CCK8S reversed DAMGO-, but not lamotrigine-induced inhibition of cell firing and this reversal was prevented with the selective CCK(B) receptor antagonist PD 135158. Although lamotrigine inhibited both brush- and cold-evoked cell firing in neuropathic animals, lamotrigine inhibition of mustard oil-evoked cell firing in the same animals was not significantly greater than that observed in controls. These results suggest that the antinociceptive properties of lamotrigine within the spinal dorsal horn are unlikely to be mediated via interactions with the opioid/CCK pathway.

Topics: Action Potentials; Analgesics, Opioid; Animals; Anti-Anxiety Agents; Calcium Channel Blockers; Cholecystokinin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Indoles; Inflammation; Lamotrigine; Male; Meglumine; Mustard Plant; Neural Pathways; Nociceptors; Opioid Peptides; Pain; Peripheral Nervous System Diseases; Plant Extracts; Plant Oils; Posterior Horn Cells; Rats; Rats, Wistar; Sincalide; Triazines

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by mu-opioid receptor agonists including morphine partially results from activating the NRM-spinal cord pathway. It is generally believed that mu-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole-cell patch-clamp recordings combined with single-cell RT-PCR analysis were used to test the hypothesis that DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD(67)). Homologous desensitization of mu-opioid receptors in NRM neurons could result in the development of morphine-induced tolerance. G protein-coupled receptor kinase (GRK) is believed to mediate mu-opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMAMGO-induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single-cell RT-PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Whole-cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to G(betagamma)-binding domain of GRK and inhibits G(betagamma) activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K(+) channels and that GRK2 mediates short-term homologous desensitization of mu-opioid receptors in NRM GABAergic neurons.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; beta-Adrenergic Receptor Kinases; Binding Sites; Cyclic AMP-Dependent Protein Kinases; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; G-Protein-Coupled Receptor Kinase 2; G-Protein-Coupled Receptor Kinase 3; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Ion Transport; Membrane Potentials; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Oligopeptides; Pain; Patch-Clamp Techniques; Phosphorylation; Potassium; Potassium Channels; Potassium Channels, Inwardly Rectifying; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spinal Cord

Spinal nociceptive transmission is mediated by glutamate and neuropeptides such as substance P (SP) and neurokinin A (NKA). The neuropeptide-mediated excitatory postsynaptic potentials (EPSPs) had a slow onset and long duration. Here, we demonstrate SP- and NKA-mediated excitatory postsynaptic currents (EPSCs) in dorsal horn neurons of young rats using whole-cell patch-clamp recording techniques. After complete blockade of glutamate receptor-mediated currents, we observed a small residual EPSC. The residual EPSCs exhibited temporal summation in response to a train of stimulation (six pulses delivered at 10-50 Hz). High intensity stimulation (the same or greater than the stimulation threshold for nociceptive fibers in vivo) was required for evoking these summated EPSCs. Summated EPSCs were attenuated or abolished by capsaicin pretreatment, which depletes SP and NKA from presynaptic terminals; SP and NKA pretreatment; NK(1) or NK(2) receptor antagonists; and inhibition of postsynaptic G proteins. EPSCs were neither blocked by a metabotropic glutamate receptor antagonist nor a gamma-aminobutyric acid(B) receptor antagonist. The summated EPSCs were also sensitive to voltage-gated calcium channel antagonists or mu-opioid receptor activation by DAMGO. The present study provides electrophysiological evidence that suggests the possible contribution of SP and NKA to sensory synaptic transmission between primary afferent fibers and dorsal horn neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Analgesics, Opioid; Animals; Calcium Channel Blockers; Calcium Channels, P-Type; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Guanosine Diphosphate; In Vitro Techniques; Neurokinin A; omega-Conotoxin GVIA; Pain; Patch-Clamp Techniques; Phosphinic Acids; Posterior Horn Cells; Propanolamines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P; Synaptic Transmission; Thionucleotides

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Topics: Analgesics; Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Methionine; Immune Sera; Injections, Spinal; Injections, Subcutaneous; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptide Fragments; Time Factors

Opioids have been thought to induce analgesia by activating the descending pain control system, especially at the level of periaqueductal gray, and regulate the neurotransmitter release through the inhibition of calcium channel. In the present study, the modulatory effects of protein kinase C and protein kinase A on the mu-opioid agonist-induced inhibition of the high-voltage activated calcium current were examined in the acutely dissociated rat periaqueductal gray neurons with the nystatin-perforated patch-clamp technique. Among 505 neurons tested, the barium current passing through the high-voltage activated calcium channels of 172 neurons (34%) were inhibited by 32+/-3% with the application of an mu-opioid agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM). The barium currents itself and the DAMGO-induced inhibitory effects were not affected by the application of either an adenylate cyclase activator (forskolin, 1 microM) or a protein kinase inhibitor (staurosporin, 10 nM) for 2 min. The DAMGO inhibition was completely and irreversibly antagonized by the application of a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 1 microM) for 2 min without any alteration of the barium current itself. However, the antagonizing effect of PMA was completely abolished by the application of 10 nM staurosporin for 2 min. After then, PMA did not show the antagonizing effect any more. Inversely, when staurosporin was applied before PMA, the antagonizing effect of PMA was also not shown. These results demonstrate that the mu-opioid agonist-induced inhibition of the periaqueductal gray neuronal high-voltage activated calcium current can be antagonized by protein kinase C activation. This finding may provide us a significant clue to understand the action mechanism of opioid-induced analgesia in the periaqueductal gray.

Topics: Analgesics, Opioid; Animals; Calcium Channels; Calcium Signaling; Carcinogens; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Female; Male; Membrane Potentials; Neurons; Pain; Periaqueductal Gray; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Staurosporine; Synaptic Transmission; Tetradecanoylphorbol Acetate

Development of tolerance in mice pretreated intracerebroventricularly with mu-opioid receptor agonist endomorphin-1, endomorphin-2, or [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) was compared between endomorphin-1- and endomorphin-2-induced antinociception with the tail-flick test. A 2-h pretreatment with endomorphin-1 (30 nmol) produced a 3-fold shift to the right in the dose-response curve for endomorphin-1. Similarly, a 1-h pretreatment with endomorphin-2 (70 nmol) caused a 3.9-fold shift to the right for endomorphin-2. In cross-tolerance experiments, pretreatment with endomorphin-2 (70 nmol) caused a 2.3-fold shift of the dose-response curve for endomorphin-1, whereas pretreatment with endomorphin-1 (30 nmol) caused no change of the endomorphin-2 dose-response curve. Thus, mice acutely tolerant to endomorphin-1 were not cross-tolerant to endomorphin-2, although mice made tolerant to endomorphin-2 were partially cross-tolerant to endomorphin-1; an asymmetric cross-tolerance occurred. Pretreatment with DAMGO 3 h before intracerebroventricular injection of endomorphin-1, endomorphin-2, or DAMGO attenuated markedly the antinociception induced by endomorphin-1 and DAMGO but not endomorphin-2. It is proposed that two separate subtypes of mu-opioid receptors are involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. One subtype of opioid mu-receptors is stimulated by DAMGO, endomorphin-1, and endomorphin-2, and another subtype of mu-opioid receptors is stimulated solely by endomorphin-2.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Oligopeptides; Pain; Pain Measurement; Time Factors

The alpha(2) adrenergic receptor (AR) class of catecholamine/imidazoline (I) agonists, such as norepinephrine and clonidine, produce spinal antinociceptive synergy when co-administered with opioids. We have observed that intrathecally administered moxonidine, a selective I(1)/alpha(2) (AR) agonist, produces antinociception. The present experiments tested moxonidine for ability to synergize with morphine, deltorphin II, and DAMGO (Tyr-D-Ala-NMe-Phe-Gly(ol)) to inhibit substance P-elicited nociceptive behavior in Institute of Cancer Research mice. Moxonidine, morphine, deltorphin II, and DAMGO inhibited substance P-elicited nociceptive behavior with full efficacy. Effective dose 50% (ED(50)) values were calculated and equi-effective dose ratios of the combinations moxonidine-morphine, moxonidine-deltorphin II, and moxonidine-DAMGO were determined. The interactions were tested by isobolographic analysis. The observed ED(50) values of the combinations were statistically compared against their respective calculated theoretical additive ED(50) values. The combinations of moxonidine-morphine and moxonidine-deltorphin II resulted in significant leftward shifts in the dose-response curves compared to those of each agonist administered separately. The ED(50) values of the dose-response curves of these combinations were significantly less than the corresponding calculated theoretical additive ED(50) values; these results indicated that moxonidine synergizes with both morphine and deltorphin II. In contrast, combining moxonidine with DAMGO did not increase the potencies of the agonists (in combination) when compared to the potencies of each agonist administered separately. These results indicated that the moxonidine-DAMGO interaction is subadditive. Collectively, these data demonstrate that moxonidine combined with some opioid agonists produces spinal antinociceptive synergy. Spinally administered moxonidine-opioid combinations may prove an effective therapeutic strategy to manage pain.

Topics: Adrenergic alpha-2 Receptor Agonists; Aggression; Animals; Antihypertensive Agents; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Imidazoles; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Morphine; Oligopeptides; Pain; Spinal Cord; Substance P

We have previously shown that activation of kappa opioid receptors within the rostral ventral medulla in lightly anesthetized rats has an anti-mu opioid analgesic action in male rats. Microinjections of the kappa opioid receptor agonist, U69593, attenuated the increase in tail-flick latency produced by activation of mu opioid receptors located within the ventrolateral periaqueductal gray. There are sex differences in the pain modulating potency of opioid analgesics, including kappa opioid agonists. In the present study, we examined whether activation of kappa opioid receptors within the rostral ventral medulla in lightly anesthetized female rats produces an anti-mu opioid analgesic effect similar to that found in males. We found that in the RVM the same dose of kappa opioid receptor agonist that reduces mu receptor-mediated increase in tail-flick latency in male rats produces a moderate increase in tail-flick latency in female rats. Additionally, we discovered that female rats are significantly more sensitive to the mu opioid agonist, DAMGO, injected into the ventrolateral periaqueductal gray. The results indicate that these two brain structures, which mediate the analgesic effects of opioids, are sexually dimorphic with regard to opioid receptor function.

Topics: Analgesics, Opioid; Animals; Brain Stem; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Injections; Male; Morphine; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sex Characteristics; Time Factors

Previous in vitro studies showed that the degradation of dynorphin-(1-8) [dyn-(1-8)] by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon. In the present investigations, effects of the three PIs on the anti-nociception induced by the intra-third-ventricular (i.t.v.) administration of dyn-(1-8) were examined. The inhibitory effect of dyn-(1-8) on the tail-flick response was increased more than 100-fold by the i.t.v. pretreatment of rats with the three PIs. The inhibition produced by dyn-(1-8) in rats pretreated with any combination of two PIs was significantly smaller than that in rats pretreated with three PIs, indicating that any residual single peptidase could inactivate significant amounts of dyn-(1-8). The antagonistic effectiveness of naloxone, a relatively selective mu-opioid antagonist, indicates that dyn-(1-8)-induced inhibition of tail-flick response in rats pretreated with three PIs is mediated by mu-opioid receptors. Furthermore, mu-receptor-mediated inhibition induced by dyn-(1-8) was significantly greater than that produced by [Met5]-enkephalin in rats pretreated with three PIs. The data obtained in the present investigations together with those obtained in previous studies strongly indicate that dyn-(1-8) not only has well-known kappa-agonist activity but also has high mu-agonist activity.

Topics: Analgesics, Opioid; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Bacterial Agents; Captopril; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glycopeptides; Injections, Intraventricular; Male; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Peptide Fragments; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Receptors, Opioid, mu

The effect of (+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5, 10-imine maleate (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) injected intrathecally (i.t.) on the inhibition of the tail-flick response induced by morphine, D-Ala(2)-NmePhe(4)-Gly-ol-enkephalin (DAMGO), beta-endorphin, D-Pen(2,5)-enkephalin (DPDPE), or ¿(trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide)¿ (U50, 488H) administered i.t. was studied in ICR mice. The i.t. injection of MK-801 (2 microg) or CNQX (1 microg) alone did not affect the basal tail-flick response. Morphine (0.2 microg), DAMGO (0.8 ng), beta-endorphin (0.1 microg), DPDPE (0.5 microg) or U50, 488H (6 microg) caused only slight inhibition of the tail-flick response. CNQX injected i.t., but not MK-801, enhanced the inhibition of the tail-flick response induced by i.t. administered morphine, DAMGO, DPDPE or U50, 488H. However, CNQX or MK-801 injected i.t. was not effective in enhancing the inhibition of the tail-flick response induced by beta-endorphin administered i.t. The potentiating effect of CNQX on tail-flick inhibition induced by morphine, DAMGO, DPDPE or U50, 488H was blocked by naloxone (from 1 to 20 microg), yohimbine (from 1 to 20 microg) or methysergide (from 1 to 20 microg) injected i.t. in a dose-dependent manner. Our results suggest that the blockade of AMPA/kainate receptors located in the spinal cord appears to be involved in enhancing the inhibition of the tail-flick response induced by stimulation of spinal mu-, delta-, and kappa-opioid receptors. Furthermore, this potentiating action may be mediated by spinal noradrenergic and serotonergic receptors. However, N-methyl-D-aspartate receptors may not be involved in modulating the inhibition of the tail-flick response induced by various opioids administered spinally.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics, Opioid; Animals; beta-Endorphin; Dizocilpine Maleate; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Pain; Spinal Cord; Time Factors

Exposure to stressful or fear-inducing environmental stimuli activates descending antinociceptive systems resulting in a decreased pain response to peripheral noxious stimuli. Stimulating mu opioid receptors in the basolateral nucleus of the amygdala (BLA) in anesthetized rats produces antinociception that is similar to environmentally induced antinociception in awake rats. Recent evidence suggests that both forms of antinociception are mediated via projections from the amygdala to the ventral periaqueductal gray (PAG). In the present study, we examined the types of neurochemicals released in the ventral PAG that may be important in the expression of antinociception produced by amygdala stimulation in anesthetized rats. Microinjection of a mu opioid receptor agonist into the BLA resulted in a time dependent increase in tail flick latency that was attenuated by preadministration of a mu opioid receptor or a neurotensin receptor antagonist into the ventral PAG. Microinjection of a delta(2) opioid receptor antagonist or an NMDA receptor antagonist into the ventral PAG was ineffective. These findings suggest that amygdala stimulation produces antinociception that is mediated in part by opioid and neurotensin release within the ventral PAG.

Topics: Amygdala; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Naltrexone; Narcotic Antagonists; Neural Pathways; Neurons; Neurotensin; Nociceptors; Pain; Peptides; Periaqueductal Gray; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotensin; Receptors, Opioid, delta; Receptors, Opioid, mu; Time Factors; Valine

kappa-opioid receptor agonists (kappa-ORAs) have been shown to modulate visceral nociception through an interaction with a peripheral, possibly novel, kappa-opioid-like receptor. We used in the present experiments an antisense strategy to further explore the hypothesis that kappa-ORA effects in the colon are produced at a site different from the cloned kappa-opioid receptor (KOR). An antisense oligodeoxynucleotide (ODN) to the cloned rat KOR was administered intrathecally (12.5 microg, twice daily for 4 d) to specifically knock-down the cloned KOR. Efficacy of the KOR antisense ODN treatment was behaviorally evaluated by assessing the antinociceptive effects of peripherally administered kappa- (EMD 61, 753 and U 69,593), mu- (DAMGO) and delta- (deltorphin) ORAs in the formalin test. Intrathecal antisense, but not mismatch ODN blocked the actions of EMD 61,753 and U 69,593 without affecting the actions of DAMGO or deltorphin; a complete recovery of antinociceptive actions of the kappa-ORA EMD 61,753 was observed 10 d after the termination of antisense ODN treatment. In contrast, the ability of EMD 61,753 to dose-dependently attenuate responses of pelvic nerve afferent fibers to noxious colonic distension was unaffected in the same rats in which the antisense ODN effectively knocked-down the KOR as assessed in the formalin test. Additionally, Western blot analysis demonstrated a significant downregulation of KOR protein in the L4-S1 dorsal root ganglia of antisense, but not mismatch ODN-treated rats. The present results support the existence of a non-kappa-opioid receptor site of action localized in the colon.

Topics: Acetamides; Analgesics, Opioid; Animals; Colon; Disinfectants; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Formaldehyde; Ganglia, Spinal; Male; Nociceptors; Oligonucleotides, Antisense; Oligopeptides; Pain; Physical Stimulation; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Rectum; Visceral Afferents

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

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.

Topics: Analgesics; Analgesics, Opioid; Animals; Autoantibodies; Biological Transport; Brain Chemistry; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Epitopes; GTP-Binding Protein alpha Subunits, Gi-Go; Immunoblotting; Immunoglobulin G; Injections, Intraventricular; Iodine Radioisotopes; Male; Mice; Mice, Inbred Strains; Microscopy, Immunoelectron; Morphine; Neurons; Nociceptors; Oligopeptides; Pain; Protein Kinase C; Signal Transduction

We studied spinal analgesic and antiallodynic effects of endomorphin-1 and endomorphin-2 administered i.t. in comparison with Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO) or morphine, during acute, inflammatory and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5, 5, 10 microg i.t.) increased the tail-flick latency and, to the lesser extent, the paw pressure latency. The range of potencies in both those models of acute pain was as follows: DAMGO > morphine = endomorphin-1 > endomorphin-2. In a model of inflammatory pain, the number of formalin-induced flinching episodes was decreased by endomorphin-1. The effect of endomorphin-2 was much less pronounced. Both DAMGO and morphine significantly inhibited the pain-related behavior evoked by formalin. In a neuropathic pain model (sciatic nerve crushing in rats), endomorphin-1 and -2 (5 microg i.t.) had a statistically significant effect on the tail-flick latency and on the cold-water tail flick latency. Morphine, 5 microg, was found to be ineffective. Endomorphin-1 and -2 (2.5 and 5 microg i.t.) dose-dependently antagonized allodynia. Those effects of endomorphins were antagonized in acute (30 microg), inflammatory (30 microg) and neuropathic pain models (60 microg) by cyprodime, a selective mu-opioid receptor antagonist. In conclusion, our results show a strong analgesic action of endomorphins at the spinal cord level. The most interesting finding is a strong, stronger than in the case of morphine, antiallodynic effect of endomorphins in rats subjected to sciatic nerve crushing, which suggests a possible use of these compounds in a very difficult therapy of neuropathic pain.

Topics: Acute Disease; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Inflammation; Injections, Spinal; Male; Morphine; Pain; Rats; Spinal Cord

We investigated the role of mu-opioid receptor subtypes in both endomorphin-1 and endomorphin-2 induced antinociception in mice using supraspinally mediated behavior. With tail pressure as a mechanical noxious stimulus, both intracerebroventricularly (i.c.v.) and intrathecally (i.t.) injected-endomorphins produced potent and significant antinociceptive activity. Antinociception induced by i.t. and i.c.v. injection of endomorphin-1 was not reversed by pretreatment with a selective mu1-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.). By contrast, antinociception induced by i.t. and i.c.v. endomorphin-2 was significantly decreased by mu1-opioid receptor antagonist. Antinociception of both i.t. and i.c.v. endomorphin-1 and -2 was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg, s.c.). The results indicate that endomorphins may produce antinociception through the distinct mu1 and mu2 subtypes of mu-opioid receptor.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptors; Oligopeptides; Pain; Receptors, Opioid, mu; Time Factors

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.

Topics: Animals; Brain; Calcium Channels; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Ganglia, Spinal; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Isoenzymes; Membrane Potentials; Mice; Mice, Knockout; Morphine; Neurons, Afferent; Pain; Phospholipase C beta; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Type C Phospholipases

The aim of this paper is to study the influence of salmon calcitonin (SCT) on opioid analgesia when opioid transduction pathways are functionally uncoupled from Gi/o proteins by treatment with pertussis toxin (PTX). The antinociceptive effect of morphine and three selective opioid agonists, [D-Ala2,N-Me-Phe2,Gly5-ol]enkephalin (DAMGO) (OP(3-mu receptor agonist), [D-Pen2.5]-enkephalin (OP-1-delta receptor agonist) and trans-( +/- )-3,4-dichloro-N-methyl-N-[2-1(-pyrrolidinyl)-cyclohexyl]-benzene-acetam ide methane sulfonate (U-50, 488H) (OP1-kappareceptor agonist) was evaluated, using the tail flick test, in mice treated with PTX or with PTX and SCT. PTX blocked the antinociceptive effect of the opioids, being the antinociception similar in control animals and in mice treated with PTX and SCT. Thus, SCT prevents the effect of the blockade of Gi/o-proteins. From this it could be suggested that calcitonin activates alternative antinociceptive mechanisms that are not dependent on Gi/o-proteins.

Topics: Analgesia; Analgesics; Analgesics, Opioid; Animals; Calcitonin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Male; Mice; Morphine; Pain; Pertussis Toxin; Receptors, Opioid; Uncoupling Agents; Virulence Factors, Bordetella

We investigated behavioral responses to noxious cold and heat stimuli in mice. Similar to the hot-plate test, mice showed licking or jumping responses on a cold-plate (0 degrees C). The sensitivity to noxious heat (55 degrees C) was not correlated to the sensitivity to noxious cold, indicating that nociceptive processing of cold and heat are different. Behavioral responses to noxious cold are inhibited by systemic morphine or intrathecal administration of morphine. Lesion of the medial frontal cortex, including the anterior cingulate cortex, or selective activation of two types of opioid receptors in the anterior cingulate cortex produces dose-dependent antinociceptive effects on behavioral responses to noxious cold stimuli. These results suggest that activation of opioid receptors in the anterior cingulate cortex can produce powerful antinociception.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Cold Temperature; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Gyrus Cinguli; Hot Temperature; Injections, Spinal; Male; Mice; Mice, Inbred C57BL; Morphine; Narcotics; Nociceptors; Pain; Reaction Time

Using the in vivo whole cell recording procedure described previously, we recorded 73 neurons in laminae I and II in the lumbar spinal cord of the rat. Input impedances averaged 332 MOmega, which indicated that prior sharp electrode recordings contained a significant current shunt. Characterization of the adequate stimuli from the excitatory hindlimb receptive field indicated that 39 of 73 neurons were nociceptive, 6 were innocuous cooling cells, 20 responded maximally to brush, and 8 cells were not excited by stimulation of the skin of the hindlimb. The locations of 15 neurons were marked with biocytin. Nociceptive neurons were mostly found in lamina I and outer II, cooling cells in lamina I, and innocuous mechanoreceptive cells were mostly found in inner II or in the overlying white matter. The mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-Enkephalin (DAMGO) hyperpolarized 7 of 19 tested neurons with a conductance increase. This hyperpolarization was reversed by naloxone in the neurons in which it was applied. DAMGO also decreased the frequency of spontaneous PSPs in 13 neurons, 7 of which were also hyperpolarized by DAMGO. Five of the seven hyperpolarized neurons were nociceptive, responding to both heat and mechanically noxious stimuli, whereas two responded to slow, innocuous brush. These results indicate that whole cell, tight seal recordings sample a similar population of lamina I and II neurons in the rat as those found with sharp electrode recordings in cat and monkey. They further indicate that DAMGO hyperpolarizes a subset of the nociceptive neurons that have input from both heat and mechanical nociceptors and that presynaptic DAMGO effects can be observed in nociceptive neurons that are not hyperpolarized by DAMGO.

Topics: Animals; Cold Temperature; Electric Stimulation; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Histocytochemistry; Hot Temperature; Lysine; Membrane Potentials; Microelectrodes; Narcotics; Neurons; Pain; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Receptors, Opioid, mu; Receptors, Presynaptic; Spinal Cord; Substantia Gelatinosa; Synaptic Membranes

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.

Topics: Adrenergic alpha-Agonists; Analgesics, Opioid; Animals; Bicuculline; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Amino Acid Agonists; GABA Antagonists; Hot Temperature; Iontophoresis; Kainic Acid; Male; Medulla Oblongata; N-Methylaspartate; Naloxone; Narcotic Antagonists; Nociceptors; Opioid Peptides; Pain; Peptide Fragments; Peptides; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Somatostatin; Spinal Cord; Tail

There is abundant evidence that opioid receptors are present on peripheral terminals of primary afferent neurons. Experimental and clinical studies have shown that activation of these peripheral opioid receptors produces potent analgesia. In addition to peripheral opioid receptors, cholecystokinin receptors are present in sensory neurons. We examined the hypothesis that cholecystokinin receptors may be present on the same primary afferent neuron and that either exogenous or endogenous cholecystokinin may modulate peripheral antinociceptive effects of mu-opioid receptor agonists. Administration of cholecystokinin into inflamed paws, of the rat, but not intravenously attenuated peripheral antinociceptive effects induced by two mu-opioid receptor agonists, [D-Ala2,N-methyl-Phe4,Gly-ol5]-enkephalin and fentanyl. Only the desulphated form of cholecystokinin produced significant and dose-dependent attenuation. Cholecystokinin alone did not alter nociceptive baseline values in inflamed or non-inflamed paws. The anti-opioid effect of cholecystokinin was dose-dependently antagonized by the cholecystokininB receptor-selective antagonist L-365260, but not by the cholecystokininA receptor-selective antagonist L-364718. Local pretreatment with the protein kinase C specific inhibitor calphostin C abolished cholecystokinin's effect. Peripheral antinociceptive effects of [D-Ala2,N-methyl-Phe4,Gly-ol5]-enkephalin and fentanyl were not altered by intraplantar L-365260 alone. These results indicate that activation of peripheral cholecystokininB but not cholecystokininA receptors attenuates the local antinociceptive effects of mu-opioid receptor agonists in inflamed tissue. This anti-opioid effect may be mediated by protein kinase C in sensory nerve terminals. Endogenous cholecystokinin does not seem to influence the efficacy of peripheral opioids under both normal and inflammatory conditions.

Topics: Analgesics, Opioid; Animals; Cholecystokinin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Inhibitors; Fentanyl; Male; Naphthalenes; Pain; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Protein Kinase C; Rats; Rats, Wistar; Receptors, Cholecystokinin

The amygdala, periaqueductal gray (PAG), and rostral ventromedial medulla (RVM) are critical for the expression of some forms of stress-related changes in pain sensitivity. In barbiturate anesthetized rats, microinjection of agonists for the mu opioid receptor into the amygdala results in inhibition of the tail flick (TF) reflex evoked by radiant heat. We tested the idea that TF inhibition following opioid stimulation of the amygdala is expressed through a serial circuit which includes the PAG and RVM. Rats were anesthetized and prepared for microinjection of DAMGO (0.5 microg/0.25 microl) into the basolateral amygdala (BLA) and lidocaine HCl (2.5%/0.4-0.5 microl) into either the ventrolateral PAG or RVM. Lidocaine did not significantly alter baseline values for TF latency or TF amplitude. When injected into the PAG prior to DAMGO application in the BLA, lidocaine significantly attenuated DAMGO-induced antinociception for the entire 40 min testing session. Similar treatment in the RVM also resulted in an attenuation of antinociception although rats showed significant recovery of TF inhibition by 40 min after lidocaine injection. Since acute injection of lidocaine into the RVM also affected baseline heart rate, separate animals were prepared with small electrolytic lesions placed in the RVM. Chronic RVM lesions also blocked TF inhibition produced by amygdala stimulation but did not affect heart rate. These results, when taken together with similar findings in awake behaving animals, suggest that a neural circuit which includes the amygdala, PAG, and RVM is responsible for the expression of several forms of hypoalgesia in the rat.

Topics: Amygdala; Analgesics, Opioid; Anesthetics, Local; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Lidocaine; Male; Medulla Oblongata; Microinjections; Pain; Pain Measurement; Periaqueductal Gray; Rats; Receptors, Opioid, mu; Reflex; Stimulation, Chemical

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

Capsaicin produces burning pain, followed by nociceptive responses, such as allodynia and hyperalgesia in humans and rodents. In the present study, when administered subcutaneously into the tail of rhesus monkeys, capsaicin (0.01-0.32 mg) dose-dependently produced thermal allodynia manifested as reduced tail-withdrawal latencies in 46 degrees C water, from a maximum value of 20 sec to approximately 2 sec. Coadministration of selective mu opioid agonists, fentanyl (0.003-0.1 mg) and (D-Ala2,N-Me-Phe4, Gly5-ol)-enkephalin (0.001-0.03 mg), dose-dependently inhibited capsaicin-induced allodynia. This local antinociception was antagonized by small doses of opioid antagonists, quadazocine (0.03 mg) and quaternary naltrexone (1 mg), applied locally in the tail. However, these doses of antagonists injected s.c. in the back did not antagonize local fentanyl. Comparing the relative potency of either agonist or antagonist after local and systemic administration confirmed that the site of action of locally applied mu opioid agonists is in the tail. These results provide evidence that activation of peripheral mu opioid receptors can diminish capsaicin-induced allodynia in primates. This experimental pain model could be a useful tool for evaluating peripherally acting antinociceptive agents without central side effects and enhance new approaches to the treatment of inflammatory pain.

Topics: Animals; Azocines; Capsaicin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Hot Temperature; Macaca mulatta; Male; Naltrexone; Pain; Receptors, Opioid, mu

The present experiments evaluated the influence of intraventricular mu and delta opioid receptors on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Defeat stress consisted of: (1) an aggressive confrontation in which the experimental intruder rat exhibited escape, defensive and submissive behaviors [i.e., upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident stimulus rat with a wire mesh screen for 10-20 min. Defeat stress was immediately followed by an experimental session with tactile startle (20 psi). The mu opioid receptor agonists morphine (0.1-0.6 microg i.c.v.) and [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO; 0.01-0.3 microg i.c.v.), and the delta opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE; 10-100 microg i.c.v.) dose-dependently decreased startle-induced USV and increased tail-flick latencies in socially inexperienced and defeated rats. Of greater interest, morphine, DAMGO and DPDPE increased the occurrence of the submissive crouch posture, and defeated rats were more sensitive than socially inexperienced rats to the startle-induced USV-suppressive and antinociceptive effects of morphine and DPDPE. The antinociceptive effects of DAMGO were likewise obtained at lower doses in defeated rats. Finally, the USV-suppressive effects of morphine and DAMGO were reversed with the mu receptor antagonist naltrexone (0.1 mg/kg i.p.), but the USV-suppressive effects produced by DPDPE were not reversed with the delta receptor antagonist naltrindole (1 mg/kg i.p.). These results confirm mu, but not delta opioid receptor activation as significant in affective vocal, passive-submissive behavior, as well as reflexive antinociception. Furthermore, similar to previous studies with restraint and electric shock stress, the facilitation of mu opioid effects on vocal responses and antinociception is consistent with the proposal that defeat stress activated endogenous opioid mechanisms.

Topics: Aggression; Analgesics; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Morphine; Narcotic Antagonists; Narcotics; Pain; Pain Threshold; Rats; Rats, Long-Evans; Receptors, Opioid, delta; Receptors, Opioid, mu; Reflex; Social Behavior; Stress, Psychological; Vocalization, Animal

Tetanic stimulation of high-threshold primary afferent fibers in the dorsal root was found to elicit intrinsic optical signals (IOSs) in transverse slices of 11- to 20-day-old rat spinal cords. The IOS, lasting for 30 s or longer, was most prominent in the lamina II of the dorsal horn. Treatment with a Na+-K+-2Cl- co-transport blocker, furosemide, abolished the IOS, suggesting that the origin of the IOS is the cellular swelling due to an activity-dependent rise in extracellular K+. Substance P antagonist spantide, glutamate antagonists 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, and the mu-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin suppressed IOSs. Thus, IOSs represent at least in part the slow excitatory response that is known to be generated in dorsal horn neurons after tetanic activation of unmyelinated afferent fibers.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Analgesics; Analgesics, Opioid; Animals; Coloring Agents; Diuretics; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Furosemide; Ganglia, Spinal; Neurons, Afferent; Pain; Rats; Substance P; Substantia Gelatinosa

Ligation injury of the L5/L6 nerve roots in rats produces behavioral signs representative of clinical conditions of neuropathic pain, including tactile allodynia and thermal and mechanical hyperalgesia. In this model, intrathecal morphine shows no antiallodynic activity, as well as decreased antinociceptive potency and efficacy. This study was designed to explore the antinociceptive activity of intrathecal clonidine alone or in combination with intrathecal morphine (1:3 fixed ratio) in nerve-injured rats. The aims, with this study, were to use nerve-injured animals to determine: (1) whether the antinociceptive potency and efficacy of intrathecal clonidine was altered, and (2) whether the combination of intrathecal morphine and clonidine would act synergistically to produce antinociception.. Unilateral nerve injury was produced by ligation of the L5 and L6 spinal roots of male Sprague-Dawley rats. Sham-operated rats underwent a similar surgical procedure but without nerve ligation. Morphine and clonidine were given intrathecally through implanted catheters alone or in a 1:3 fixed ratio. Nociceptive responses were measured by recording tail withdrawal latency from a 55 degrees C water bath, and data were calculated as % maximal possible effect (%MPE).. Morphine produced a dose-dependent antinociceptive effect in both sham-operated and nerve-injured rats. The doses calculated to produce a 50 %MPE (i.e., A50) (+/-95% confidence intervals [CI]) were 15 +/- 4.9 micrograms and 30 +/- 18 micrograms, respectively. Though morphine was able to produce a maximal response (100%) in sham-operated rats, the maximal response achieved in nerve-injured animals was only 69 +/- 21.9 %MPE. Clonidine produced a dose-dependent effect, with an A50 (+/-95% CI) of 120 +/- 24 micrograms in sham-operated rats. In nerve-ligated rats, clonidine produced a maximal effect that reached a plateau of 55 +/- 10.9 %MPE and 49 +/- 10.2 %MPE at 100 and 200 micrograms, respectively, preventing the calculation of an A50. In sham-operated rats, a morphine-clonidine mixture produced maximal efficacy, with an A50 (+/-95% CI) of 15 +/- 9.2 micrograms (total dose), significantly less than the theoretical additive A50 of 44 +/- 10 micrograms. In L5/L6 nerve-ligated rats, the morphine-clonidine combination produced maximal efficacy, with an A50 (+/-95% CI) of 11 +/- 5.4 micrograms (total dose), which was significantly less than the theoretical additive A50 of 118 +/- 73 micrograms, indicating a synergistic antinociceptive interaction. The intrathecal injection of [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) produced A50 values of 0.23 microgram (range, 0.09-0.6) and 0.97 microgram (range, 0.34-2.7) in sham-operated and ligated rats, respectively. Phentolamine (4 mg/kg, intraperitoneally) produced no antinociceptive effect alone and attenuated, rather than enhanced, the effect of morphine in both groups of rats.. These data show that: (1) clonidine, like morphine, loses antinociceptive potency and efficacy after nerve ligation injury, and (2) strongly suggest that a spinal combination of morphine and clonidine synergize under conditions of nerve injury to elicit a significant antinociceptive action when either drug alone may be lacking in efficacy. It is unlikely that the synergy of morphine with clonidine is due to an attenuation of spinal sympathetic outflow by clonidine, because the sympatholytic agent phentolamine produced an opposing effect on morphine antinociception. The data suggest that combinations of morphine and clonidine may prove useful in controlling pain in patients with neuropathic conditions.

Topics: Adrenergic alpha-Agonists; Analgesics; Animals; Clonidine; Dose-Response Relationship, Drug; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraperitoneal; Injections, Spinal; Male; Morphine; Pain; Phentolamine; Rats; Rats, Sprague-Dawley; Spinal Nerve Roots

The effects of enantiomorphs of TAN-67 (2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino[2,3,3-g]isoquinoline), (-)TAN-67 and (+)TAN-67, given intrathecally (i.t.) on antinociceptive response with the tail-flick test were studied in male ICR mice. (-)TAN-67 at doses from 17.9 to 89.4 nmol given i.t. produced a dose- and time-dependent inhibition of the tail-flick response, whereas its enantiomer (+)TAN-67 even at smaller doses (1.8, 4.5 and 8.9 nmol) given i.t. decreased the latencies of the tail-flick response. In addition, (+)TAN-67 at higher doses (17.9-89.4 nmol) given i.t. produced scratching and biting pain-like responses. The antinociceptive response induced by i.t.-administered (-)TAN-67 was mediated by the stimulation of delta-1 but not by delta-2, mu or kappa opioid receptors, because the effect was blocked by the i.t. pretreatment with BNTX, but not by naltriben, [D-Phe-Cys-Tyr-[D-Try-Orn-Thr-Pen-Thr-NH2 or nor-binaltorphimine dihydrochloride. Pretreatment with (-)TAN-67 given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of (-)TAN-67 and by [D-Pen2,5]enkephalin (DPDPE). However, the tail-flick inhibition induced by [D-Ala2]deltorphin II, [D-Ala2,NMePhe4,Gly5-ol]enkephalin and U50,488H were not affected by (-)TAN-67 pretreatment. Conversely, pretreatment with DPDPE given i.t. 3 hr earlier attenuated the tail-flick inhibition induced by subsequent i.t. administration of (-)TAN-67 and by DPDPE. However, the tail-flick inhibition induced by [D-Ala2]deltorphin II was not affected by i.t. DPDPE pretreatment. It is concluded that (-)TAN-67 given i.t. produces delta-1 opioid receptor-mediated antinociception; on the other hand, its enantiomer (+)TAN-67 produces hyperalgesia. Present studies provide other evidence that delta-1 opioid receptors exist separated from delta-2 opioid receptor.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Hyperalgesia; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Pain; Pain Measurement; Pyrrolidines; Quinolines; Reaction Time; Receptors, Opioid, delta; Spinal Cord

The changes in the latency for tail withdrawal in response to noxious heating of the skin induced by microinjection of opioid or serotonergic agonists into the anterior pretectal nucleus (APtN) was studied in rats. The mu-opioid agonist DAMGO (78 and 156 picomol), but not the delta-opioid agonist DADLE (70 and 140 pmol), the kappa-opioid agonist bremazocine (0.24 and 0.48 nanomol) or the sigma-opioid agonist N-allylnormetazocine (0.54 nanomol), produced a dose-dependent antinociceptive effect. The 5-HT1 agonist 5-carboxamidotryptamine (19 and 38 nanomol) and the 5-HT1B agonist, CGS 12066B (1.12 and 2.24 nanomol), but not the non-selective 5-HT agonist m-CPP (41 to 164 nanomol), 5-HT2 agonist alpha-methylserotonin (36 and 72 nanomol) and 5-HT3 agonist 2-methylserotonin (36 and 72 nanomol), produced a dose-dependent antinociceptive effect. These results indicate that the antinociceptive effects of opioid or serotonergic agonists microinjected into the APtN depend on drug interaction with local mu or 5-HT1B receptors, respectively.

Topics: Analgesics; Animals; Benzomorphans; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Microinjections; Pain; Phenazocine; Piperazines; Quinoxalines; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Serotonin; Serotonin Receptor Agonists

Microinjections of excitatory amino acids made into the ventrolateral midbrain periaqueductal gray of the rat have revealed that neurons in this region integrate a reaction characterised by quiescence, hyporeactivity, hypotension and bradycardia. Microinjections of both excitatory amino acids and opioids into the ventrolateral periaqueductal gray have shown also that it is a key central site mediating analgesia. The effects of injections of opioids into the ventrolateral periaqueductal gray on arterial pressure and heart rate or behaviour are unknown. In this study we first mapped in the rat the extent of the ventrolateral periaqueductal gray hypotensive region as revealed by microinjections of excitatory amino acids. We found that ventrolateral periaqueductal gray depressor region extended more rostrally than previously thought into the tegmentum ventrolateral to the periaqueductal gray. Subsequently we studied for the first time, the effects of microinjections of mu-, delta-, and kappa-opioid agonists made into the ventrolateral periaqueductal grey depressor region. In contrast to the effects of excitatory amino acid injections, microinjections of the mu-opioid agonist ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin) evoked hypertension and tachycardia at approximately 50% of sites. Similar to excitatory amino acid injections, microinjections of both the delta-opioid agonist ([D-Pen2,D-Pen5]enkephalin), and the kappa-opioid agonist ((5,7,8)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-y l]-benzeneacetamide) evoked either a hypotension and bradycardia, or had no effect. These results indicate that different opiate receptor subtypes are present on a distinct population of ventrolateral periaqueductal gray neurons, or at different ventrolateral periaqueductal gray synaptic locations (pre- or post-synaptic).

Topics: Analgesics; Animals; Benzeneacetamides; Blood Pressure; Bradycardia; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Excitatory Amino Acids; Heart Rate; Homocysteine; Hypertension; Hypotension; Male; Microinjections; Neural Inhibition; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Our study was designed to determine involvement of nitric oxide (NO) in the antinociception mediated by mu, delta and kappa opioid receptors in acute and prolonged pain in the rat spinal cord. The effect of intrathecally (i.t.) injected NO synthase inhibitors and opioid receptor agonists was evaluated in acute pain using a tail-flick and a paw pressure tests, and in prolonged pain by quantification the pain-related behavior after peripheral formalin injection. It was found that the neuronal NO synthase inhibitor 7-nitroindazole (50-400 microg), used in inactive doses, dose-dependently enhanced antinociception induced by morphine (0.5 microg) in the tail-flick and paw pressure. Moreover, coadministration of N(G)-nitro-L-arginine methyl ester (50 microg) another NO synthase inhibitor, with morphine (0.05-0.5 microg) as well as with specific agonists of mu ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin 0.1-2.5 ng) and delta ([D-Pen(2,5)]enkephalin 0.02-0.5 microg) opioid receptors, enhanced dose-dependent antinociception in the tail-flick and paw pressure. Coadministration of N(G)-nitro-L-arginine methyl ester with specific kappa opioid receptor agonist 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg), produced antinociception in the paw pressure only. Additionally, N(G)-nitro-L-arginine methyl ester (100 microg) profoundly potentiated the antinociception induced by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (0.5, 15 ng) and [D-Pen(2,5)]enkephalin (2, 10 microg) in the dose-related manner in the formalin test. N(G)-nitro-L-arginine methyl ester (100 microg) also enhanced the antinociception induced by 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg) but only at the last two time points of the second phase of the formalin test. These data show that inhibition of the spinal NO synthase potentiates the mu-, delta- and to a lesser extent, kappa-mediated spinal antinociception in both acute and prolonged pain.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Enzyme Inhibitors; Formaldehyde; Injections, Spinal; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Pain; Pyrrolidines; Rats; Rats, Wistar; Receptors, Opioid; Spinal Cord

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glycopeptides; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Jejunum; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Structure-Activity Relationship; Synaptosomes

he effect of intrathecally administered [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) on withdrawal latencies evoked by noxious heat applied to either cervical or lumbar dermatomes was studied in awake rats. Administration of DAMGO to the lumbar intrathecal space produces a dose-dependent suppression of withdrawals evoked by noxious thermal stimulation in either lumbar or cervical dermatomes. Administration of the same doses of DAMGO to the cervical spinal cord produces a suppression of withdrawals evoked by stimulation in cervical but not lumbar dermatomes. Control experiments provide evidence that the drugs administered intrathecally to either enlargement do not spread to the other enlargement.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Spinal; Lumbosacral Region; Male; Neck; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley

Several laboratories recently identified a 17 amino-acid peptide, termed "nociceptin" or "orphanin FQ (OFQ)", as the endogenous ligand for the LC132 (or "opioid receptor-like1") receptor. Taken together with the fact that the cellular effects of OFQ are to a large extent opioid-like, the close relationship between the LC132 receptor and known opioid receptors raised expectations that the behavioral effects of this peptide would resemble those of opioids. However studies of the role of OFQ in nociception have not provided a unified view. The aim of the present study was to use a combination of electrophysiological and pharmacological techniques to characterize the actions of OFQ in a brain region in which the circuitry mediating the analgesic actions of opioids has been relatively well characterized, the rostral ventromedial medulla (RVM). Single-cell recording was combined with opioid administration and local infusion of OFQ in the RVM of rats lightlyanesthetized with barbiturates. The tail flick reflex was used as a behavioral index of nociceptive responsiveness. Two classes of physiologically identifiable RVM neurons with distinct responses to opioids have been characterized. -cells are activated, although indirectly, by opioids, and there is strong evidence that this activation is crucial to opioid antinociception. -cells, thought to enable nociception, are directly inhibited by opioids. Cells of a third class, cells, do not respond to opioids and whether or not they have any role in nociceptive modulation remains an open question. OFQ infused within the RVM profoundly suppressed the firing of all classes of RVM neurons, blocking opioid-induced activation of -cells. The antinociceptive effects of a micro-opioid agonist infused at the same site were significantly attenuated in these animals. Those of systemically administered morphine, which can produce its antinociceptive effects by acting at a number of CNS sites, were not blocked by RVM OFQ. Inasmuch as activation of -cells can account for the antinociceptive action of opioids within the RVM, these results demonstrate that, at least within the medulla, OFQ can exert a functional "antiopioid" effect by suppressing firing of this cell class. However to the extent that antinociceptive and pronociceptive outflows from various brain regions involved in both transmission and modulation of nociception are active under different conditions, focal application of OFQ in different regions could potentially prod

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Medulla Oblongata; Morphine; Narcotic Antagonists; Neural Pathways; Nociceptin; Opioid Peptides; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid

The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor-binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta-funaltrexamine. Additionally, antisera to [Leu5]enkephalin and dynorphin also resulted in a significant increase in formalin-induced flinching, whereas antisera to [Met5]enkephalin had no effect. On the basis of significant increases in formalin-induced flinching produced by 1) receptor-selective doses of delta and kappa, but not mu, opioid antagonists and 2) antisera to [Leu5]enkephalin and dynorphin A, but not [Met5]enkephalin, these data suggest the presence of an opioid inhibitory tone which acts to limit the intensity of the pain signal. This tone appears to be mediated via activation of delta and kappa receptors, possibly by a [Leu5]enkephalin- and dynorphin-like substance, respectively.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Formaldehyde; Immune Sera; Male; Naloxone; Naltrexone; Narcotic Antagonists; Opioid Peptides; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa

In previous studies, we reported that supraspinally administered DAMGO, a mu-opioid agonist, produces a dose-related, naloxone-reversible inhibition of formalin-evoked pain behaviors and spinal cord Fos-like immunoreactivity (FLI) in the rat spinal cord. Although these results support the hypothesis that activation of supraspinal mu-opioid receptors produces antinociception by increasing the activity of bulbospinal inhibitory pathways, other studies suggest that supraspinal morphine decreases rather than increases descending inhibitory control. In the present study, we specifically examined the effect of intracerebroventricular (i.c.v.) injection of morphine in the rat. Supraspinal morphine produced a dose-related, naloxone-reversible inhibition of both formalin-evoked behaviors nd spinal cord FLI. Although the magnitude of the antinociception produced by i.c.v. morphine in the formalin test was significantly correlated with the numbers of FLI neurons in the spinal cord, the lowest dose of i.c.v. morphine tested (0.70 nmol) produced a significant reduction of FLI in the superficial laminae without producing behavioral antinociception, which is consistent with our hypothesis that noxious stimulus-evoked Fos expression in the superficial laminae is a poor predictor of the magnitude of pain behavior. These data support the hypothesis that the antinociceptive effects of supraspinally administered morphine result from an increase in descending inhibitory control.

Topics: Analgesics; Analgesics, Opioid; Animals; Area Under Curve; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Immunohistochemistry; Injections, Intraventricular; Male; Morphine; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Physical Stimulation; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Spinal Cord

The effects of local treatment with opioid receptor agonists on the early (0-10 min) and late (20-40 min) behavioural response and extravasation induced by intraplantar injection of 1% formalin in rats were examined. The mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) depressed pain behaviour in the late phase, and extravasation in both phases. The kappa-opioid receptor agonist trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] benzeneacetamide methanesulfonate (U50,488H) suppressed the behavioural response in both phases, but extravasation was enhanced in the early phase and not altered in the late phase. The delta-opioid receptor agonist [D-Pen2,5]enkephalin (DPDPE) enhanced the behavioural response in the late phase, but inhibited extravasation in the both early and late phases. Systemic injection of the agonists had no effects, and pretreatment with s.c. naloxone methiodide reversed the effects of locally administered agonists. These data (1) support the notion that different pathophysiological mechanisms underlie the two phases of the formalin test, and (2) indicate that depending on the receptor specificity, opioid receptor agonists have both pro- and antinociceptive effects, as well as pro- and antiinflammatory activity.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Extravasation of Diagnostic and Therapeutic Materials; Formaldehyde; Inflammation; Injections, Intraperitoneal; Injections, Subcutaneous; Male; Narcotics; Pain; Pyrrolidines; Rats; Receptors, Opioid

Studies in mice and rats have shown that antinociception produced by intrathecal (i.t.) administration of opioids can be partially inhibited by intracerebroventricular (i.c.v.) administration of naloxone. In this study we tested the hypothesis that this inhibition by i.c.v. naloxone results from antagonism of supraspinal endogenous opioid-mediated antinociception produced by the action of i.t. opioids on an ascending antinociceptive pathway. In rats lightly anesthetized with urethane/alpha-chloralose, i.t. DAMGO, i.t. lidocaine, or spinal transection at T5-T6 all attenuated the trigeminal jaw opening reflex (JOR) (i.e., were antinociceptive), an effect that was antagonized in each case by i.c.v. naloxone. These findings support the suggestion that there exists a pathway that ascends from the spinal cord to a supraspinal site that tonically inhibits antinociception mediated by supraspinal opioids. When activity in this ascending pathway is suppressed (e.g., by i.t. opioids or local anesthetics or by spinal cord transection), antinociception mediated by supraspinal opioids is disinhibited. To determine the supraspinal site(s) at which endogenous opioid-dependent antinociception is evoked by i.t. opioids, we microinjected naloxone methiodide into several supraspinal sites. Microinjection of naloxone methiodide into nucleus accumbens but not into the rostral ventral medulla (RVM) or the periaqueductal gray matter (PAG) antagonized the suppression of the JOR produced by i.t. DAMGO or lidocaine. The possibility that this ascending pathway may represent a source of spinal input to mesolimbic circuitry involved in setting the state of sensorimotor reactivity to noxious stimuli is discussed.

Topics: Analgesics; Animals; Brain Stem; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Lidocaine; Male; Mice; Microinjections; Models, Neurological; Naloxone; Pain; Rats; Rats, Sprague-Dawley; Reflex; Spinal Cord; Synapses; Trigeminal Nerve

Spinally administered mu opioid agonists produce potent antinociception and inhibition of gastrointestinal transit. Blockade of 5-hydroxytryptamine (5-HT) or norepinephrine (NE) uptake potentiates intrathecal (i.t.) DAMGO antinociception. To determine whether 5-HT and NE uptake blockade will also potentiate the gastrointestinal inhibition, mice were treated with zimelidine, desipramine or saline, followed by i.t. DAMGO and tested for tailflick antinociception or inhibition of gastrointestinal transit. DAMGO produced antinociception dose-dependently (ED50 = 4.6 ng). Zimelidine (10 mg/kg, s.c., 1 hr before DAMGO) produced a 6.2-fold leftward shift in the antinociceptive dose-response curve (ED50 = 0.73 ng). Desipramine produced a 5.3-fold shift (ED50 = 1.4 ng). DAMGO also produced a dose-dependent inhibition of gastrointestinal transit (ED50 = 117 ng). However, zimelidine or desipramine treatment did not affect DAMGO inhibition of gastrointestinal transit (ED50 = 80 ng.).

Topics: Animals; Desipramine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gastrointestinal Motility; Injections, Spinal; Male; Mice; Norepinephrine; Pain; Receptors, Opioid, mu; Serotonin; Zimeldine

Intrathecal pretreatment of mice with an antisense oligodeoxynucleotide directed against the kappa-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as delta 9-THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 or delta 9-THC. Furthermore, kappa-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using kappa-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Base Sequence; Dronabinol; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Oligonucleotides, Antisense; Pain; Psychotropic Drugs; Pyrrolidines; Receptors, Opioid, kappa; Spinal Cord

The antinociceptive efficacy of different opioid-receptor agonists following their intrathecal (i.t.) administration was examined in awake, unanesthetized rats in a model of visceral pain. Cumulative i.t. doses of the mu-preferring opioid-receptor agonist morphine produced dose-dependent attenuation of the change (increase) in mean arterial pressure (delta MAP) and elevation of the visceromotor threshold to colorectal distension (CRD). Similar dose-dependent antinociceptive effects were produced after i.t. administration of the mu opioid-receptor-selective agonist DAMPGO. Morphine and DAMPGO were equipotent against the delta MAP to phasic CRD (80 mm Hg, 20 sec), but DAMPGO was more than 6 times more potent than morphine in elevating the visceromotor threshold to an incrementing CRD. Intrathecal administration of the delta opioid-receptor-selective agonist DPDPE produced, like morphine and DAMPGO, a dose-dependent attenuation of the delta MAP to CRD; DPDPE was one-tenth as potent as morphine or DAMPGO. DPDPE also dose-dependently elevated the visceromotor threshold to CRD, but its efficacy was only half that of morphine or DAMPGO. The kappa opioid-receptor-selective agonist U 50488H was without antinociceptive efficacy after i.t. administration, but did attenuate responses to CRD after systemic administration. The antinociceptive effects produced by morphine and DAMPGO were antagonized by i.t. pretreatment with naloxone and the effects produced by DPDPE were antagonized by i.t. pretreatment with the delta opioid-receptor-selective antagonist naltrindole. These data indicate that local mu and delta, but not kappa, opioid receptors can modulate visceral nociceptive transmission in the spinal cord.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Catheterization; Colon; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Morphine; Pain; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Rectum

Two cyclic analogues of the brain peptide Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) were synthesized and tested for analgesic activity in the rat tail flick test after intracerebroventricular (ICV) injection. The analogues were about 200-fold more potent than the parent peptide. Analgesia was dose dependent, and at 1 microgram the two analogues, the mu-selective enkephalin analogue DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol), and morphine, all produced analgesia lasting between 40 and 60 min. Analgesia of longer duration was evident at higher doses of the analogues and lasted more than 6 h after 100 micrograms, the highest dose tested. The results show that peptide analogues based on the structure of the endogenously occurring Tyr-W-MIF-1 can produce potent and long-lasting effects on nociception.

Topics: Analgesia; Analgesics; Animals; Cerebral Ventricles; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Male; Morphine; MSH Release-Inhibiting Hormone; Narcotic Antagonists; Pain; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Time Factors

We have studied prostaglandin E1-induced mechanical hyperalgesia in the rat hindpaw, by assessing paw-withdrawal thresholds, before and after injecting prostaglandin E1 alone or with other agents, in normal and streptozotocin-induced diabetic rats. In normal and diabetic rats, prostaglandin E1 (1-1000 ng) produced a dose-dependent decrease in mechanical nociceptive threshold. In diabetic rats, prostaglandin E1 was more potent than in normal rats, in producing hyperalgesia, whereas prostaglandin E2 hyperalgesia was not changed in normal and diabetic rats. Prostaglandin E1-induced hyperalgesia was not inhibited by E-type 1 prostaglandin receptor antagonists, SC19220 or SC51089, either in normal or diabetic rats. In fact, in the presence of SC19220, prostaglandin E1 produced enhanced hyperalgesia, in normal rats. Prostaglandin E1 hyperalgesia was not significantly modified by sympathectomy or indomethacin. Unlike prostaglandin E2, prostaglandin E1 hyperalgesia was not blocked by the inhibitor of the stimulatory guanine nucleotide-binding regulatory protein, guanosine 5'-O-(2-thiodiphosphate). It is suggested that prostaglandin E1 decreases primary afferent nociceptive threshold directly, by activating a prostaglandin receptor other than the E-type 1 prostaglandin receptor, and that this receptor is not coupled to a stimulatory guanine nucleotide-binding regulatory protein.

Topics: Alprostadil; Analgesics; Animals; Diabetes Mellitus, Experimental; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hindlimb; Hydrazines; Hyperalgesia; Male; Oxazepines; Pain; Rats; Rats, Sprague-Dawley

This study examined the influence of acute and repeated administration of the kappa-opioid receptor antagonist, nor-binaltorphimine, upon opioid-induced antinociception as measured by the tail-pressure test. A single intracerebroventricular (i.c.v.) injection of nor-binaltorphimine (30 micrograms) administered 1, 10 or 30 days prior to algesiometric testing prevented the analgesic effect of the kappa-opioid receptor agonist, (5 alpha, 7 alpha, 8 beta)-(-)-N- methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzenacet amide (U69593). The analgesic effect of the mu-opioid receptor agonist, [D-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin (DAMGO), and the delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), was not modified. In contrast, when nor-binaltorphimine was administered repeatedly (twice daily i.c.v. administration of 30 micrograms nor-binaltorphimine for 10 days), the analgesic effect of DAMGO, DPDPE as well as U69593 was abolished. In the case of mu- and delta-opioid receptor agonists, this abolition was apparent when testing occurred 1 or 2, but not 5 days after termination of nor-binaltorphimine treatment. This treatment regimen also resulted in a long-lasting antagonism (e.g. 20 days) of U69593-induced analgesia. These data show that, depending on the treatment regimen employed, nor-binaltorphimine can function as a selective kappa-opioid receptor antagonist, or as an antagonist at multiple opioid receptor subtypes. Further, they demonstrate that nor-binaltorphimine functions as a long-lasting kappa-opioid receptor antagonist in vivo.

Topics: Amino Acid Sequence; Analgesics; Animals; Benzeneacetamides; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Injections, Intraventricular; Male; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pain; Pain Threshold; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Somatostatin

Microinjection of [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO) into either the periaqueductal gray (PAG) or the rostral ventral medulla (RVM) elicits analgesia in the tailflick assay in the rat. Co-administration of DAMGO into both regions together results in a profound synergistic interaction similar to that we previously reported with morphine. U50,488H and DPDPE are inactive when given into either region. [D-Ala2,Glu4]Deltorphin (deltorphin), on the other hand, elicits an analgesic response, although the maximal response is less than than mu agonists. Co-administration of DAMGO into one region with deltorphin in the other also results in a significant synergy. However, co-administration of DAMGO and deltorphin together in the same region gives only additive effects. These results confirm the existence of mu/mu synergy between the PAG and RVM. kappa 1 and delta 1 agents are inactive, but the delta 2 agonist deltorphin is active in both regions. Our results indicate the presence of mu/delta 2 synergy between the PAG and RVM which appears to involve interactions of pathways rather than receptor interactions at the cellular level.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Analgesics; Animals; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Medulla Oblongata; Molecular Sequence Data; Oligopeptides; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu

Intracerebroventricular (i.c.v.) administration to mice of IgGs raised against alpha subunits of Gi2 or Gx/z transducer proteins lessened the activation of low Km GTPase induced by morphine, DAMGO and DADLE in P2 membranes from mouse periaqueductal grey matter (PAG). In mice injected with anti Gi2 alpha, DADLE, DPDPE and [D-Ala2] Deltorphin II, but not beta-endorphin-(1-31), antagonized the analgesic activity of morphine. Conversely, following anti Gx/z alpha, morphine antagonized the antinociceptive potency of DADLE. It is concluded that opioids display diverse efficacy at mu-Gi2 and mu-Gx/z complexes to produce supraspinal analgesia in mice.

Topics: Amino Acid Sequence; Analgesia; Analgesics, Opioid; Animals; Cell Membrane; Cerebral Ventricles; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; GTP Phosphohydrolases; GTP-Binding Proteins; Immunoglobulin G; Injections, Intraventricular; Kinetics; Male; Mice; Molecular Sequence Data; Morphine; Naloxone; Pain; Peptide Fragments; Receptors, Opioid, mu; Signal Transduction

Chlornaltrexamine (beta-CNA, 0.5 micrograms) alone or beta-CNA plus either mu-agonist, D-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO, 500 ng) or delta-agonist, D-Pen2-D-Pen5-enkephalin (DPDPE, 10 micrograms) was injected intrathecally (i.t.) to protect mu- or delta-opioid receptors, respectively, for 24 h in male ICR mice. The antinociception was assessed by the tail-flick and hot-plate test. DPDPE or DAMGO injected i.t. increased inhibition of the tail-flick and hot-plate response in a dose-dependent manner. The dose-response curve for tail-flick and hot-plate response induced by DPDPE or DAMGO in i.t. saline-treated group significantly shifted to the right in i.t. beta-CNA alone treated group but returned to the control level in the group treated with i.t. beta-CNA coadministered with DPDPE or DAMGO, respectively. The effects of protection of mu- and delta-opioid receptor in the spinal cord on inhibition of the tail-flick and hot-plate response induced by beta-endorphin and morphine administered intracerebroventricularly (i.c.v.) were then studied. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with DAMGO attenuated inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. However, i.t. treatment with beta-CNA coadministered with DPDPE did not affect inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with either DPDPE or DAMGO did not alter inhibition of the hot-plate response induced by beta-endorphin administered i.c.v.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Analgesics; Animals; beta-Endorphin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pain; Pain Measurement; Spinal Cord

These studies were designed to investigate how the aging process alters the spinal antinociceptive efficacy of mu (mu), delta (delta) and epsilon (epsilon) opioid receptor agonists administered intrathecally (i.t.) in rats. Various doses of the mu agonist DAGO, the delta agonist DPDPE or the putative epsilon beta-endorphin were injected i.t. in young (5-6-month-old), mature (15-16-month-old) and aged (25-26-month-old) Fischer 344 rats. Antinociception was measured using the rat tail-flick analgesiometric assay. The data demonstrated a decline in spinal opioid-induced antinociception as a function of age. For instance, the i.t. dose of DPDPE or beta-endorphin needed to produce antinociception in the 25-26-month-old rats was higher than that needed to elevate tail-flick latency in the young and mature animals. We also noted that the i.t. doses of the opioid agonists needed to produce 'antinociception' in the aged cohort were within a range of spinal doses that produced motor impairment. Apparently, the aging process alters the ability of opioid receptors to mediate antinociception. Perhaps an age-related decrease in the number and/or affinity of opioid receptor sites in the rat spinal cord accounts for these observations.

Topics: Aging; Analgesics; Analysis of Variance; Animals; beta-Endorphin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Pain; Rats; Rats, Inbred F344; Spine

U-50,488, (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide hydrochloride), is a selective kappa-opioid receptor agonist. In this study, we found that U-50,488 antagonized morphine-induced antinociception in morphine-naive guinea pigs at doses which did not have any antinociceptive effect by themselves (0.01-3 mg/kg). On the other hand, U-50,488 (3 mg/kg) partially restored morphine-induced antinociception in morphine-tolerant guinea pigs (8 mg/kg/day i.p. morphine HCl for 6 days). Furthermore, the development of tolerance to morphine antinociception was completely blocked by coadministration of U-50,488 at a very low dose (0.003 mg/kg i.p.) which neither exerted an antinociceptive effect by itself nor affected the antinociception induced by 8 mg/kg of morphine HCl. The withdrawal signs induced by 8 mg/kg (i.p.) naloxone HCl on the 7th day were also depressed by coadministration of 0.003 mg/kg U-50,488 with morphine HCl (8 mg/kg i.p.) every day for 7 days. These effects of U-50,488 could be applied to humans to prevent morphine tolerance and dependence.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Dose-Response Relationship, Drug; Drug Synergism; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Male; Morphine; Morphine Dependence; Nociceptors; Pain; Pyrrolidines; Receptors, Opioid, kappa

A polyclonal antiserum directed against the first 16 amino acids of the N-terminal sequence of the murine delta opioid receptor was raised in rabbits. The intracerebroventricular (i.c.v.) injection to mice of the anti delta receptor IgGs impaired the antinociception produced by DPDPE, [D-Ala2]-Deltorphin II, DADLE and beta-endorphin-(1-31) when studied 24 h later in the tail-flick test. Antinociception produced by morphine and DAMGO was fully expressed in mice undergoing this treatment. The selective delta antagonist ICI 174864 (0.8 nmols/mouse, i.c.v.) significantly reduced the antinociceptive activity of opioids to the extent observed after giving the antibodies. ICI 174864 did not decrease further the antinociception that remained after the anti delta receptor serum. The specific binding displayed by 3 nM [3H]-DPDPE was reduced in membranes pre-incubated with the antiserum, whereas no change could be detected for 0.6 nM [3H]-DAMGO labelling mu receptors. This experimental approach revealed the delta component of opioid-evoked supraspinal antinociception in mice.

Topics: Amino Acid Sequence; Analysis of Variance; Animals; beta-Endorphin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; GTP-Binding Proteins; Immunoglobulin G; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Molecular Sequence Data; Morphine; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Protein Binding; Reaction Time; Receptors, Opioid, delta

Intravenous (i.v.) administration of morphine produces a dose-dependent inhibition of the tail-flick (TF) reflex, depressor response, and bradycardia in the rat. Some of these effects depend on interactions of i.v. morphine with peripheral opioid receptors and the integrity of cervical vagal afferents. The present studies used the relatively specific mu, delta, and kappa opioid receptor agonists (DAGO, DPDPE or U-50,488H) and the relatively specific mu, delta, and kappa opioid receptor antagonists (beta-FNA, naloxonazine, naltrindole or nor-BNI) in either intact rats or rats with bilateral cervical vagotomy (CVAG) to delineate the vagal afferent/opioid-mediated components of these effects. I.v. administration of DAGO in intact rats produced a dose-dependent inhibition of the TF reflex, depressor response, and bradycardia virtually identical to those produced by i.v. morphine. All of these effects of either i.v. DAGO or i.v. morphine were significantly attenuated by either bilateral CVAG or pre-treatment with the mu 2 opioid receptor antagonist beta-FNA. Pre-treatment with the mu 1 opioid receptor antagonist naloxonazine affected i.v. DAGO-induced inhibition of the TF reflex and bradycardia, but had no significant effects on i.v. morphine-produced responses. I.v. administration of DPDPE produced a dose-dependent pressor response, but had no marked effects on the either the TF reflex or heart rate (HR). The pressor response was unaffected by either bilateral CVAG or pre-treatment with naltrindole, naloxone, hexamethonium, or bertylium. i.v. administration of U-50,488H produced a depressor response and bradycardia, but had no significant effect on the TF reflex. The depressor response and bradycardia produced by i.v. U-50,488H were unaffected by bilateral CVAG, but could be antagonized by pre-treatment with either nor-BNI or naloxone. These studies suggest that the vagal afferent-mediated antinociceptive and cardiovascular effects of i.v. morphine are primarily mediated by interactions with low affinity mu 2 opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analysis of Variance; Animals; Blood Pressure; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Heart Rate; Hexamethonium; Hexamethonium Compounds; Indoles; Injections, Intravenous; Male; Morphinans; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Pain; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reference Values; Time Factors; Vagotomy; Vagus Nerve

In recent studies, the superfusion of the corium side of the skin with inflammatory mediators failed to produce sensitization of nociceptors to mechanical stimuli. We have studied the effects of intradermal (i.d.) and subcutaneous (s.c.) injections of prostaglandin E2 (PGE2) and bradykinin (BK) in a behavioral model of hyperalgesia. PGE2 or BK was injected into the rat hind-paw, and paw-withdrawal thresholds in response to noxious mechanical stimulation before and after the drug were compared. Subcutaneous injection of PGE2 (1-1000 ng), a hyperalgesic inflammatory mediator, did not significantly alter paw-withdrawal thresholds, under the same conditions in which i.d. injections dose-dependently lowered paw-withdrawal thresholds. Similarly, BK (1-1000 ng), another hyperalgesic mediator, given s.c. failed to significantly alter paw-withdrawal thresholds while i.d. injections dose-dependently lowered paw-withdrawal thresholds. The prostaglandin E-type, EP1 receptor antagonist SC19220 (750 ng), given s.c. prior to PGE2 (i.d.) did not significantly change PGE2-induced hyperalgesia. However, SC19220 significantly attenuated PGE2 hyperalgesia when both were injected i.d. Also, s.c. administration of the mu-opioid antagonist, DAMGO, before PGE2 did not inhibit PGE2-induced hyperalgesia as opposed to i.d. injection. These results suggest that the inability of s.c. injection of PGE2 or BK to reach its receptor site on the terminals of primary afferent nociceptors may be responsible for the ineffectiveness of these hyperalgesic mediators to sensitize cutaneous nociceptors to mechanical stimuli in the rat and underscore the importance of the site of application and site of action of hyperalgesic agents in the study of hyperalgesic mechanisms.

Topics: Analgesics; Animals; Bradykinin; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Inflammation; Injections, Intradermal; Injections, Subcutaneous; Male; Pain; Pain Threshold; Rats; Rats, Sprague-Dawley

The antinociceptive potency of morphine and the morphine metabolite morphine-6-glucuronide (M6G) was examined after injection into the substantia nigra and periaqueductal gray (PAG) of rats. Both drugs produced antinociception in both sites. The antinociceptive potency of M6G was significantly greater than morphine in the nigra. There was no difference in the antinociceptive potency of M6G in the nigra and PAG. M6G and other opioids were also examined for motivational effects after intranigral injection. A high dose of intranigral morphine (10.0 nmol) produced a conditioned place preference. No significant motivational effects were produced by 1.0 nmol of M6G, D-Ala2, N-Me-Phe4,Gly5-ol-enkephalin (DAGO), D-Pen2,D-Pen5-enkephalin (DPDPE), or U-50,488H. It is concluded that the substantia nigra plays an important role in opioid antinociception. The role of the nigra in opioid reward is questionable.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Analysis of Variance; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Microinjections; Morphine; Morphine Derivatives; Motivation; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reward; Stereotaxic Techniques; Substantia Nigra; Time Factors

The antinociception of opiates is mediated through the activation of opioid receptors in several mid brain and brain stem areas. This paper reports that the forebrain area termed area tempestas (AT), first identified as a convulsant trigger area, is also a component of the endogenous pain suppression system. Unilateral AT application of DAMGO, morphine and U-50,488H in rats at doses in the nanogram range produced marked and dose-dependent increases in the latency to respond to nociceptive stimuli. A lower effect is found after application of DPDPE and DADLE. Antinociception is more evident in the hot plate than in the tail flick test. In the former test, the effect was restricted to the paws contralateral to the hemisphere of injection. Unilateral AT application of naltrexone (4 ng) reduced in the contralateral paws the antinociceptive effect that the bilateral AT application of morphine (20 ng/hemisphere) had induced in both body sides. Unilateral application of naltrexone, (20 ng) ICI 154, 129 (20 ng) and Win 44,441-3 (8 ng) antagonized the antinociceptive effect elicited by the systemic injection of morphine (2.5 mg/kg s), DPDPE (20 mg/kg s) and U-50,488H (20 mg/kg s), respectively. In the hot plate test, the antagonism was found in the paws ipsilateral and contralateral to the hemisphere of injection of the antagonists.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Anticonvulsants; Azocines; Bicuculline; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Escape Reaction; Foot; Hot Temperature; Male; Morphine; Naltrexone; Narcotic Antagonists; Olfactory Pathways; Pain; Phenazocine; Pressure; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid; Seizures; Tail

We examined whether streptozotocin-induced diabetes can modulate beta-endorphin-induced antinociception in mice. While beta-endorphin administered i.c.v. produced a dose-dependent inhibition of the tail-flick response in both diabetic and non-diabetic mice, the antinociceptive response was greater in diabetic mice than in non-diabetic mice. The ED50 value of beta-endorphin administered i.c.v. in diabetic mice was significantly lower than that in non-diabetic mice. The antinociceptive effects of beta-endorphin administered i.c.v. in both diabetic and non-diabetic mice were significantly antagonized by s.c. administration of naltrindole, a selective delta-opioid receptor antagonist. beta-Endorphin administered i.t. also produced a dose-dependent antinociception in both diabetic and non-diabetic mice. However, the ED50 value of kappa-opioid receptor antagonist. On the other hand, the antinociceptive potency of DPDPE, a selective delta-opioid agonist, administered i.t. is significantly increased in diabetic mice, as compared with non-diabetic mice, whereas, the antinociceptive potency of U-50,488H, a kappa-opioid receptor agonist, administered i.t. is significantly less than in non-diabetic mice. These results suggest that diabetes may modulate beta-endorphin-induced antinociception differently at the spinal and supraspinal levels.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; beta-Endorphin; Cerebral Ventricles; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Pain; Pyrrolidines; Reference Values

Two novel oxime derivatives of naltrexone, 6-[2-phenylethyl]-oximino naltrexone (NPC 831) and 6-[3-phenylpropyl]-oximino naltrexone (NPC 836) were potent agonists at opioid receptors. Both compounds inhibited binding to all three opioid receptor subtypes with nanomolar affinities. In vivo, NPC 831 and NPC 836 were equipotent to morphine and more potent than the kappa-selective agonist U-50,488H to produce analgesia. ED50 values of 4.02 mg/kg for NPC 831 and 2.24 mg/kg for NPC 836 were generated for inhibition of the tail-flick response in the rat, and ED50 values of 0.05 mg/kg for NPC 831 and 0.02 mg/kg for NPC 836 were calculated for inhibition of the writhing response in the mouse. Bombesin-induced scratching was used to evaluate NPC 831 and NPC 836 for kappa-agonist properties, and the A50, defined as the percent antagonism of the bombesin-induced response, was 1.86 mg/kg for NPC 831 and 0.08 mg/kg for NPC 836, compared to an A50 of 1.54 mg/kg for U-50,488H. These data suggest that NPC 831 and NPC 836 possess potent mu- and kappa-agonist properties in vivo, with NPC 836 being approximately twice as potent as NPC 831 to produce analgesia and 20 times as potent as NPC 831 to inhibit the scratching response produced by bombesin.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Acetates; Analgesics; Animals; Binding, Competitive; Bombesin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Guinea Pigs; Ligands; Male; Mice; Naltrexone; Oximes; Pain; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid; Receptors, Opioid, kappa

This study evaluated the antinociceptive effects produced when different combinations of supraspinal mu- and delta-opioid agonists were co-administered with spinal mu-, delta-, and kappa-opioid agonists. Using the Randall-Selitto paw-withdrawal test, in the rat, changes in nociceptive thresholds were measured following co-administration of sequentially increasing i.c.v. doses of either DAMGO or DPDPE with a low-antinociceptive dose of intrathecal DAMGO, DPDPE, or U50,488H. Antinociceptive synergy (i.e. a more than additive antinociceptive effect) was demonstrated with all of the combinations tested except for supraspinal DPDPE co-administered with spinal DAMGO. The results of this study provide support for the suggestion that supraspinal and spinal antinociceptive mechanisms share, in part, common neural circuits. Marked differences in the overall magnitude of the antinociceptive effects produced by the various combinations of opioid agonists were demonstrated through a secondary analysis of the data. When sequentially increasing i.c.v. doses of DAMGO were administered, significantly larger increases in nociceptive thresholds were observed with co-administration of intrathecal injections of low antinociceptive doses of either DAMGO or U50,488H compared to DPDPE. In contrast, when DPDPE was administered supraspinally, the largest increases in nociceptive thresholds were demonstrated with co-administration of DPDPE at the spinal site. The results of the secondary analysis provide support for the hypothesis that descending antinociceptive control systems activated by supraspinal administration of selective mu- and delta-opioid agonists interact, differently, with spinal mu-, delta-, and kappa-opioidergic mechanisms.

Topics: Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Male; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Spinal Cord

This study evaluated the antinociceptive effects of systemically administered selective opioid agonists of mu (DAMGO), delta (BUBU) and kappa (U 69593) receptors on the vocalization threshold to paw pressure in a rat model of peripheral unilateral mononeuropathy produced by loose ligatures around the common sciatic nerve. DAMGO (0.5-2 mg/kg), BUBU (1.5-6 mg/kg) and U 69593 (0.75-3 mg/kg) injected intravenously (i.v.) produced a potent long-lasting antinociceptive effect on both hind paws. The effects on the lesioned paw were clearly and statistically more potent than for the non-lesioned paw. The selective antinociceptive effect of 2 mg/kg DAMGO, 3 mg/kg BUBU and 1.5 mg/kg U 69593 were completely prevented by prior administration of the appropriate antagonists: 0.1 mg/kg naloxone, 1 mg/kg naltrindole and 0.4 mg/kg MR 2266. The present data clearly show that an acute i.v. injection of these selective opioid agonists induces potent antinociceptive effects in a rat model of peripheral neuropathy. These data are discussed with regard to the classical view that there is opioid resistance in neuropathic pain.

Topics: Analgesics; Animals; Benzeneacetamides; Benzomorphans; Disease Models, Animal; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vocalization, Animal

1. Much evidence in the literature supports the idea that cholecystokinin (CCK) interacts with opioids in pain mechanisms. In this work, we have investigated the supraspinal interactions between enkephalins and CCK, using the hot plate test in mice. 2. Intracerebroventricular (i.c.v.) administration of BDNL (a mixed CCKA/CCKB agonist) induced dose-dependent antinociceptive responses on both paw lick and jump responses. In contrast, using the same test, the i.c.v. injection of BC 264 (a selective CCKB agonist) induced a hyperalgesic effect, which was restricted to paw licking and occurred only at a high dose of 2.5 nmol. 3. In addition, i.c.v. administration of BDNL potentiated the antinociceptive effects of the mixed inhibitor of enkephalin degrading enzymes, RB 101 and of the mu-agonist, DAMGO, while BC 264 reduced these effects. 4. Furthermore, at a dose where it interacts selectively with delta-opioid receptors, the opioid agonist BUBU reversed the hyperalgesic responses of BC 264 (2.5 nmol) but was unable to modify the effects induced by BDNL. 5. Taken together, these results suggest the existence of regulatory mechanisms between CCK and enkephalin systems in the control of pain. These regulatory loops could enhance the antinociceptive effects of morphine allowing the opiate doses used to be reduced and thus, possibly, the side-effects to be minimized.

Topics: Amino Acid Sequence; Analgesics; Animals; Cholecystokinin; Disulfides; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Male; Mice; Molecular Sequence Data; Oligopeptides; Pain; Pain Measurement; Peptide Fragments; Phenylalanine; Sincalide; Spinal Cord

A series of trans-3-(6- and 7-substituted-decahydro-4a-isoquinolinyl)phenols and trans-3-(octahydro-4a-isoquinolinyl)phenols have been synthesized as potential opioid analgesics. Using a combination of in vitro and in vivo test systems, the receptor profiles of selected compounds have been assessed and in some instances distinguish between mu- and kappa-receptor agonists. In general, introduction of a 6-exocyclic methylene group into the trans-3-(decahydro-4a-isoquinolinyl)phenol system enhanced both antinociceptive activity and kappa-opioid receptor selectivity. For each series, analogues bearing an N-cyclopropylmethyl substituent exhibited greater kappa-receptor selectivity while N-methyl derivatives showed greater mu-receptor selectivity. The 7-substituted compounds (3b) were significantly less potent antinociceptive agents than their 6-substituted counterparts (3a), the octahydroisoquinoline analogues exhibiting intermediate activity. The axial 8-methyl-6-exocyclic methylene isoquinoline (20) is the most potent compound in the mouse abdominal constriction assay (ED50 = 0.05 mg/kg sc), whereas the equatorial 8-methyl isomer (16) was significantly less potent (ED50 = 3.3 mg/kg sc).

Topics: Analgesics; Animals; Guinea Pigs; Isoquinolines; Male; Mice; Pain; Phenols; Rabbits; Receptors, Opioid; Stereoisomerism; Structure-Activity Relationship

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered mu-opioid agonists, such as morphine (10 micrograms) and [D-Ala2,N-Me Phe4,Gly-ol5]enkephalin (DAMGO, 0.5 micrograms). However, i.c.v. administration of [D-Pen2,5]enkephalin (DPDPE, 5 micrograms), a delta-opioid agonist, and U-50,488H (50 micrograms), a kappa-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 microgram), DAMGO (10 micrograms), DPDPE (0.5 micrograms) or U-50,488H (50 micrograms) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal mu-opioid receptor-mediated antinociception, but they are normally responsive to activation of delta- and kappa-opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Cerebral Ventricles; Diabetes Mellitus, Experimental; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Injections, Subcutaneous; Male; Mice; Mice, Inbred ICR; Morphine; Pain; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord

Recently, an experimental model of monoarthritis was described in the rat induced by injection with Freund's adjuvant of the tibio-tarsal joint of one hindlimb. After injection, the clinical and behavioural signs of arthritis are stable from weeks 2 to 6 post-injection. Our purpose was to study the regulation of mu-, delta- and kappa-opioid binding sites in the superficial layers (laminae I-II) of the lumbar and cervical enlargements of the spinal cord 2, 4 and 6 weeks post-injection. Using quantitative receptor autoradiography and highly selective opioid ligands, we found complex changes consisting of a bilateral increase in specific [3H]DAMGO (Tyr*-D-Ala-Gly-NMe-Phe-Gly-ol) and [3H]pCl-DPDPE (Tyr*-D-Pen-Gly-Cl-Phe-D-Pen) binding at 2 weeks post-injection and a bilateral decrease in [3H]U-69593 ((5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl]) specific binding at 4 weeks post-injection. These changes were restricted to the lumbar level. At 6 weeks post-injection, there was a bilateral increase in [3H]pCl-DPDPE specific binding at both lumbar and cervical levels. Altogether, these results suggest that, after probable local changes in endogenous opioid peptides, the three types of opioid binding sites are differentially involved in the development of the pathological process. These results contrast with the lack of significant modification in mu-, delta- and kappa-opioid binding classically reported at various levels of the spinal cord in polyarthritic rats at 3 weeks post-injection and verified for 2, 4 and 6 weeks post-injection in the present study.

Topics: Animals; Arthritis; Arthritis, Experimental; Autoradiography; Benzeneacetamides; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Freund's Adjuvant; Male; Pain; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord

Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Anura; Binding, Competitive; Brain; Catalepsy; Cerebral Ventricles; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Injections, Intraventricular; Kinetics; Molecular Sequence Data; Myenteric Plexus; Naloxone; Nociceptors; Oligopeptides; Opioid Peptides; Pain; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Skin; Skin Physiological Phenomena; Structure-Activity Relationship

The s.c. administration of [Met5]-enkephalin to 10-day-old rats pretreated with the mixture of 3 peptidase inhibitors, amastatin, captopril and phosphoramidon, produced the inhibition of tail-flick response and loss of righting reflex. When infant rats were pretreated with the mixture of any combination of two peptidase inhibitors, however, the change in both the response and the reflex were not produced at all by enkephalin injection, indicating that 3 kinds of enzymes, amastatin-sensitive aminopeptidase(s), captopril-sensitive peptidyl dipeptidase A and phosphoramidon-sensitive endopeptidase 24.11, played an important role in the inactivation of enkephalin after its systemic administration. Additionally, the fact that the two enkephalin-induced effects were more effectively antagonized by naloxone, a relatively selective mu-opioid antagonist, than by naltrindole, a specific delta-antagonist, or by nor-binaltorphimine, a specific kappa-antagonist, showed that these two effects were produced by the interaction of enkephalin with mu receptors. Moreover the involvement of mu receptors in the production of these two effects was shown by the fact that the s.c. administration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a selective mu agonist, also produced these two effects which were more effectively antagonized by naloxone than by naltrindole or nor-binaltorphimine. Since the magnitude of the two effects induced by enkephalins in 15-day-old rats was significantly lower than that in 10-day-old rats, and the two enkephalin-induced effects were not produced at all in 20-day-old rats, a maturation-induced decrease in the permeability of the blood-brain barrier against opioid peptides was indicated.

Topics: Aging; Animals; Anti-Bacterial Agents; Captopril; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Female; Glycopeptides; Injections, Subcutaneous; Male; Oligopeptides; Pain; Peptides; Posture; Protease Inhibitors; Rats; Rats, Wistar; Reflex

The effects of naloxone on the analgesic response were examined using the tail-flick test, in mice with streptozotocin-induced diabetes. Subcutaneous injection of naloxone (5 mg/kg, s.c.) produced a marked analgesia in diabetic mice but not in age-matched non-diabetic mice. Naloxone-induced analgesia in diabetic mice was significantly reduced by pretreatment with naltrindole (0.1 mg/kg, s.c.), a selective antagonist of delta-opioid receptors. By contrast, no significant naloxone-induced increase in tail-flick latency in diabetic mice was observed after chronic treatment with naloxone (5 mg/kg, s.c.) for 5 days. However, the tail-flick latency was significantly increased by chronic treatment with naloxone in non-diabetic mice. Furthermore, the significant naloxone-induced increase in tail-flick latency in non-diabetic mice that had been chronically treated with naloxone was also antagonized by pretreatment with naltrindole. Chronic pretreatment with 5 mg/kg of naloxone for 5 days markedly attenuated the analgesic effect of the delta-agonist DPDPE in diabetic mice, whereas this pretreatment significantly enhanced the effect of DPDPE in non-diabetic mice. These results suggest that naloxone-induced 'paradoxical' analgesia in mice may be mediated predominantly by delta-opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesia; Animals; Diabetes Mellitus, Experimental; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Hot Temperature; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Naloxone; Pain; Pain Measurement; Pyrrolidines; Reaction Time; Time Factors

1. It has recently been suggested that opioid antagonists may be divided into those possessing negative intrinsic activity (e.g. naloxone) and those with neutral intrinsic activity (e.g. MR2266). 2. MR2266 was chronically administered to rats by subcutaneous infusion at a dose of 0.3 mg kg-1 h-1 for 1 week. 3. This dose reduced ingestive behaviour and blocked the antinociceptive effects of a kappa-agonist, indicating occupation of opioid receptors in vivo. 4. No supersensitivity could be detected to the antinociceptive actions of mu or kappa agonists, either one or two days after cessation of treatment. 5. No up-regulation of mu, delta or kappa binding sites was observed. 6. Since naloxone induces both supersensitivity and receptor up-regulation under equivalent conditions, the results suggest that negative intrinsic activity may be required for these phenomena to occur.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Benzomorphans; Binding, Competitive; Drinking; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Narcotic Antagonists; Pain; Pain Measurement; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Sensory Thresholds; Up-Regulation

Previously, we demonstrated the enhanced affinity of opioid receptors for naloxone in striatal slices from morphine-dependent mice. In our present study, binding characteristics of the mu opioid receptor agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) and dihydromorphine, the delta opioid receptor agonist, [D-Ala2, D-Leu5]enkephalin (DADLE), and the opioid antagonist, naloxone, were examined in striatal slices from morphine-tolerant and -dependent mice. Striatal slices from mice that were implanted with a morphine pellet for 3,7 and 21 days displayed significant decreases in Kd values (5.1, 4.6 and 5.5 nM, respectively) of [3H]DAMGO when compared to those in slices from control animals that were not implanted or implanted with placebo pellets (9.6 and 9.3 nM, respectively). Also, a significant increase in the binding affinity of naloxone, but not that of dihydromorphine, was observed in striatal slices of mice that were implanted with a morphine pellet for 3 days. Significant increases in the Bmax of delta binding sites in striatal slices of mice that were implanted with a morphine pellet for 3, 7 and 21 days (20.7, 18.1 and 17.7 pmol/mg tissue, respectively) were observed when compared to that in slices from control mice that were implanted with placebo pellets (11.4 pmol/mg tissue). The enhancement in the binding affinity of DAMGO and naloxone and the increased density of DADLE binding sites paralleled the development of morphine tolerance and dependence and [D-Pen2,D-Pen5]enkephalin cross-tolerance in whole animals. An antinociceptive potentiation between morphine and DAMGO was observed in morphine-tolerant and -dependent mice whereas in naive animals the effects of the two drugs were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Binding Sites; Corpus Striatum; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Mice; Morphine; Morphine Dependence; Naloxone; Pain; Receptors, Opioid

Morphine has been considered to be primarily a mu opiate receptor agonist. The present study was designed to determine if opiate receptor subtypes in addition to mu contribute to morphine analgesia at the level of the spinal cord. Extracellular activity of single wide dynamic range (WDR) neurons in the feline lumbar spinal cord were studied. Intrathecal administration of DAGO (selective mu agonist) or DPDPE (selective delta agonist) suppressed the noxiously (51 degrees C radiant heat) evoked activity of WDR neurons. Pretreatment with spinal beta-FNA (selective mu antagonist) antagonized the suppressive effects of spinal DAGO, but not that of DPDPE. Two doses of spinal morphine (200 and 400 micrograms) suppressed the noxiously evoked activity of WDR neurons confirming our previous report. Following beta-FNA pretreatment, the suppressive effects of morphine were reduced, however, when ICI 174,864 (selective delta antagonist) was co-administered with morphine on the spinal cord of the animals pretreated by beta-FNA, there was an even greater reduction in the neuronal suppression by morphine. Intravenous ICI 174,864 also reversed the suppressive effects of morphine in beta-FNA pretreated animals. beta-FNA antagonism of spinal morphine is evidence of the well-known mu receptor-mediating antinociception. However, antagonism by ICI 174,864 of morphine suppression in beta-FNA-pretreated animals demonstrates that morphine is capable of suppressing noxiously evoked activity of WDR neurons as a result of an interaction with delta receptors in addition to mu receptors at the level of spinal cord.

Topics: Analgesia; Animals; Cats; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Evoked Potentials; Female; Hot Temperature; Injections, Spinal; Male; Morphine; Naltrexone; Narcotic Antagonists; Neurons; Pain; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

In an earlier report, we demonstrated that subcutaneous injection of formalin in the rat hindpaw evokes a characteristic pattern of expression of the fos protein product of the c-fos protooncogene in spinal cord neurons, and that systemic morphine reversed the fos-like immunoreactivity in a dose-dependent, naloxone-reversible manner. The present study compared the effects of intracerebroventricular administration of the mu-selective opioid ligand [D-Ala2, NMe-Phe4, Gly-ol5] enkephalin, on the pain behavior and spinal cord fos-like immunoreactivity produced by subcutaneous formalin. Formalin injection produced a biphasic pain behavioral response which lasted about 1 h. There was a significant correlation between the formalin pain score and overall fos-like immunoreactivity in the lumbar enlargement. The greatest numbers of labeled cells and most intense fos-like immunoreactivity were found in laminae I, IIo and V of the L4-5 segments, ipsilateral to the formalin-injected paw. Considerable staining was also found in the ipsilateral ventral horn laminae VII and VIII. [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin produced a dose-related, naloxone-reversible inhibition of both the formalin-evoked pain behavior and fos expression in the cord. The behavioral response to formalin, however, could be completely blocked without eliminating the expression of fos in spinal neurons. Moreover, subpopulations of neurons were differentially regulated. Thus, 100% inhibition of pain behavior was produced at a dose of [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin which reduced fos-like immunoreactivity in the superficial laminae by only 64% and in the neck and ventral cord by 85%. Furthermore, the dose of [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin which produced approximately 50% inhibition of fos-like immunoreactivity in the neck and ventral regions of the spinal cord was without effect in the superficial dorsal horn. Since the potencies for inhibition of pain behavior and fos-like immunoreactivity in the neck and ventral horn were comparable, these data suggest that the activity of neurons in these regions is directly related to the pain behavior produced by nociceptive inputs. Finally, we found that bilateral, midthoracic lesions of the dorsal part of the lateral funiculus blocked both the antinociception and fos suppression produced by intracerebroventricular [D-Ala2, NMe-Phe4, Gly-ol5]enkephalin. These results are consistent with the hypothesis that the analgesic action of supraspinally adm

Topics: Amino Acid Sequence; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Foot; Formaldehyde; Gene Expression Regulation; Injections, Intraventricular; Male; Molecular Sequence Data; Naloxone; Neurons; Pain; Pain Measurement; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Single-Blind Method; Spinal Cord

Studies of various strains of mice revealed marked differences in their analgesic sensitivity towards morphine (mu), U50,488H (kappa 1) and naloxone benzoylhydrazone (NalBzoH; kappa 3). Sensitivity to mu and kappa analgesia varied independently of the other. Analgesic sensitivity to morphine remained relatively consistent among 3 different nociceptive assays for each strain. However, the sensitivity of an individual strain to NalBzoH remained highly dependent upon the assay used. CD-1 mice were sensitive to NalBzoH in all 3 assays, but in BALB/c mice NalBzoH produced analgesia only in the hot plate and cold water tail-flick assays. In Swiss-Webster mice, NalBzoH was active in the radiant heat and cold water tail-flicks but inactive in the hot plate. Although the levels of mu, kappa 1 and kappa 3 binding in whole brain homogenates did vary somewhat, they did not correlate with analgesic sensitivity. These results suggests that the genetic controls over mu and kappa analgesia operate independently and further illustrate the many difficulties in evaluating potential analgesics.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesia; Analgesics; Animals; Benzeneacetamides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Mice, Inbred Strains; Morphine; Naloxone; Pain; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Species Specificity

Cholera toxin, an agent that impairs the function of Gs transducer proteins, was injected (0.5 microgram/mouse, icv) and the antinociceptive activity of opioids and clonidine was studied 24h later in the tail-flick test. In these animals, an enhancement of the analgesic potency of morphine, beta-endorphin and clonidine could be observed. Cholera toxin did not modify the antinociception evoked by the enkephalin derivatives DAGO and DADLE. Pertussis toxin that catalyses the ADP ribosylation of alpha subunits of Gi/Go regulatory proteins was given icv (0.5 microgram/mouse). This treatment reduced the analgesic effect of opioids and clonidine. However, while the analgesia elicited by DAGO, DADLE and clonidine was greatly decreased, the effect of morphine and beta-endorphin was reduced to a moderate extent. It is concluded that Gi/Go regulatory proteins functionally coupled to opioid and alpha 2 receptors are implicated in the efficacy displayed by opioids and clonidine to produce supraspinal analgesia. Moreover, these two receptors are susceptible to regulation by a process that might involve a Gs protein.

Topics: Analgesia; Animals; beta-Endorphin; Cerebral Ventricles; Cholera Toxin; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; GTP-Binding Proteins; Injections, Intraventricular; Male; Mice; Morphine; Narcotics; Pain; Pertussis Toxin; Receptors, Adrenergic, alpha; Virulence Factors, Bordetella

This study compared the antinociceptive and motor effects produced by intrathecal administration of selective mu-, delta-, and kappa-opioid receptor agonists in the rat. Changes in nociceptive threshold were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Each opioid agonist produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. In the motor coordination studies, DAMGO and DPDPE, but not U50,488H, produced statistically significant decreases in rotarod performance scores compared to vehicle controls. The results of these studies suggest that motor side-effects produced by opioid agonists need to be considered when interpreting the results of antinociceptive tests that are dependent on a normally functioning motor system.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Narcotic Antagonists; Pain; Postural Balance; Psychomotor Performance; Pyrrolidines; Rats; Rats, Inbred Strains; Sensory Thresholds

alpha N-acetyl human beta-endorphin-(1-31) injected icv to mice antagonized the analgesic activity of beta-endorphin-(1-31) and morphine whereas the analgesia evoked by DADLE and DAGO was enhanced by this treatment. The modulatory activity of alpha N-acetyl beta-endorphin-(1-31) was exhibited at remarkable low doses (fmols) reaching a maximum that persisted even though the dose was increased 100,000 times. The regulatory effect of a single dose of the acetylated neuropeptide lasted for 24h. The activity of alpha N-acetyl human beta-endorphin-(1-31) was partially retained by the shorter peptide alpha N-acetyl human beta-endorphin-(1-27) and to a lesser extent by beta-endorphin-(1-27), beta-endorphin-(1-31) lacked this regulatory activity on opioid analgesia. Acetylated beta-endorphin-(1-31) displayed a biphasic curve when competing with 5 pM [125I]-Tyr27 human beta-endorphin-(1-31) specific binding, the first step (20 to 30% of the binding) was abolished with an apparent IC50 of 0.35 nM, and the rest with an IC50 of 200 nM. It is suggested that alpha N-acetyl beta-endorphin-(1-31) changed the efficiency of the opioid analgesics by acting upon a specific substrate that is functionally coupled to the opioid receptor, presumably the guanine nucleotide binding regulatory proteins Gi/Go.

Topics: Acetylation; Animals; beta-Endorphin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Mice; Pain; Structure-Activity Relationship; Tail; Time Factors

1. In view of the presence of mu, delta and kappa opioid receptors in the spinal dorsal horn and their apparent involvement in behavioural analgesia, the present experiments addressed the action of selective agonists ionophoresed in the vicinity of rat dorsal horn neurones which were located either in lamina I or in laminae III-V. 2. In laminae III-V, kappa agonists (U50488H and dynorphin A) caused a selective inhibition of the nociceptive responses of multireceptive cells, whilst mu and delta agonists [( D-Ala2, MePhe4, Gly-ol]enkephalin and [D-Pen2, D-Pen5]enkephalin respectively) failed to alter either the spontaneous activity or the response to noxious and innocuous cutaneous stimuli and to D,L-homocysteic acid or glutamate. Nocispecific neurones were encountered too rarely in laminae III-V to study their properties. 3. In lamina I, agonists had no effects on either nocispecific or multireceptive neurones. In contrast, the mu agonist [D-Ala2, MePhe4, Gly-ol]enkephalin consistently inhibited nociceptive responses of both multireceptive and nocispecific lamina I cells. The delta agonist [D-Pen2, D-Pen5]enkephalin consistently caused selective inhibition of the nociceptive responses of multireceptive cells but had a mixed profile of action on nocispecific cells. 4. These results suggest that mu, delta and kappa opioid receptors mediate different antinociceptive actions in both laminae III-V and lamina I. The study reveals a distinct physiological role for delta receptors in modulating nociceptive inputs to lamina I neurones. In contrast to mu and kappa receptor actions, delta receptors heterogeneously influence subpopulations of neurones.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Neurons; Pain; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord

An in vivo preparation of the rat spinal cord was used to investigate the electrophysiological actions of two non-opioid peptides, cholecystokinin (CCK8) and FLFQPQRF-NH2 (FMRFamide-like peptide) applied intrathecally. These compounds were examined alone and as a pretreatment before DAGO, a mu opioid agonist, and DSTBULET, a delta opioid agonist, both which selectively reduce C-fibre evoked dorsal horn neurone activity elicited by transcutaneous electrical stimulation. Given alone, CCK8 (1 microgram) elicited a modest enhancement of C-fibre induced activity which returned to control levels after 20 min, while FLFQPQRF-NH2 (10 micrograms) had no significant effect on C-fibre evoked firing. As a pretreatment, however, both peptides selectively prevented the inhibition of C-fibre evoked activity normally resulting from intrathecal DAGO, while having no effect on that resulting from DSTBULET. Further, CCK8 enhanced the facilitation of C-fibre evoked firing normally observed with low doses of DAGO. These data indicate that the anti-opioid roles suggested for CCK8 and FLFQPQRF-NH2 may be specific for neural elements utilizing the mu opioid receptor.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Evoked Potentials; Injections, Spinal; Male; Nerve Fibers; Neurons; Oligopeptides; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sincalide; Spinal Cord

Mice treated chronically with opioid antagonists have increased receptor density in brain and are supersensitive to the pharmacodynamic action of morphine. In the present study mice were implanted subcutaneously with naltrexone or placebo pellets for 8 days. During implantation mice received daily injections of morphine (100 or 250 mg/kg) or saline. Morphine analgesia was completely blocked in mice that were implanted with naltrexone at the low dose of morphine; while some analgesic action was observed at the higher dose. Mice implanted with placebo were analgesic following the daily morphine treatment. At the end of 8 days the pellets were removed and 24 h later some mice were tested for morphine analgesia while others were examined in binding studies. Naltrexone treatment increased [3H]naloxone, 3H[D-Ala2-D-Leu5]enkephalin (DADLE) and 3H[D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) binding compared to controls and increased the analgesic potency of morphine. Daily treatment with morphine did not alter brain opioid binding or naltrexone-induced receptor upregulation. Mice injected daily with morphine were significantly less sensitive to morphine (tolerant) than their respective saline control group for both the placebo and the naltrexone-treated groups. However, naltrexone-treated mice were more sensitive to morphine than placebo controls regardless of whether they were injected daily with morphine or not. These results indicate that if naltrexone-induced opioid receptor upregulation occurs in the presence of repeated agonist administration, the new binding sites mediate tolerance via desensitization to morphine.

Topics: Analgesia; Animals; Brain; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Kinetics; Male; Mice; Morphine; Naloxone; Naltrexone; Pain; Receptors, Opioid; Reference Values; Up-Regulation

Although an opioid-mediated mechanism appears to be involved in the alteration of pain perception during feeding behavior, little is known about macronutrient effects on nociception. In this report we show that prolonged sucrose feeding alters responsiveness to painful stimuli and the analgesic potency of morphine. Male Sprague-Dawley rats maintained on ad lib laboratory chow with continuous access to a 20% sucrose solution displayed a significant decrease in tail-flick latency as early as 20 hours after introduction of the sucrose. The differences in pain threshold were naloxone sensitive. After 25 days on the diet, morphine sulfate, 8 mg/kg administered IP, proved to be significantly more potent in the sucrose-fed animals. The results indicate that sucrose feeding alters endogenous opioid-mediated nociception.

Topics: Animals; Blood Glucose; Body Composition; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Insulin; Male; Medulla Oblongata; Morphine; Pain; Periaqueductal Gray; Rats; Rats, Inbred Strains; Sensory Thresholds; Serotonin; Sucrose

By employing a constant-rate (1 microliter/hr) constant-dose intrathecal infusion pump in rats, groups received a 7-day infusion of one of three concentrations (in nmol/hr) of each of the following receptor preferring ligands: morphine (mu: 2, 6, 20), sufentanil (mu: 0.06, 0.2, 0.6), DAMGO (mu: 0.1, 0.3, 1.0), DADLE (delta: 2, 6, 20) or ST-91 (alpha-2: 3, 10, 30). On day 7 the magnitude of tolerance was assessed by establishing i.t. dose-response curves for the effect of the chronic drug given as a bolus. Each infused animal was used for one bolus injection. It was observed that for all drugs, a parallel rightward shift in the dose-response curve was produced with the degree of shift proportional to the log of the infusion dose. Of particular significance was the finding that drugs differed significantly in the magnitude of the shift produced by the chronic infusion of equieffective concentrations. Thus, at the intermediate infusion dose, the degree of the shift in the bolus dose-response curve (i.e., magnitude of tolerance) for morphine, DADLE, ST-91, DAMGO and sufentanil was 55, 46, 29, 7 and 2, respectively. These data are interpreted to suggest that agents with high potency and a significant receptor reserve will produce a smaller shift in the dose-response curve (e.g., sufentanil and DAMGO) than the similar infusion of an equieffective dose of another agent with less potency and a smaller population of spare receptors (morphine).

Topics: Analgesics; Animals; Clonidine; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Fentanyl; Infusions, Parenteral; Male; Morphine; Pain; Rats; Rats, Inbred Strains; Reference Values; Sufentanil

Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 is an extraordinarily potent and highly mu-selective opioid heptapeptide isolated from amphibian skin. It is unique among peptides synthesized by animal cells in having an amino acid residue in the D-configuration. At least two different preprodermorphin cDNAs were cloned from skin of Phylomedusa sauvagei; their predicted amino acid sequences contained four to five homologous repeats of 35 amino acids, each repeat including one copy of the dermorphin progenitor sequence. Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly, flanked by Lys-Arg at the amino end and by Glu-Ala-Lys-Lys at the carboxyl end [Science (Wash. D. C.) 238:200-202 (1987)]. The D-Ala in position 2 in dermorphin is encoded by a usual Ala codon in the precursor sequence. Of the two prodermorphin molecules, one has a dermorphin copy replaced with a distinct heptapeptide same processing signals. Assuming the same pathway as for the release of dermorphin, processing of this precursor may yield, beside dermorphin, a copy of a new peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2. We have synthetized this peptide together with its (L-Met2)-counterpart and evaluated their respective opioid receptor selectivity in the mouse vas deferens and guinea pig ileum assays and in rat brain membrane binding assays. Overall, the data collected demonstrate that the putative prodermorphin product Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 named dermenkephalin, behaves as a potent delta opioid agonist exhibiting high affinity and high selectivity for the delta opioid receptor. Prodermorphin, thus, offers a surprising example of an opioid biosynthetic precursor that might simultaneously generate highly potent and fully selective agonists for the mu- (morphine) and the delta (enkephalin) opioid receptors, respectively. In addition, because dermenkephalin has no structural features in common with the sequence of all the hitherto known opioid peptides, it should be a useful tool for identifying conformational determinants for high affinity and selective binding of opioids to the delta receptor.

Topics: Amino Acid Sequence; Analgesia; Animals; Binding, Competitive; Brain; Cell Membrane; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Kinetics; Male; Mice; Oligopeptides; Opioid Peptides; Pain; Rats; Receptors, Opioid; Receptors, Opioid, delta

It has been demonstrated in experiments on unrestrained and unanesthetized curarized cats that periaqueductal gray matter stimulation produce sympathetic-activating action, raise arterial pressure and heart rate, but at the same time is not effective enough to suppress the nociceptive shifts of haemodynamic reactions. Opioid mechanisms of spinal cord plays an essential role in sympathetic-activating action of periaqueductal gray matter. It is suggested that the influence of antinociceptive areas of the brain stem on sympathetic haemodynamic regulation is one of the causes of resistance of nociceptive haemodynamic reactions to narcotic analgetics.

Topics: Animals; Blood Pressure; Cats; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Kidney; Morphine; p-Chloroamphetamine; Pain; Periaqueductal Gray; Receptors, Opioid; Serotonin; Sympathetic Nervous System

The antinociceptive properties of intrathecally (i.t.) administered [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin (DAMPGO) and [D-Pen2, D-Pen5]enkephalin (DPDPE), selective opioid agonists for mu (mu) and delta (delta) sites, respectively, were compared in rats. DAMPGO and DPDPE elevated tail-flick latency (TFL) in a dose-dependent manner, and the spinal antinociceptive actions of both drugs were reversed by the opiate antagonist naloxone. These findings suggest that both DAMPGO and DPDPE interact with spinal opiate receptors to elevate TFL. Another set of experiments was done to determine the involvement of local spinal serotonin (5-HT) or norepinephrine (NE) in DAMPGO and DPDPE-induced spinal analgesia. Both the alpha 1 noradrenergic receptor antagonist WB-4101 and the alpha 2 blocker yohimbine failed to alter the antinociceptive actions of DAMPGO and DPDPE. Similarly, the 5-HT receptor antagonists pindolol, ritanserin and ICS 205-930 (selective for 5-HT1, 5-HT2 and 5-HT3 sites, respectively) failed to inhibit opioid-induced spinal analgesia. Thus, while DAMPGO and DPDPE produce antinociception via an interaction with spinal opioid receptors, apparently neither drug activates endogenous monoaminergic systems.

Topics: Anesthetics; Animals; Biogenic Monoamines; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Spinal; Male; Naloxone; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Spinal Cord

Thirteen patients with pain from various causes were treated by electroacupuncture for 30 min. Cerebrospinal fluid (CSF) was obtained before and after treatment. Opioid-like substances in the CSF were fractionated by high pressure liquid chromatography and assayed by competitive receptor binding using a mu-specific radioligand, [D-ala2, MePhe4, gly-ol5]-enkephalin (DAGO). Opioid activity, associated with a fraction, eluted at 18-20% acetonitrile, consistently showed an increase in level after acupuncture. Two other fractions eluted at larger concentrations of acetonitrile also increased significantly after acupuncture; however the increase was not consistently observed in every patient. Measurements of beta-endorphin and dynorphin by radioimmunoassay indicated that 80 and 60% of the patients, respectively, had a higher level of these peptides after acupuncture. The nature of the opioid activity, eluted at 18-20% acetonitrile is unknown; however a small amount of it could be found in various parts of the brain of rat.

Topics: beta-Endorphin; Chromatography, High Pressure Liquid; Chronic Disease; Dynorphins; Electroacupuncture; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Pain; Pain Management; Radioimmunoassay

Selective agonists for mu- and kappa-opioid receptor types were infused, bilaterally, into the intralaminar central lateral nucleus of the rat. Subcataleptic doses of the mu-agonist, DAGO (0.25 and 1.0 microgram), elevated tailshock threshold for eliciting pain vocalization and motor responses. The hyperalgesic effect of U50,488 is not likely to be the result of antagonist action at a mu 2-isoreceptor; the general mu-antagonist, naloxone, and its less lipophilic quaternary analogue, both failed to produce a significant reduction in pain thresholds. Paralleling their effects on pain, DAGO and U50,488 elevated and reduced, respectively, lateral hypothalamic electrical stimulation threshold for positive reinforcement. These results suggest that medial thalamic opioid mechanisms are not exclusively involved in pain modulation but may generally regulate the responsiveness of the organism to motivating stimuli. Moreover, mu- and kappa-receptors may mediate opposite behavioral effects of opioid peptides.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Electric Stimulation; Electroshock; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Infusions, Parenteral; Male; Microinjections; Naloxone; Pain; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reward; Self Stimulation; Thalamus

Topics: Acute Disease; Animals; Cerebral Ventricles; Chronic Disease; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraperitoneal; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Pain

The effects of the calcium channel antagonists diltiazem, nifedipine and the calcium channel agonist, BAY K 8644, on immobilization-induced opioid analgesia and locomotor activity were examined in CF-1 and C57BL strains of mice, respectively. The calcium channel antagonists enhanced the experimenatlly induced analgesia and activity, whereas the agonist reduced these responses. In addition, the calcium channel antagonists augmented, while the agonist attenuated, the analgesic effects of the specific mu and delta opioid agonists, DAGO and [D-Pen2,D-Pen5]-enkephalin (DPDPE), respectively. These results indicate that calcium channel antagonists have facilitatory and/or modulatory effects on the behavioral and physiological consequences of endogenous mu and delta opioid activity.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Channel Blockers; Diltiazem; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Immobilization; Mice; Mice, Inbred C57BL; Motor Activity; Naloxone; Nifedipine; Pain; Reaction Time; Stress, Physiological; Verapamil

The possibility that the opioid delta-receptor mediates antinociception in tests where heat is the noxious stimulus was investigated using highly selective mu- and delta-agonist and -antagonists. Antinociceptive dose-response curves were constructed for mu ([D-Ala2,NMePhe4,Gly-ol]enkephalin, DAGO; morphine) and delta ([D-Pen2,D-Pen5]enkephalin, DPDPE)-agonists in the absence, and in the presence of the mu non-surmountable antagonist, beta-funaltrexamine (beta-FNA) or the delta-antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, where Aib is alpha-amino-isobutyric acid). Agonists and ICI 174,864 were given alone in the same intracerebroventricular (i.c.v.) or intrathecal (i.th.) injection to mice 20 min prior to testing in the warm-water (55 degrees C) tail-withdrawal test (+10 min for i.th. DPDPE); beta-FNA was given as a single i.c.v. or i.th. pretreatment dose (20 and 0.01 nM, respectively) 4 h prior to testing. I.c.v. pretreatment with beta-FNA resulted in a rightward displacement of the DAGO and morphine antinociceptive dose-response lines, but failed to displace the i.c.v. DPDPE curve. Similarly, i.th. pretreatment with beta-FNA displaced the i.th. morphine dose-response curve to the right without affecting the i.th. DPDPE antinociceptive dose-response line. ICI 174,864 (1 and 3 micrograms) produced a dose-related antagonism of i.c.v. or i.th. DPDPE, but did not alter the antinociceptive effects of DAGO or morphine given by the same routes. Co-administration of ICI 174,864 (3 micrograms) with i.c.v. morphine in beta-FNA pretreated (but not control) mice resulted in a further rightward displacement of the morphine dose-response line.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Dose-Response Relationship, Drug; Drug Combinations; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Hot Temperature; Injections, Intraventricular; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Morphine; Naltrexone; Nociceptors; Pain; Receptors, Opioid; Receptors, Opioid, delta; Spinal Cord

The spontaneous discharges in 15 out of 19 small-diameter afferent units from inflamed knee joints of anaesthetized cats were significantly inhibited by one or several opiates (morphine in the dose range 1.0-5.0 mg/kg; gly-ol 0.5-5.0 mg/kg; U50488 1.0-10.0 mg/kg; ethylketocyclazacine 0.5-4.0 mg/kg administered by close arterial injection into the joint). In the majority of cases a subsequent injection of naloxone (1 mg/kg i.a.) significantly reversed this effect. These data provide an electrophysiological demonstration that opiates may act on opiate receptors located at peripheral sites of primary afferent fibres and hence exert a peripheral 'analgesic' effect.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Animals; Arthritis; Cats; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Ethylketocyclazocine; Morphine; Naloxone; Nociceptors; Pain; Peripheral Nerves; Pyrrolidines

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Oligopeptides; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord; Synaptic Transmission

Large baseline differences were found in open field activity and rearing behavior between 2 psychogenetically selected rat lines, as well as differences in the effects of peripherally administered morphine (2,4 and 10 mg/kg) on those behaviors and on body temperature. The 2 lines also showed different capacities of tritiated DAGO binding to preparations of striatal membranes.

Topics: Animals; Body Temperature; Cell Membrane; Corpus Striatum; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Morphine; Motor Activity; Pain; Rats; Rats, Mutant Strains; Receptors, Opioid; Species Specificity

Stress (e.g. restraint) potentiates analgesia and alters changes in body temperature induced by morphine administered either systemically or intracerebroventricularly (i.c.v.) in rats. In order to extend the generality of this phenomenon to opioid peptides, we determined whether the analgesic and thermic effects of i.c.v. D-Ala2-D-Leu5-enkephalin (DADLE) or D-Ala2-N-MePhe4-Gly5(ol)-enkephalin (DAGO), agonists selective for delta- and mu-opioid receptors, respectively, were affected by restraint stress. Analgesia was measured in the tail-flick test and core body temperature by rectal probe. The unstressed rats exhibited a dose-dependent increase in tail-flick latencies after administration of either DAGO or DADLE. Restrained rats treated with DAGO or DADLE had a greater analgesic response to each dose of peptide than did unstressed rats; both the magnitude and duration of the drug effect were increased. The unstressed group of rats responded to all doses of DAGO and DADLE with an increase of core temperature. In contrast, restrained rats showed a decrease of core temperature following injection with either DAGO or DADLE. Thus, restraint stress can significantly modify the effects of DAGO and DADLE on analgesia and body temperature in a manner that is qualitatively and quantitatively similar to that observed previously for morphine administered by the i.c.v. route.

Topics: Animals; Body Temperature; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Injections, Intraventricular; Male; Morphine; Pain; Rats; Rats, Inbred Strains; Receptors, Opioid; Restraint, Physical; Stress, Physiological

In the present investigation the effects of selective agonists for mu (Tyr-D-Ala-Me-Phe-Gly-ol (DAGO)) and delta (Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET)) opioid receptors on neuronal activities induced by noxious cutaneous stimuli in the rat ventrobasal (VB) thalamus were analyzed. The two agonists produced a clear depressive action on thermal as well as mechanical noxious stimuli. The depressive action of DTLET (3 mg/kg i.v.) was lower and of shorter duration than that of DAGO (2 mg/kg i.v.). However, this effect is unambiguously related to the selective stimulation of opioid receptors since a consistent effect was also observed for a dose as low as 1.5 mg/kg i.v. of DTLET. Moreover, DTLET effect needs a high concentration of naloxone (0.5 mg/kg i.v.) to be reversed, while DAGO effect is totally reversed with 0.1 mg/kg i.v.

Topics: Action Potentials; Animals; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hot Temperature; Male; Naloxone; Neurons; Oligopeptides; Pain; Physical Stimulation; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Thalamus

The analgesic activity of the prototypic opioid peptides for the mu (D-Ala2-Me-Phen4-Gly-ol5-enkephalin [DAGO]) kappa (Dynorphin 1-13), delta (D-Ala2-D-Leu5-enkephalin [DADLE]), or epsilon (beta-endorphin) receptor was assessed in a rat tooth pulp stimulation procedure. All opioid peptides tested and the opioid alkaloid U50, 488H (kappa receptor agonist) significantly elevated response thresholds. The rank order of potency based on the Minimum Effective Dose values was beta-endorphin greater than DAGO = dynorphin A (1-13) amide greater than DADLE greater than dynorphin A (1-13) greater than U50,488H. Based on absolute magnitude, the rank order of dose response slopes was DAGO greater than U50,488H greater than dynorphin A (1-13) amide greater than beta-endorphin greater than DADLE. Dynorphin A (1-13) produced the shallowest dose response slope and the magnitude of response threshold was the lowest for all compounds tested. Finally, the general conclusion that mu agonists are effective against noxious stimuli derived from thermal, chemical, and mechanical is extended by our data to include electrical sources derived from tooth pulp stimulation; kappa agonists are effective against noxious stimuli derived from chemical, mechanical, and electrical sources (tooth pulp stimulation) and delta agonists are effective analgesics against thermal, chemical and electrical stimuli (tooth pulp stimulation).

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; beta-Endorphin; Dental Pulp; Dynorphins; Electric Stimulation; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Pain; Peptide Fragments; Pyrrolidines; Rats; Rats, Inbred Strains

Peptides based in the stabilised tetrapeptide HTyr-D-Ala-Gly-MePheOH have been synthesised and shown to have substantial opioid activity both in vitro and in vivo. The selectivity of these compounds of different receptor populations has been investigated using both isolated tissue assays and binding studies. Results suggest that the compounds are potent agonists at mu-receptors with little or no affinity for the delta-receptor population. One of the compounds, RX783006 (HTyr-D-Ala-Gly-MePhe-NH(CH2)2OH), has been tritiated to high specific radioactivity and may prove to be a useful probe in the elucidation of the function of the heterogenous opiate receptor population.

Topics: Animals; Behavior, Animal; beta-Lipotropin; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Muscle Contraction; Naloxone; Pain; Peptide Fragments; Radioligand Assay; Receptors, Opioid; Structure-Activity Relationship; Vas Deferens