ACTIVATION OF PERIPHERAL kappa/delta OPIOID RECEPTORS MEDIATES 15-DEOXY-(Delta 12,14)-PROSTAGLANDIN J(2) INDUCED-ANTINOCICEPTION IN RAT TEMPOROMANDIBULAR JOINT

This study assessed the effect of the agonist 15d-PGJ(2) administered into the rat temporomandibular joint (TMJ) on nociceptive behavioral and the anti-inflammatory potential of this prostaglandin on TMJ. It was observed that 15-deoxy-(Delta 12,14)-prostaglandin J(2) (15d-PGJ(2)) significantly reduced formalin-induced nociceptive behavior in a dose dependent manner, however injection of 15d-PGJ(2) into the contralateral TMJ failed to reduce such effects. This antinociceptive effect is dependent on peroxisome proliferator-activated receptors-gamma (PPAR-gamma) since pre-treatment with GW9662 (PPAR-gamma receptor antagonist) blocked the antinociceptive effect of 15d-PGJ(2) in the TMJ. In addition, the antinociceptive effect of 15d-PGJ(2) was also blocked by naloxone suggesting the involvement of peripheral opioids in the process. Confirming this hypothesis pre-treatment with kappa, delta, but not mu receptor antagonists significantly reduced the antinociceptive effect of 15d-PGJ(2) in the TMJ. Similarly to opioid agonists, the 15d-PGJ(2) antinociceptive action depends on the nitric oxide (NO)/guanilate cyclase (cGMP)/ATP-sensitive potassium channel blocker(K(ATP)(+)) channel pathway since it was prevented by the pre-treatment with the inhibitors of nitric oxide synthase (NOS; aminoguanidine), cGMP (ODQ), or the K(ATP)(+) (glibenclamide). In addition, 15d-PGJ(2) (100 ng/TMJ) inhibits 5-HT-induced TMJ hypernociception. Besides, TMJ treated with 15d-PGJ(2) showed lower vascular permeability, assessed by Evan`s Blue extravasation, and also lower neutrophil migration induced by carrageenan administration. Taken together, these results demonstrate that 15d-PGJ(2) has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-gamma activation. The results also suggest that 15d-PGJ(2) induced-peripheral antinociceptive response in the TMJ is mediated by kappa/delta opioid receptors by the activation of the intracellular L-arginine/NO/cGMP/K(ATP)(+) channel pathway. The pharmacological properties of the peripheral administration of 15d-PGJ(2) highlight the potential use of this PPAR-gamma agonist on TMJ inflammatory pain conditions. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6 μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1 h after treatment and lasted for up to 48 h. Intraplantar injection of nor-binaltorphimine (50 g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10 μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20 g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain. © 2013 Elsevier Inc.

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.

Plasticity of Opioid Receptors in the Female Periaqueductal Gray: Multiparity-Induced Increase in the Activity of Genes Encoding for Mu and Kappa Receptors and a Post-Translational Decrease in Delta Receptor Expression

The periaqueductal gray (PAG) has been reported as a potential site for opioid regulation of behavioral selection. Opioid-mediated behavioral and physiological responses differ between nulliparous and multiparous females. This study addresses the effects of multiple reproductive experiences on mu-, kappa- and delta-opioid receptor (Oprm1, Oprk1, and Oprd1 respectively) gene activity and mu, kappa and delta protein expression (MOR, KOR and DOR respectively) in the PAG of the female rats. This was done by evaluating the opioid gene expression using real-time (RT-PCR) and quantification of each protein receptor by Western blot analysis. The RT-PCR results show that multiple reproductive experiences increase Oprm1 and Oprk1 gene expression. Western blot analysis revealed increased MOR and KOR while DOR protein was decreased in multiparous animals. Taken together, these data suggest that multiple reproductive experiences influence both gene activity and opioid receptor expression in the PAG. Post-translational mechanisms seem particularly relevant for DOR expression. Thus, opioid transmission in the PAG might be modulated by different mechanisms of multiparity-induced plasticity according to the opioid receptor type.

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

Role of preoptic opioid receptors in the body temperature reduction during hypoxia

Evidence indicates that endogenous opioids play a role in body temperature (Tb) regulation in mammals but no data exist about the involvement of the specific opioid receptors, mu, kappa and delta, in the reduction of Tb induced by hypoxia. Thus, we investigated the participation of these opioid receptors in the anteroventral preoptic region (AVPO) in hypoxic decrease of Th. To this end, Th of unanesthetized Wistar rats was monitored by temperature data loggers before and after intra-AVPO microinjection of the selective kappa-opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 0.1 and 1.0 mu g/100 nL/animal), the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) cyclic (CTAP; 0.1 and 1.0 mu g/100 nL/animal), and the selective delta-opioid receptor antagonist Naltrindole (0.06 and 0.6 mu g/100 nL/animal) or saline (vehicle, 100 nu animal), during normoxia and hypoxia (7% inspired O(2)). Under normoxia, no effect of opioid antagonists on Th was observed. Hypoxia induced Th to reduce in vehicle group, a response that was inhibited by the microinjection intra-AVPO of nor-BNI. In contrast, CTAP and Naltrindole did not change Th during hypoxia but caused a longer latency for the return of Th to the normoxic values just after low O(2) exposure. Our results indicate the kappa-opioid receptor in the AVPO is important for the reduction of Th during hypoxia while the mu and delta receptors are involved in the increase of Th during normoxia post-hypoxia. (C) 2009 Elsevier B.V. All rights reserved.

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing.

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.

Role of preoptic opioid receptors in the body temperature reduction during hypoxia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.

The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures-the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala(2) N-Me-Phe(4) Gly(5)-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 mu l) - a selective mu-receptor agonist - or nor-binaltorphimine (BNI; 2.5 and 5 mu g/0.2 mu l) - a selective K-receptor antagonist with tardive action - on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region. (C) 2009 Elsevier B.V. All rights reserved.

Behavioral effects of acute stimulation of kappa-opioid receptors during lactation

The behavioral effects of the K-opioid receptor agonist U69593 were examined in lactating rats. On day 5 of lactation, animals were treated with 0.1 mg/kg of U69593 to determine whether it influences general activity and maternal latencies toward pups. Because little attention has been given to the possibility that pre-mating treatment with morphine may modulate the response to K-opioid receptor stimulation, another group of animals was submitted to the same acute challenge after abrupt withdrawal from repeated treatment with morphine sulfate during the pre-mating period (5 mg/kg on alternate days for a total of five doses). Acute F;opioid stimulation reduced total locomotion, rearing frequency, and time spent self-grooming and increased immobility duration. These K agonist effects were not observed in animals pretreated with morphine. Similarly, latencies to retrieve pups were longer only in animals pretreated with saline and challenged acutely with U69593. None of these effects were observed in morphine sulfate-pretreated animals. The present results suggest that pre-mating repeated exposure to morphine produces a tolerance-like effect on behavioral responses to low-dose K-opioid receptor stimulation in active reproductive females. (c) 2008 Elsevier Inc. All rights reserved.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

Predicting the Pose of β-Casomorphin-5 and 7 in the Opioid Receptors

The opioid receptors consist of three main subtypes; μ, δ, and κ. Previous binding studies have shown that fragments of the milk protein, β-casein, known as β-casomorphins are agonists of these receptors which are selective for the μ receptor subtype. Using the crystal structures of these three receptors, computational molecular docking studies were done using the software GOLD to determine the conformation of β-casomorphin-5 and 7 when they bind to these three opioid receptors. GOLD was able to discriminate among the three receptors when docking the rigid ligands co-crystalized with the receptors. However, GOLD could not discriminate among the three receptors for either of the highly flexible β-casomorphins. A per amino acid scoring method was developed to overcome this problem. This method was used to predict the conformation of both β-casomorphin-5 and 7 in the μ receptor and determine that the two amino acid residues, Lys303 and Trp318 of the μ receptor are responsible for discriminating among the three receptor subtypes for binding of the β-casomorphin-5 and 7.

Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction, of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2-5 mu g/kg) and the crude venom (400-1600 mu g/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 mu g/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs. (C) 2008 Elsevier B.V. All rights reserved.