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1.
PLoS One ; 15(12): e0232864, 2020.
Article in English | MEDLINE | ID: mdl-33373369

ABSTRACT

Activation of the kappa opioid receptor (KOR) contributes to the aversive properties of stress, and modulates key neuronal circuits underlying many neurobehavioral disorders. KOR agonists directly inhibit ventral tegmental area (VTA) dopaminergic neurons, contributing to aversive responses (Margolis et al. 2003, 2006); therefore, selective KOR antagonists represent a novel therapeutic approach to restore circuit function. We used whole cell electrophysiology in acute rat midbrain slices to evaluate pharmacological properties of four novel KOR antagonists: BTRX-335140, BTRX-395750, PF-04455242, and JNJ-67953964. Each compound concentration-dependently reduced the outward current induced by the KOR selective agonist U-69,593. BTRX-335140 and BTRX-395750 fully blocked U-69,593 currents (IC50 = 1.2 ± 0.9 and 1.2 ± 1.3 nM, respectively). JNJ-67953964 showed an IC50 of 3.0 ± 4.6 nM. PF-04455242 exhibited partial antagonist activity asymptoting at 55% blockade (IC50 = 6.7 ± 15.1 nM). In 3/8 of neurons, 1 µM PF-04455242 generated an outward current independent of KOR activation. BTRX-335140 (10 nM) did not affect responses to saturating concentrations of the mu opioid receptor (MOR) agonist DAMGO or the delta opioid receptor (DOR) agonist DPDPE, while JNJ-67953964 (10 nM) partially blocked DAMGO and DPDPE responses. Importantly, BTRX-335140 (10 nM) rapidly washed out with complete recovery of U-69,593 responses within 10 min. Collectively, we show electrophysiological evidence of key differences amongst KOR antagonists that could impact their therapeutic potential and have not been observed using recombinant systems. The results of this study demonstrate the value of characterizing compounds in native neuronal tissue and within circuits implicated in the neurobehavioral disorders of interest.


Subject(s)
Dopaminergic Neurons/drug effects , Membrane Potentials/drug effects , Receptors, Opioid, kappa/antagonists & inhibitors , Analgesics, Opioid/pharmacology , Animals , Benzamides/pharmacology , Biphenyl Compounds/pharmacology , Dopaminergic Neurons/metabolism , Electrophysiology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Male , Mesencephalon/metabolism , Narcotic Antagonists/pharmacology , Oxadiazoles/pharmacology , Patch-Clamp Techniques/methods , Piperidines/pharmacology , Pyrrolidines/pharmacology , Quinolines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/metabolism , Sulfonamides/pharmacology , Ventral Tegmental Area/drug effects
2.
Reprod Toxicol ; 93: 211-218, 2020 04.
Article in English | MEDLINE | ID: mdl-32145291

ABSTRACT

The endogenous opioid peptides have been reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with sentences around the harmful effect of opioids in male fertility but, actually, there is only one study regarding the real fertility potential of spermatozoa that have been exposed to mu specific opioids. The aim of the present study was to see if the modulation of delta (OPRD1) and kappa (OPRK1) opioid receptors in mouse sperm during capacitation was able to vary the embryo production after in vitro fertilization (IVF). The presence of OPRD1 and OPRK1 in mouse mature spermatozoa was analyzed by RT-PCR and immunofluorescence. Incubating the sperm with, on one hand, the delta specific agonist DPDPE and/or antagonist naltrindole, and, on the other hand, the kappa specific agonist U-50488 and antagonist nor-binaltorphimine, we analyzed the involvement of OPRD1 and OPRK1 on IVF and preimplantational embryo development. We verified the presence of OPRD1 and OPRK1 in mouse mature spermatozoa, not only at the mRNA level but also at protein level. Moreover, the sperm incubation with DPDPE, before the IVF, had an effect on the fertilization rate of sperm and reduced the number of reached blastocysts, which was reverted by naltrindole. Instead, the use of the kappa agonist U-50488 and the antagonist nor-binaltophimine did not have any effect on the amount and the quality of the achieved blastocysts. Although nowadays the pure delta or kappa opioid ligands are not used for the clinic, clinical trials are being conducted to be used in the near future, so it would be interesting to know if the modulation of these receptors in sperm would generate any consequence in relation to fertilization capacity.


Subject(s)
Fertilization in Vitro , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/metabolism , Spermatozoa/physiology , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Animals , Blastocyst/physiology , Embryo, Mammalian , Embryonic Development , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Male , Mice , Naltrexone/analogs & derivatives , Naltrexone/pharmacology , Narcotic Antagonists/pharmacology , Oocytes/physiology , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, delta/genetics , Receptors, Opioid, kappa/genetics , Sperm Capacitation
3.
Sci Rep ; 9(1): 5771, 2019 04 08.
Article in English | MEDLINE | ID: mdl-30962495

ABSTRACT

Here, we report the chemical synthesis of two DPDPE analogues 7a (NOVA1) and 7b (NOVA2). This entailed the solid-phase synthesis of two enkephalin precursor chains followed by a CuI-catalyzed azide-alkyne cycloaddition, with the aim of improving in vivo analgesic efficacy versus DPDPE. NOVA2 showed good affinity and selectivity for the µ-opioid receptor (KI of 59.2 nM, EC50 of 12.9 nM, EMax of 87.3%), and long lasting anti-nociceptive effects in mice when compared to DPDPE.


Subject(s)
Analgesics/chemical synthesis , Click Chemistry/methods , Enkephalin, D-Penicillamine (2,5)-/analogs & derivatives , Analgesics/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Cycloaddition Reaction/methods , Enkephalin, D-Penicillamine (2,5)-/chemical synthesis , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Humans , Male , Mice , Protein Binding , Receptors, Opioid, mu/metabolism
4.
Am J Physiol Regul Integr Comp Physiol ; 316(6): R727-R734, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30943058

ABSTRACT

A reflex arising from contracting hindlimb muscle is responsible in part for the increases in arterial pressure and heart rate evoked by exercise. The afferent arm of this reflex comprises group III and IV afferents. δ-Opioid receptors are expressed predominately on the spinal endings of group III afferents, whereas µ-opioid receptors are expressed predominately on the spinal endings of group IV afferents. Using stimuli that activated group III afferents, namely static contraction, calcaneal tendon stretch, and lactic acid injection into the superficial epigastric artery, we tested the hypothesis that, in rats with either patent or ligated femoral arteries, activation of pre- and postsynaptic δ-opioid receptors in the dorsal horn attenuated pressor reflex responses to these stimuli. In rats with patent arteries or ligated femoral arteries, [d-Pen2,5]enkephalin (DPDPE), a δ-opioid agonist injected intrathecally (10 µg in 10 µl), significantly attenuated the pressor responses to contraction, stretch, and lactic acid (all P < 0.05). Naltrindole, a δ-opioid receptor antagonist, prevented the attenuation. In contrast, DPDPE did not attenuate the pressor response to capsaicin injection into the superficial epigastric artery in either group of rats (both P > 0.05). Intrathecal injection of saline (10 µl), the vehicle for DPDPE, had no effect on the pressor responses in either group of rats. We conclude that activation of spinal δ-opioid receptors attenuates reflexes evoked by group III afferents in both freely perfused and ligated rats.


Subject(s)
Enkephalin, D-Penicillamine (2,5)-/pharmacology , Physical Conditioning, Animal/physiology , Receptors, Opioid, delta/drug effects , Reflex/physiology , Animals , Decerebrate State/physiopathology , Femoral Artery/physiopathology , Heart Rate/physiology , Male , Muscle Contraction/physiology , Muscle, Skeletal/physiology , Physical Exertion/physiology , Rats, Sprague-Dawley , Receptors, Opioid, mu/drug effects
5.
Eur J Pharmacol ; 837: 88-95, 2018 Oct 15.
Article in English | MEDLINE | ID: mdl-30086266

ABSTRACT

The nucleus accumbens contains delta-opioid receptors that may decrease inhibitory neurotransmission. As GABAB receptors inhibit dopamine release, decrease in activation of GABAB receptors may be a mediator of delta-opioid receptor-induced accumbal dopamine efflux. If so, accumbal dopamine efflux induced by delta-opioid receptor activation should be suppressed by stimulating GABAB receptors. As delta-opioid receptors are further subdivided into delta1- and delta2-opioid receptors, we analysed the effects of the GABAB receptor agonist baclofen on delta1- and delta2-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds show total amount administered (mol) during 25-50 min infusions. Baclofen (2.5 and 5.0 nmol), which did not alter basal dopamine levels, inhibited the delta1-opioid receptor agonist DPDPE (5.0 nmol)-induced dopamine efflux. Baclofen (2.5 and 5.0 nmol) also inhibited the delta2-opioid receptor agonist deltorphin II (25.0 nmol)-induced dopamine efflux. A low dose of the GABAB receptor antagonist 2-hydroxysaclofen (100.0 pmol), which failed to alter basal accumbal dopamine levels, counteracted the inhibitory effects of baclofen (5.0 nmol) on DPDPE (5.0 nmol)- and deltorphin II (25.0 nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABAB receptor-mediated inhibition of accumbal dopaminergic activity facilitates activation of delta1- and delta2-opioid receptor-induced increases in accumbal dopamine efflux. This study suggests that activation of delta1- and delta2-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABAB receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux.


Subject(s)
Dopamine/metabolism , Nucleus Accumbens/metabolism , Receptors, GABA-B/physiology , Receptors, Opioid, delta/physiology , Animals , Baclofen/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Male , Oligopeptides/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology
6.
Brain Res ; 1697: 53-58, 2018 10 15.
Article in English | MEDLINE | ID: mdl-29902466

ABSTRACT

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.


Subject(s)
Amniotic Fluid/physiology , Pain Measurement/methods , Pain Threshold/drug effects , Receptors, Opioid, delta/agonists , Amniotic Fluid/metabolism , Analgesics/pharmacology , Analgesics, Opioid/pharmacology , Animals , Cold Temperature/adverse effects , Eating , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Enkephalins/pharmacology , Female , Pain/metabolism , Pregnancy , Rats , Rats, Long-Evans , Receptors, Opioid, delta/drug effects , Water
7.
J Physiol Pharmacol ; 69(5)2018 Oct.
Article in English | MEDLINE | ID: mdl-30683824

ABSTRACT

Steroid hormones play an important role in the regulation of cyclic changes in the uterus and preparation of intrauterine environment for the egg fertilization, embryo implantation and maintenance of pregnancy. Their secretion by porcine uterus has been demonstrated. The present study aimed to establish the effect of opioid receptors (µ, δ and κ) activation by selective agonists (DAMGO, DPLPE and U 50.488, respectively) on in vitro secretion of steroid hormones (during 6-h and 24-h incubations) by the endometrial explants of gilts on days 2 - 3, 10 - 11, 12 - 3, 15 - 16, 18 - 20 of the estrous cycle and 10 - 11, 12 - 13, 15 - 16 of pregnancy. The agonists at certain of tested concentrations (10-9, 10-8 and 10-7 M) affected secretion of steroid hormones. Progesterone secretion was increased by µ-opioid receptor agonist on days 18 - 20 (6 h) and by δ-agonist on days 2 - 3 and 18 - 20 (24 h) of the cycle. During pregnancy (days 15 - 16), κ-agonist increased it (6 h), but µ-opioid agonist decreased (24 h). Androstenedione secretion was decreased during shorter incubation; by µ- and δ-receptor agonists on days 2 - 3, by all agonists on days 12 - 13, and by κ-receptor agonist on days 18 - 20 of the cycle. However, it was increased during longer incubation with agonists of κ- and µ-opioid receptors on days 10 - 11 and 18 - 20 of the cycle, respectively. Estradiol secretion was elevated by κ- and µ-agonists (6 h) on days 2 - 3 and 15 - 16 of the cycle, respectively, as well as following 24-h incubation with µ-agonist on days 15 - 16, and µ- and κ-agonists on days 18 - 20 of the cycle. During pregnancy, its secretion was increased (24 h) ondays 15 - 16 by µ- and κ-opioid agonists. Cortisol secretion did not significantly change (versus control) in response to applied treatments. These results indicate a potential involvement of EOPs in the modulation of endometrial steroidogenesis in the pig during the estrous cycle and pregnancy.


Subject(s)
Endometrium/metabolism , Estrous Cycle/metabolism , Pregnancy/metabolism , Receptors, Opioid/agonists , Steroids/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Analgesics, Opioid/pharmacology , Animals , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Female , Swine
8.
ACS Chem Neurosci ; 8(11): 2549-2557, 2017 11 15.
Article in English | MEDLINE | ID: mdl-28796483

ABSTRACT

In an effort to expand the structure-activity relationship (SAR) studies of a series of mixed-efficacy opioid ligands, peptidomimetics that incorporate methoxy and hydroxy groups around a benzyl or 2-methylindanyl pendant on a tetrahydroquinoline (THQ) core of the peptidomimetics were evaluated. Compounds containing a methoxy or hydroxy moiety in the o- or m-positions increased binding affinity to the kappa opioid receptor (KOR), whereas compounds containing methoxy or hydroxy groups in the p-position decreased KOR affinity and reduced or eliminated efficacy at the mu opioid receptor (MOR). The results from a substituted 2-methylindanyl series aligned with the findings from the substituted benzyl series. Our studies culminated in the development of 8c, a mixed-efficacy MOR agonist/KOR agonist with subnanomolar binding affinity for both MOR and KOR.


Subject(s)
Analgesics, Opioid/chemistry , Peptidomimetics/chemistry , Receptors, Opioid, delta/agonists , Receptors, Opioid, kappa/agonists , Receptors, Opioid, mu/agonists , Analgesics, Opioid/chemical synthesis , Analgesics, Opioid/pharmacology , Animals , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Drug Evaluation, Preclinical , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , Mice , Naloxone/pharmacology , Peptidomimetics/chemical synthesis , Peptidomimetics/pharmacology , Protein Binding , Protein Conformation , Rats , Structure-Activity Relationship
9.
PLoS One ; 12(7): e0180998, 2017.
Article in English | MEDLINE | ID: mdl-28700700

ABSTRACT

The involvement of heme oxygenase 1 (HO-1) in the modulation of the antinociceptive effects of opioids in type 1 diabetes has been demonstrated but the role played by the transcription factor Nrf2 in the regulation of painful neuropathy and in the effects and expression of δ-opioid receptors (DOR) in type 2 diabetes, has not been studied. In male BKS.Cg-m+/+Leprdb/J (db/db) mice, the anti-allodynic effects produced by a Nrf2 transcription factor activator, sulforaphane (SFN) administered alone and combined with two DOR agonists, [d-Pen(2),d-Pen(5)]-Enkephalin (DPDPE) and (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N diethylbenzamide (SNC-80), were evaluated. The effects of SFN on glucose levels and body weight as well as on the proteins levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), MAPKs (JNK) and DOR in sciatic nerve from db/db mice were also assessed. This study showed that the administration of SFN dose dependently reversed mechanical allodynia, reduced hyperglycemia and body weight gain associated to type 2 diabetes and significantly increased the anti-allodynic effects of DPDPE and SNC-80 in db/db mice. This treatment normalized the down regulation of Nrf2 and NQO1 and enhanced the protein levels of HO-1 in db/db mice. Moreover, the administration of SFN also inhibited the JNK phosphorylation and DOR down-regulation in the sciatic nerve of diabetic mice. Our data indicated that SFN treatment is effective in reversing mechanical allodynia and enhancing DOR antinociceptive effects in db/db mice which effects might be mediated by activating Nrf2 signaling, reducing hyperglycemia, inhibiting JNK phosphorylation and avoiding DOR down-regulation in the sciatic nerve of these animals. These results propose SFN, alone and/or combined with DOR agonists, as interesting approaches for the treatment of painful diabetic neuropathy associated to type 2 diabetes in mice.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , NF-E2-Related Factor 2/metabolism , Receptors, Opioid, delta/metabolism , Animals , Benzamides/pharmacology , Blood Glucose/drug effects , Blood Glucose/metabolism , Blotting, Western , Body Weight/drug effects , Body Weight/genetics , Body Weight/physiology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetic Neuropathies/metabolism , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Hyperalgesia/metabolism , Isothiocyanates/pharmacology , Male , Mice , NF-E2-Related Factor 2/genetics , Piperazines/pharmacology , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/genetics , Sulfoxides
10.
Sci Rep ; 6: 32799, 2016 09 08.
Article in English | MEDLINE | ID: mdl-27605249

ABSTRACT

Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides ß-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment.


Subject(s)
Analgesics, Opioid/pharmacology , Neuralgia/drug therapy , Neuralgia/etiology , Opioid Peptides/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Animals , Corticotropin-Releasing Hormone/pharmacology , Dynorphins/genetics , Dynorphins/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Enkephalin, Methionine/genetics , Enkephalin, Methionine/metabolism , Enkephalin, Methionine/pharmacology , Hot Temperature/adverse effects , Male , Mice, Inbred C57BL , Mice, Knockout , Neuralgia/genetics , Opioid Peptides/genetics , Peripheral Nervous System/metabolism , Receptors, Opioid/agonists , beta-Endorphin/genetics , beta-Endorphin/metabolism , beta-Endorphin/pharmacology
11.
Eksp Klin Farmakol ; 78(1): 27-9, 2015.
Article in Russian | MEDLINE | ID: mdl-25826871

ABSTRACT

It is established that activation of delta1 opioid receptors with their selective agonist DPDPE(100 µg/kg) significantly increases IgM immune response not only in C57BL/6J mice with unchanged psychoemotional state, but also in mice displaying aggressive or depressive-like behavior in the social stress model (10 days of agonistic confrontations). SCH-23390 (1.0 mg/kg), selective antagonist of dopamine D1 receptors, and selective D2 receptor blocker haloperidol (1 mg/kg) prevented the immunostimulating effect of DPDPE in animals not subjected to social stress. At the same time, both SCH-23390 and haloperidol did not affect DPDPE-induced immunostimulation in mice engaged in aggressive or depressive-like behaviors.


Subject(s)
Aggression/drug effects , Analgesics, Opioid/pharmacology , Depression/immunology , Dopamine Antagonists/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Immunoglobulin M/biosynthesis , Stress, Psychological/immunology , Aggression/psychology , Animals , Benzazepines/pharmacology , Depression/drug therapy , Depression/psychology , Erythrocytes/immunology , Haloperidol/pharmacology , Immunization , Male , Mice , Mice, Inbred C57BL , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/metabolism , Receptors, Opioid, delta/metabolism , Sheep , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Stress, Psychological/drug therapy
12.
Brain Res ; 1609: 1-11, 2015 Jun 03.
Article in English | MEDLINE | ID: mdl-25801116

ABSTRACT

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.


Subject(s)
Analgesics, Opioid/pharmacology , Enkephalins/pharmacology , Neuroprotective Agents/pharmacology , Stroke/drug therapy , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/chemistry , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Analgesics, Opioid/chemistry , Brain Edema/drug therapy , Brain Edema/pathology , Brain Edema/physiopathology , Brain Infarction/drug therapy , Brain Infarction/pathology , Brain Infarction/physiopathology , Brain Ischemia/drug therapy , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Cells, Cultured , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/chemistry , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/chemistry , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Enkephalins/chemistry , Glutamic Acid/toxicity , Ischemia/drug therapy , Ischemia/pathology , Motor Activity/drug effects , Motor Activity/physiology , Neurons/drug effects , Neurons/pathology , Neuroprotective Agents/chemistry , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/agonists , Receptors, Opioid, kappa/metabolism , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/metabolism , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Severity of Illness Index , Stroke/pathology , Stroke/physiopathology
13.
J Pharmacol Exp Ther ; 353(1): 44-51, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25637601

ABSTRACT

The regulation of opioid receptor system function in peripheral sensory neurons is not well understood. Opioid agonist efficacy to inhibit nociceptor function and to promote antinociception is generally weak under basal conditions and frequently no response occurs. However, in response to a cyclooxygenase-dependent metabolite of arachidonic acid (AA) after exposure to inflammatory mediators, such as bradykinin (BK) or exogenous AA, peripheral opioid receptor systems become much more responsive to opioid agonists. In this study, we examined the time course for the induction and maintenance of functional competence of the δ-opioid receptor (DOR) system in adult rat nociceptors in culture and in vivo. We found that the responsive state of DOR after pretreatment with BK or exogenous AA is transient (30-60 minutes) and persists for 15-30 minutes after a 15-minute exposure of nociceptors to BK or AA. Interestingly, whereas functional competence of the DOR system could be reinduced with a second application of BK 60 minutes after the first, responsiveness of the DOR system could not be reinduced after an initial exposure to AA. This nonresponsive state of DOR after exogenous AA was mediated by a lipoxygenase (LOX)-dependent metabolite of AA. Intraplantar carrageenan also produced transient DOR functional competence and responsiveness was also reinduced by inhibition of LOX. Thus, the DOR system expressed by peripheral sensory neurons is under dual regulation by cyclooxygenase- and LOX-dependent metabolites of AA.


Subject(s)
Arachidonic Acid/metabolism , Receptors, Opioid, delta/metabolism , Sensory Receptor Cells/metabolism , Animals , Arachidonic Acid/pharmacology , Bradykinin/pharmacology , Cells, Cultured , Cyclic AMP/metabolism , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Hyperalgesia/metabolism , Hyperalgesia/physiopathology , Lipoxygenase/metabolism , Lipoxygenase Inhibitors/pharmacology , Male , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Sensory Receptor Cells/drug effects , Trigeminal Ganglion/cytology
14.
J Pharmacol Exp Ther ; 351(1): 224-32, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25204546

ABSTRACT

The administration of µ-opioid receptor (MOR), δ-opioid receptor (DOR), and cannabinoid 2 receptor (CB2R) agonists attenuates inflammatory pain. We investigated whether treatment with the heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX (CoPP), could modulate the local effects and expression of MOR, DOR, or CB2R during chronic inflammatory pain. In mice with inflammatory pain induced by the subplantar administration of complete Freund's adjuvant, we evaluated the effects of the intraperitoneal administration of 10 mg/kg CoPP on the antiallodynic and antihyperalgesic actions of locally administered MOR (morphine), DOR (DPDPE {[d-Pen(2),d-Pen(5)]-enkephalin}), or CB2R [JWH-015 {(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone}] agonists and its reversion with the HO-1 inhibitor, tin protoporphyrin IX (SnPP). The effect of CoPP treatment on the dorsal root ganglia expression of HO-1, MOR, DOR, and CB2R was also assessed. The results show that treatment with CoPP increased the local antinociceptive effects produced by morphine, DPDPE, or JWH-015 during chronic inflammatory pain, and these effects were blocked by the subplantar administration of SnPP, indicating the participation of HO-1 in the antinociceptive actions. CoPP treatment, apart from inducing the expression of HO-1, also enhanced the expression of MOR, did not alter CB2R, and avoided the decreased expression of DOR induced by inflammatory pain. This study shows that the HO-1 inducer (CoPP) increased the local antinociceptive effects of MOR, DOR, and CB2R agonists during inflammatory pain by altering the peripheral expression of MOR and DOR. Therefore, the coadministration of CoPP with local morphine, DPDPE, or JWH-015 may be a good strategy for the management of chronic inflammatory pain.


Subject(s)
Analgesics/pharmacology , Heme Oxygenase-1/antagonists & inhibitors , Nociceptive Pain/drug therapy , Protoporphyrins/pharmacology , Analgesics/therapeutic use , Animals , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Male , Mice , Mice, Inbred C57BL , Morphine/pharmacology , Nociception/drug effects , Nociceptive Pain/metabolism , Protoporphyrins/therapeutic use , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/metabolism , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Receptors, sigma/agonists , Receptors, sigma/genetics , Receptors, sigma/metabolism
15.
Am J Physiol Cell Physiol ; 307(3): C266-77, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24898587

ABSTRACT

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved µ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined µ {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and δ ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 µM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.


Subject(s)
Chromaffin Cells/metabolism , KATP Channels/biosynthesis , Potassium Channels, Inwardly Rectifying/biosynthesis , Receptors, Opioid, delta/agonists , Receptors, Opioid, mu/agonists , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Adrenal Cortex/cytology , Adrenal Cortex/metabolism , Adrenal Medulla/cytology , Adrenal Medulla/metabolism , Analgesics, Opioid/pharmacology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carbonic Anhydrase I/biosynthesis , Carbonic Anhydrase II/biosynthesis , Cell Hypoxia , Cell Line , Chromaffin Cells/cytology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Dopamine/metabolism , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Enzyme Inhibitors/pharmacology , Female , Hypercapnia , Indoles/pharmacology , Isoquinolines/pharmacology , KATP Channels/genetics , Maleimides/pharmacology , Morphine/pharmacology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Norepinephrine/metabolism , Oligopeptides/pharmacology , Potassium Channels, Inwardly Rectifying/genetics , Pregnancy , Promoter Regions, Genetic , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar , Sulfonamides/pharmacology
16.
PLoS One ; 9(4): e82315, 2014.
Article in English | MEDLINE | ID: mdl-24691128

ABSTRACT

BACKGROUND: An opioid peptide neuron/humoral feedback regulation might be involved in changes of intraocular pressure (IOP). The aims of this study are to investigate the effects of arcuate nucleus (ARC) and opioid peptides on intraocular pressure (IOP). METHODS: Fifty-four healthy purebred New Zealand white rabbits (108 eyes) were randomly divided into 4 groups, including control group, electrical stimulation group, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) group, and [D-Pen 2, D-Pen5]- enkephalin (DPDPE) group. Bilateral IOP was measured after unilateral electrical stimulation of the ARC or unilateral microinjection into the ARC of the selective µ-opioid receptor agonist DAMGO or the selective δ opioid receptor agonist DPDPE, both alone and after pre-administration of either the non-selective opioid receptor antagonist naloxone or saline. RESULTS: Both electrical stimulation in ARC and micro-injection either or opioid receptor agonists, DAMGO or DPDPE, respectively, caused a significant bilateral reduction in IOP (P<0.05) which was more pronounced in the ipsilateral than in the contralateral eye. Pretreatment with naloxone prevented some, but not all IOP reductions. CONCLUSION: The ARC takes part in the negative regulation of IOP, an action that may involve opioid neurons.


Subject(s)
Arcuate Nucleus of Hypothalamus/physiology , Intraocular Pressure/physiology , Opioid Peptides/pharmacology , Animals , Arcuate Nucleus of Hypothalamus/anatomy & histology , Arcuate Nucleus of Hypothalamus/drug effects , Electric Stimulation , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/administration & dosage , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Injections , Intraocular Pressure/drug effects , Rabbits , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/metabolism
17.
Am J Physiol Regul Integr Comp Physiol ; 306(4): R265-72, 2014 Feb 15.
Article in English | MEDLINE | ID: mdl-24401991

ABSTRACT

The central opioid system is involved in a broadly distributed neural network that regulates food intake. Here, we show that activation of central δ-opioid receptor not only stimulated normal diet intake but conversely suppressed high-fat diet intake as well. [D-Pen(2,5)]-enkephalin (DPDPE), an agonist selective for the δ-receptor, increased normal diet intake after central administration to nonfasted male mice. The orexigenic activity of DPDPE was inhibited by blockade of cyclooxygenase (COX)-2, lipocalin-type prostaglandin D synthase (L-PGDS), D-type prostanoid receptor 1 (DP(1)), and neuropeptide Y (NPY) receptor type 1 (Y1) for PGD(2) and NPY, respectively, suggesting that this was mediated by the PGD(2)-NPY system. In contrast, DPDPE decreased high-fat diet intake in mice fed a high-fat diet. DPDPE-induced suppression of high-fat diet intake was blocked by antagonists of melanocortin 4 (MC(4)) and corticotropin-releasing factor (CRF) receptors but not by knockout of the L-PGDS gene. These results suggest that central δ-opioid receptor activation suppresses high-fat diet intake via the MC-CRF system, independent of the orexigenic PGD(2) system. Furthermore, orally administered rubiscolin-6, an opioid peptide derived from spinach Rubisco, suppressed high-fat diet intake. This suppression was also blocked by centrally administered naltrindole, an antagonist for the δ-receptor, suggesting that rubiscolin-6 suppressed high-fat diet intake via activation of central δ-opioid receptor.


Subject(s)
Analgesics, Opioid/pharmacology , Eating/drug effects , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Receptors, Opioid, delta/agonists , Animals , Celecoxib , Cyclooxygenase Inhibitors/pharmacology , Diet, High-Fat , Eating/physiology , Male , Mice , Pyrazoles/pharmacology , Receptors, Prostaglandin/antagonists & inhibitors , Sulfonamides/pharmacology
18.
Eur J Pain ; 18(1): 29-38, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23740773

ABSTRACT

BACKGROUND: Although the efficacy of peripherally administered opioid has been demonstrated in preclinical and clinical studies, the underlying mechanisms of its anti-hyperalgesic effects are poorly understood. G protein-coupled inwardly rectifying potassium (GIRK) channels are linked to opioid receptors in the brain. However, the role of peripheral GIRK channels in analgesia induced by peripherally administered opioid, especially in trigeminal system, is not clear. METHODS: Expression of GIRK subunits in rat trigeminal ganglia (TG) was examined with reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. Chemical profiles of GIRK-expressing neurons in TG were further characterized. Behavioural and Fos experiments were performed to examine the functional involvement of GIRK channels in δ-opioid receptor (DOR)-mediated anti-hyperalgesia under an acute myositis condition. RESULTS: TG expressed mRNA and proteins for GIRK1 and GIRK2 subunits. Majority of GIRK1- and GIRK2-expressing neurons were non-peptidergic afferents. Inhibition of peripheral GIRK using Tertiapin-Q (TPQ) attenuated antinociceptive effects of peripherally administered DOR agonist, [D-Pen(2), D-Pen(6) ]-enkephalin (DPDPE), on mechanical hypersensitivity in masseter muscle. Furthermore, TPQ attenuated the suppressive effects of peripheral DPDPE on neuronal activation in the subnucleus caudalis of the trigeminal nucleus (Vc) following masseteric injection of capsaicin. CONCLUSIONS: Our data indicate that peripheral DOR agonist-induced suppression of mechanical hypersensitivity in the masseter muscle involves the activity of peripheral GIRK channels. These results could provide a rationale for developing a novel therapeutic approach using peripheral GIRK channel openers to mimic or supplement the effects of peripheral opioid agonist.


Subject(s)
G Protein-Coupled Inwardly-Rectifying Potassium Channels/physiology , Hyperalgesia/physiopathology , Masseter Muscle/physiopathology , Receptors, Opioid, delta/physiology , Analgesics, Opioid/pharmacology , Animals , Behavior, Animal/physiology , Blotting, Western , Brain Stem/cytology , Brain Stem/drug effects , Capsaicin/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , G Protein-Coupled Inwardly-Rectifying Potassium Channels/drug effects , Genes, fos , Immunohistochemistry , Male , Physical Stimulation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Sensory System Agents/pharmacology , Trigeminal Ganglion/cytology , Trigeminal Ganglion/drug effects , Trigeminal Ganglion/physiology
19.
Am J Physiol Heart Circ Physiol ; 305(8): H1246-55, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23934854

ABSTRACT

In rats with ligated femoral arteries, the exercise pressor reflex is exaggerated, an effect that is attenuated by stimulation of peripheral µ-opioid receptors on group IV metabosensitive afferents. In contrast, δ-opioid receptors are expressed mostly on group III mechanosensitive afferents, a finding that prompted us to determine whether stimulation of these opioid receptors could also attenuate the exaggerated exercise pressor reflex in "ligated" rats. We found femoral arterial injection of [D-Pen2,D-Pen5]enkephalin (DPDPE; 1.0 µg), a δ-opioid agonist, significantly attenuated the pressor and cardioaccelerator components of the exercise pressor reflex evoked by hindlimb muscle contraction in both rats with ligated and patent femoral arteries. DPDPE significantly decreased the pressor responses to muscle mechanoreflex activation, evoked by tendon stretch, in ligated rats only. DPDPE (1.0 µg) had no effect in either group on the pressor and cardioaccelerator responses to capsaicin (0.2 µg), which primarily stimulates group IV afferents. DPDPE (1.0 µg) had no effect on the pressor and cardioaccelerator responses to lactic acid (24 mM), which stimulates group III and IV afferents, in rats with patent femoral arteries but significantly decreased the pressor response in ligated rats. Western blots revealed the amount of protein comprising the δ-opioid receptor was greater in dorsal root ganglia innervating hindlimbs with ligated femoral arteries than in dorsal root ganglia innervating hindlimbs with patent femoral arteries. Our findings support the hypothesis that stimulation of δ-opioid receptors on group III afferents attenuated the exercise pressor reflex.


Subject(s)
Mechanoreceptors/physiology , Muscle, Skeletal/innervation , Physical Conditioning, Animal/physiology , Receptors, Opioid, delta/physiology , Reflex/physiology , Analgesics, Opioid/pharmacology , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Femoral Artery/surgery , Heart Rate/drug effects , Heart Rate/physiology , Hemodynamics/drug effects , Hemodynamics/physiology , Ligation , Male , Mechanoreceptors/drug effects , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Reflex/drug effects
20.
J Pharmacol Exp Ther ; 346(3): 535-44, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23820126

ABSTRACT

Activation of opioid receptors on peripheral sensory neurons has the potential for safe pain control, as it lacks centrally mediated side effects. While this approach often only partially suppressed neuropathic pain in animal models, opioids were mostly applied to animal paws although neuropathy was induced at the nerve trunk. Here we aimed to identify the most relevant peripheral site of opioid action for efficient antinociception in neuropathy. On days 2 and 14 following a chronic constriction injury (CCI) of the sciatic nerve in mice, we evaluated dose and time relationships of the effects of µ-, δ-, and κ-opioid receptor agonists injected either at the CCI site or intraplantarly (i.pl.) into the lesioned nerve-innervated paw, on spontaneous paw lifting and heat and mechanical hypersensitivity (using Hargreaves and von Frey tests, respectively). We found that neither agonist diminished spontaneous paw lifting, despite the application site. Heat hypersensitivity was partially attenuated by i.pl. µ-receptor agonist only, while it was improved by all three agonists applied at the CCI site. Mechanical hypersensitivity was slightly diminished by all agonists administered i.pl., whereas it was completely blocked by all opioids injected at the CCI site. These antinociceptive effects were opioid receptor type-selective and site-specific. Thus, opioids might not be effective against spontaneous pain, but they improve heat and mechanical hypersensitivity in neuropathy. Importantly, efficient alleviation of hypersensitivity is governed by peripheral opioid receptors at the injured nerve trunk rather than at its peripheral terminals. Identifying the primary action site of analgesics is important for the development of adequate pain therapies.


Subject(s)
Analgesics, Opioid/pharmacology , Nerve Endings/drug effects , Neuralgia/drug therapy , Peripheral Nerve Injuries/drug therapy , Peripheral Nerves/pathology , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Analgesics, Opioid/administration & dosage , Animals , Behavior, Animal/drug effects , Constriction, Pathologic/pathology , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Foot/innervation , Foot/pathology , Hot Temperature , Hyperalgesia/drug therapy , Hyperalgesia/psychology , Male , Mice , Mice, Inbred C57BL , Neuralgia/pathology , Pain Measurement/drug effects , Peripheral Nerve Injuries/pathology , Peripheral Nerves/drug effects , Physical Stimulation , Receptors, Opioid/agonists
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