RESUMO
Studies have shown that long-term (5alpha,6alpha)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol (morphine) treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia). The cellular adaptations contributing to sustained morphine-mediated pain sensitization are not fully understood. It was shown previously (J Neurosci 22:6747-6755, 2002) that sustained morphine exposure augments pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] release in the dorsal horn of the spinal cord in response to the heat-sensing transient receptor potential vanilloid 1 receptor agonist 8-methyl-N-vanillyl-6-nonenamide (capsaicin). In the present study, we demonstrate that sustained morphine-mediated augmentation of CGRP release from isolated primary sensory dorsal root ganglion neurons is dependent on protein kinase A and Raf-1 kinase. Our data indicate that, in addition to neural system adaptations, sustained opioid agonist treatment also produces intracellular compensatory adaptations in primary sensory neurons, leading to augmentation of evoked pain neurotransmitter release from these cells.
Assuntos
Analgésicos Opioides/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Morfina/farmacologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/biossíntese , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Indóis/farmacologia , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Opioides/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismoRESUMO
Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD=0.2+/-0.03 nM and 2+/-0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB(1) receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212-bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD=27+/-8 nM and KD=10.7+/-4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.
Assuntos
Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/química , Transdução de Sinais , Animais , Benzoxazinas/metabolismo , Células Cultivadas , Cicloexanóis/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Morfolinas/metabolismo , Naftalenos/metabolismo , Conformação Proteica , Receptor CB1 de Canabinoide/fisiologia , SpodopteraRESUMO
Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/efeitos dos fármacos , Morfina/farmacologia , Entorpecentes/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Gânglios Espinais/enzimologia , Gânglios Espinais/metabolismo , Indóis/farmacologia , Isoquinolinas/farmacologia , Neurônios Aferentes/enzimologia , Neurônios Aferentes/metabolismo , Fenóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Regulação para CimaRESUMO
The utility of morphine for the treatment of chronic pain is limited by the development of analgesic tolerance. Adenylyl cyclase (AC) superactivation, induced by chronic opioid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present work, we tested the role of Raf-1 in morphine-mediated AC superactivation in CHO cells stably expressing the human micro-opioid receptor. We found that pretreatment of CHO cells stably expressing the human micro-opioid receptor with the selective Raf-1 inhibitor, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one (GW5074, 10 microM, 60 min) completely abolished chronic morphine-mediated AC superactivation (P < 0.01). This finding indicates that Raf-1 may have a crucial role in compensatory feedback regulation of cellular cAMP levels by clinically important opioid analgesics.
Assuntos
Adenilil Ciclases/metabolismo , Indóis/farmacologia , Morfina/farmacologia , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Analgésicos Opioides/farmacologia , Animais , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , TransfecçãoRESUMO
We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein gamma2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the gamma2 subunit (Ggamma2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a delta-opioid receptor agonist. In the present study, we examined the involvement of Ggamma2 in antinociception mediated by other (mu- or kappa-opioid, cannabinoid, alpha2-adrenoreceptor) analgesic agents in a warm (55 degrees C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein gamma2 subunits in supraspinal antinociception.
Assuntos
Analgésicos/administração & dosagem , Encéfalo/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Oligonucleotídeos Antissenso/administração & dosagem , Analgésicos/uso terapêutico , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Animais , Benzoxazinas , Encéfalo/efeitos dos fármacos , Clonidina/administração & dosagem , Clonidina/uso terapêutico , Proteínas de Ligação ao GTP/genética , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfina/administração & dosagem , Morfina/uso terapêutico , Morfolinas/administração & dosagem , Morfolinas/uso terapêutico , Naftalenos/administração & dosagem , Naftalenos/uso terapêutico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Oligopeptídeos/administração & dosagem , Oligopeptídeos/uso terapêutico , Dor/prevenção & controle , Medição da Dor , Fatores de TempoRESUMO
After prolonged stimulation, the delta-opioid receptor becomes desensitized by regulatory mechanisms such as receptor phosphorylation, internalization and down-regulation. In this study, we demonstrate that morphine treatment causes phosphorylation of S363 in the C-terminus of the human delta-opioid receptor. Morphine-mediated phosphorylation reached 53+/-8% of maximum deltorphin II-mediated phosphorylation. Phosphorylation of S363 may contribute to delta-opioid receptor desensitization by morphine.
Assuntos
Morfina/farmacologia , Receptores Opioides delta/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Mutação , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Serina/genética , Serina/metabolismoRESUMO
A 23-year-old woman at 34 weeks' gestation developed recurrent syncope due to profound sinus arrest captured on electrocardiography. Syncopal events occurred in the same sitting position. An echocardiogram revealed severe collapse of the inferior vena cava each time the patient changed her posture from a supine to a sitting position, which was related to the syncope.
Assuntos
Postura/fisiologia , Complicações Cardiovasculares na Gravidez/fisiopatologia , Terceiro Trimestre da Gravidez/fisiologia , Síncope Vasovagal/fisiopatologia , Adulto , Feminino , Humanos , GravidezRESUMO
Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser(363) in human delta-opioid receptor down-regulation by a delta-selective peptide- and non-peptide agonist. Cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human delta-opioid receptor down-regulation is agonist-specific.
Assuntos
Substituição de Aminoácidos/genética , Regulação para Baixo/genética , D-Penicilina (2,5)-Encefalina/agonistas , Mutação , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Alanina/genética , Benzamidas/farmacologia , Regulação para Baixo/efeitos dos fármacos , D-Penicilina (2,5)-Encefalina/farmacologia , Humanos , Piperazinas/farmacologia , Serina/genéticaRESUMO
Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.
Assuntos
Adenilil Ciclases/metabolismo , Colforsina/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Receptores Opioides delta/agonistas , Animais , Células CHO , Cricetinae , Ativação EnzimáticaRESUMO
Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.
Assuntos
Benzamidas/farmacologia , Naltrexona/análogos & derivados , Piperazinas/farmacologia , Receptores Opioides delta/agonistas , Sequência de Aminoácidos , Analgésicos Opioides/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Regulação para Baixo , D-Penicilina (2,5)-Encefalina/farmacologia , Humanos , Dados de Sequência Molecular , Naltrexona/metabolismo , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Ensaio Radioligante , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , TrítioRESUMO
Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.
Assuntos
Analgésicos Opioides/farmacologia , Receptores Opioides delta/agonistas , Animais , Arrestinas/metabolismo , Regulação para Baixo , Humanos , Fosforilação , beta-ArrestinasRESUMO
Opioid receptor agonists mediate their analgesic effects by interacting with Gi/o protein-coupled opioid receptors. Acute treatment with opioid agonists is thought to mediate analgesia by hyperpolarization of presynatic neurons, leading to the inhibition of excitatory (pain) neurotransmitters release. After chronic treatment however, the opioid receptors gradually become less responsive to agonists, and increased drug doses become necessary to maintain the therapeutic effect (tolerance). Analgesic tolerance is the result of two, partially overlapping processes: a gradual loss of inhibitory opioid function is accompanied by an increase in excitatory signaling. Recent data indicate that chronic opioid agonist treatment simultaneously desensitizes the inhibitory-, and augments the stimulatory effects of the opioids. In the present paper we review the molecular mechanisms that may have a role in the augmentation of the excitatory signaling upon chronic opioid agonist treatment. We also briefly review our recent experimental data on the molecular mechanism of chronic opioid agonist-mediated functional sensitization of forskolin-stimulated cAMP formation, in a recombinant Chinese hamster ovary cell line stably expressing the human delta-opioid receptor (hDOR/CHO). To interpret the experimental data, we propose that chronic hDOR activaton leads to activation of multiple redundant signaling pathways that converge to activate the protein kinase, Raf-1. Raf-1 in turn phosphorylates and sensitizes the native adenylyl cyclase VI isoenzyme in hDOR/CHO cells, causing a rebound increase in forskolin-stimulated cAMP formation upon agonist withdrawal.
Assuntos
Analgésicos Opioides/farmacologia , Receptores Opioides/agonistas , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Canais Iônicos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Receptores Acoplados a Proteínas G/efeitos dos fármacosRESUMO
Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1ß, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.
Assuntos
Analgésicos Opioides/efeitos adversos , Analgésicos/administração & dosagem , Hiperalgesia/tratamento farmacológico , Indóis/administração & dosagem , Morfina/efeitos adversos , Neuroglia/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Medula Espinal/efeitos dos fármacos , Animais , Canabinoides/administração & dosagem , Hiperalgesia/induzido quimicamente , Inflamação/tratamento farmacológico , Interleucina-1beta/análise , Masculino , Neuroglia/fisiologia , Dor/induzido quimicamente , Dor/tratamento farmacológico , Piperidinas/administração & dosagem , Pirazóis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/fisiologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK1 receptor antagonist (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 µg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK1 antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK1 receptor antagonists may provide a novel paradigm for long-term pain management.
Assuntos
Analgésicos Opioides/efeitos adversos , Astrócitos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1 , Medula Espinal/patologia , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Triptofano/análogos & derivados , Analgésicos Opioides/administração & dosagem , Animais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Microglia/metabolismo , Morfina/administração & dosagem , Morfina/efeitos adversos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Triptofano/administração & dosagem , Triptofano/farmacologia , Triptofano/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Tissue damage leads to pain sensitization due to peripheral and central release of excitatory mediators such as prostaglandin E2 (PGE2). PGE2 sensitizes spinal pain neurotransmitter such as calcitonin gene-related peptide (CGRP) release via activation of cyclic AMP (cAMP)/protein kinase A (PKA)-dependent signaling mechanisms. Our previous data demonstrate that sustained morphine pretreatment sensitizes adenylyl cyclase(s) (AC) toward the direct stimulator, forskolin, in cultured primary sensory neurons (AC superactivation). In the present work we investigated the hypothesis that morphine pretreatment also sensitizes ACs toward Gs-protein-coupled excitatory modulators (such as PGE2), leading to augmented PKA-dependent CGRP release from PGE2-stimulated primary sensory dorsal root ganglion (DRG) neurons. Our results show that sustained morphine treatment potentiated PGE2-mediated cAMP formation and augmented PGE2-evoked CGRP release from cultured primary sensory neurons in a PKA-dependent manner. Our data suggest that attenuation of AC superactivation in primary sensory neurons may prevent the development of opioid-induced hyperalgesia.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Morfina/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/biossíntese , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Neurotransmissores/metabolismo , Dor/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/enzimologia , Fatores de TempoRESUMO
Studies have shown that sustained cannabinoid treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia) and innocuous mechanical stimuli (tactile allodynia). It has been suggested that augmented release of pain neurotransmitters (such as calcitonin gene-related peptide, CGRP) might be responsible for this abnormal pain sensitization. We hypothesize that intracellular adaptations upon sustained cannabinoid treatment causes augmented release of CGRP from primary nociceptors leading to increased pain sensitivity. We show that sustained (24 h) cannabinoid agonist [(+)WIN 55,212-2] treatment of 7-day-old neonatal rat dorsal root ganglion neurons significantly augments basal CGRP release from these cells in a protein kinase A-dependent manner. Our results indicate that these intracellular compensatory adaptations may play a crucial trigger role in further neuronal system adaptations for modulation of pain.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Canabinoides/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Gânglios Espinais/efeitos dos fármacos , Dor/fisiopatologia , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Benzoxazinas/farmacologia , Células Cultivadas , Esquema de Medicação , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Dor/metabolismo , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Fatores de Tempo , Regulação para Cima/fisiologiaRESUMO
Studies have demonstrated that long-term opioid treatment leads to an increased sensitivity to painful (hyperalgesia) or normally innocuous (allodynia) stimuli. The molecular mechanisms that lead to paradoxical pain sensitization upon chronic opioid treatment are not completely understood. Enhanced excitatory pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release in the dorsal horn of the spinal cord may play a role in sustained morphine-mediated paradoxical pain. Recently we have demonstrated that inhibition of Raf-1 attenuates sustained morphine treatment-mediated augmentation of CGRP release in vitro, in cultured primary sensory neurons. In the present study, we show that knockdown of spinal Raf-1 levels in vivo by intrathecal administration of Raf-1-specific siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats.
Assuntos
Analgésicos Opioides/efeitos adversos , Morfina/efeitos adversos , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Analgésicos Opioides/administração & dosagem , Animais , Esquema de Medicação , Temperatura Alta , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Injeções Espinhais , Masculino , Morfina/administração & dosagem , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human delta opioid receptors (hDOR) by measuring guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of beta-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of beta-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced beta-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.
Assuntos
Modelos Biológicos , Oligopeptídeos/farmacologia , Receptores Opioides delta/metabolismo , Alanina/genética , Animais , Arrestinas/metabolismo , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Mutação/genética , Fosforilação/efeitos dos fármacos , Serina/genética , Fatores de Tempo , beta-ArrestinasRESUMO
Stimulation of cardiac beta(2)-adrenergic receptor (beta(2)-AR) or delta-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that delta-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective delta-opioid agonist D-[Pen(2,5)]enkephalin (DPDPE) to ICA cells increased [Ca(2+)](i) transients in a concentration-dependent manner. Such an effect was abolished by the Ca(2+) channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in delta-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 +/- 15% or myocyte death by 26 +/- 4%, respectively. beta(2)-AR blockade markedly attenuated delta-opioid-initiated infarct size-limiting effect and abolished delta-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, delta-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that delta-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/beta(2)-AR signaling pathway as a result of ICA cell activation.
Assuntos
Cálcio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides delta/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cardiotônicos/administração & dosagem , Células Cultivadas , D-Penicilina (2,5)-Encefalina/administração & dosagem , Humanos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
The constrained opioid peptide (2S,3R)beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the delta-opioid receptors (). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTP gamma S) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. delta- (SNC80; (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide) or mu- (DAMGO; [D-Ala(2), Me-Phe(4),Gly(ol)(5)]enkephalin) selective opioid full agonists stimulated [(35)S]GTP gamma S binding in mouse brain membranes 150 +/- 4.5% and 152 +/- 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [(35)S]GTP gamma S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a K(e) value of 3.6 +/- 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the mu-selective opioid agonist, DAMGO. Warm water (55 degrees C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGO-mediated antinociception (40 +/- 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral delta-opioid antagonist in mouse brain.