RESUMEN
Little is known about the cellular effects induced by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), although changes in gene expression have been observed following treatments with other psychostimulants. Thus, the aim of this study was to investigate in mice, the relationships between the ras-dependent protein kinase ERK and MDMA-induced reinforcement using the conditioned place preference (CPP) and locomotor activity measurements. This was completed using real-time quantitative PCR method by a study of immediate early-genes (IEGs) transcription known to be involved in neuronal plasticity. A significant CPP was observed after repeated MDMA treatment in CD-1 mice at a dose of 9 mg kg-1 i.p. but not at 3 and 6 mg kg-1. This rewarding effect was abolished by the selective inhibitor of ERK activation, SL327 (50 mg kg-1; i.p.). Similar results were obtained on MDMA-induced locomotor activity, clearly suggesting a role of ERK pathway in these behavioral responses. Following acute i.p. injection, MDMA induced a strong c-fos transcription in brain structures, such as caudate putamen, nucleus accumbens and hippocampus, whereas egr-1 and egr-3 transcripts were only increased in the caudate putamen. MDMA-induced IEGs transcription was selectively suppressed by SL327 in the caudate putamen, suggesting a role for other signaling pathways in regulation of IEGs transcription in the other brain structures. In agreement with these results, MDMA-induced c-fos protein expression was blocked by SL327 in the caudate putamen. This study confirms and extends to mice the reported role of ERK pathway in the development of addiction-like properties of MDMA. This could facilitate studies about the molecular mechanism of this process by using mutant mice.
Asunto(s)
Encéfalo/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Locomoción/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , N-Metil-3,4-metilenodioxianfetamina/farmacología , Animales , Encéfalo/enzimología , Condicionamiento Psicológico/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Genes Inmediatos-Precoces/efectos de los fármacos , Genes Inmediatos-Precoces/fisiología , Locomoción/fisiología , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/fisiologíaRESUMEN
The discovery that the endogenous morphine-like peptides named enkephalins are inactivated by two metallopeptidases, neutral endopeptidase and aminopeptidase N, which can be blocked by dual inhibitors, represents a promising way to develop 'physiological' analgesics devoid of the side effects of morphine. A new series of dual aminophosphinic inhibitors of the two enkephalin-catabolizing enzymes has been recently designed. In this study, one of these inhibitors, RB3007, was tested in various assays commonly used to select analgesics (mouse hot-plate test, rat tail-flick test, writhing and formalin tests in mice, and paw pressure test in rats), and the extracellular levels of the endogenous enkephalins in the ventrolateral periaqueductal grey have been measured by microdialysis after systemic administration of RB3007. In the mouse hot-plate test, the dual inhibitor induced long-lasting (2 h) antinociceptive effects with a maximum of 35% analgesia 60 min after i.v. or i.p. administration. These antinociceptive responses were antagonized by prior injection of naloxone (0.1 mg/kg, s.c.). Similar long lasting effects were observed in the other animal models used. Very interestingly, injection of RB3007 (50 mg/kg, i.p.) significantly increased (82%) the extracellular levels of Met-enkephalin with a peak 60 min after i.p. injection. This increase parallels the antinociceptive responses observed. In addition, strong facilitatory effects of subanalgesic doses of the CCK(2) receptor antagonist, PD-134,308 or the synthetic opioid agonist, methadone on RB3007-induced antinociceptive responses were observed. These findings may constitute promising data for future development of a new class of analgesics that could be of major interest in a number of severe and persistent pain syndromes.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Analgésicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Meglumina/análogos & derivados , Metadona/uso terapéutico , Dolor/tratamiento farmacológico , Receptores de Colecistoquinina/antagonistas & inhibidores , Analgésicos/farmacología , Animales , Manejo de la Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Indoles/uso terapéutico , Masculino , Meglumina/farmacología , Meglumina/uso terapéutico , Metadona/farmacología , Ratones , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Profármacos/farmacología , Profármacos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/fisiologíaRESUMEN
We have recently shown concurrent changes in behavioural responses and c-Fos protein expression in the central nervous system in both naive and morphine-dependent rats after systemic administration of the opioid antagonist naloxone. However, because naloxone acts on the three major types of opioid receptors, the present study aimed at determining, in the same animals, both changes in behaviour and c-Fos-like immunoreactivity after intravenous injection of selective opioid antagonists, such as mu (beta-funaltrexamine, 10 mg/kg), delta (naltrindole, 4 mg/kg) or kappa (nor-binaltorphimine, 5 mg/kg) opioid receptor antagonists, in naive or morphine-dependent rats. In a first experimental series, only beta-funaltrexamine increased c-Fos expression in the eight central nervous system structures examined, whereas no effect was seen after naltrindole or nor-binaltorphimine administration in naive rats. These results suggest a tonic activity in the endogenous opioid peptides acting on mu opioid receptors in normal rats. A second experimental series in morphine-dependent rats showed that beta-funaltrexamine had the highest potency in the induction of classical signs of morphine withdrawal syndrome, as well as the increase in c-Fos expression in the 22 central nervous system structures studied, suggesting a major role of mu opioid receptors in opioid dependence. However, our results also demonstrated that naltrindole and, to a lesser extent, nor-binaltorphimine were able to induce moderate signs of morphine withdrawal and relatively weak c-Fos protein expression in restricted central nervous system structures. Therefore, delta and kappa opioid receptors may also contribute slightly to opioid dependence.
Asunto(s)
Sistema Nervioso Central/metabolismo , Dependencia de Morfina/metabolismo , Naltrexona/análogos & derivados , Antagonistas de Narcóticos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Recuento de Células , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Masculino , Morfina/farmacología , Dependencia de Morfina/fisiopatología , Naltrexona/farmacología , Narcóticos/farmacología , Ratas , Ratas Sprague-Dawley , Síndrome de Abstinencia a Sustancias/fisiopatologíaRESUMEN
The effects of the S enantiomer of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, alone or in combination with a functional NMDA receptor antagonist, (+)-HA966 were studied on the spinal c-Fos protein expression in the carrageenan model of inflammatory nociception. One hour 30min after intraplantar carrageenan in awake rats, c-Fos immunoreactive (c-Fos-IR) nuclei were preferentially located in the laminae I-II and V-VI of the spinal dorsal horn, i.e., spinal areas containing numerous neurons responding exclusively, or not, to peripheral nociceptive stimuli. RB101(S) (5, 10, 20 and 40mg/kg i.v.) dose-dependently reduced the total number of carrageenan-evoked c-Fos-IR nuclei (r=0.63, P<0.01), with 49+/-3% reduction (P<0.001) for the highest dose. Two highest doses of RB101(S) (20 and 40mg/kg) significantly reduced the number of carrageenan-evoked c-Fos-IR nuclei in both superficial I-II (32+/-7% and 36+/-5% reduction, respectively, P<0.05 for both) and deep V-VI (42+/-6% and 61+/-2% reduction, respectively, P<0.001 for both) laminae. The effects of RB101(S) were naloxone-reversible. Combination of low doses of RB101(S) (2.5 or 10mg/kg i.v.) and an inactive dose of (+)-HA966 (2.5mg/kg s.c.) produced supra-additive effects (39+/-4% and 51+/-5% reduction of the total number of c-Fos-IR nuclei, respectively, P<0.001 for both). These effects were partially reversed by naloxone. These results provide evidence for the potent effects of combination of RB101(S) and (+)-HA966. Considering the absence of major opioid side effects of RB101(S) and the marked increase of its antinociceptive effects by NMDA receptor antagonist, this type of drug combination could have beneficial therapeutical application.
Asunto(s)
Analgésicos/farmacología , Disulfuros/farmacología , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Inflamación/tratamiento farmacológico , Dolor/tratamiento farmacológico , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Pirrolidinonas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Médula Espinal/efectos de los fármacos , Analgésicos/uso terapéutico , Animales , Carragenina , Disulfuros/uso terapéutico , Quimioterapia Combinada , Encefalinas/agonistas , Encefalinas/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Inmunohistoquímica , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Dolor/metabolismo , Fenilalanina/uso terapéutico , Pirrolidinonas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Médula Espinal/metabolismoRESUMEN
We have previously shown that RB101, a dual inhibitor of enkephalin-degrading enzymes, decreased carrageenin-evoked c-Fos protein expression at the spinal cord level in awake rats. Moreover, we have also shown that c-Fos expression is a useful marker of the possible direct or indirect interactions between neural pathways, such as opioid and cholecystokinin systems. We now investigated the respective roles of the three main types of opioid receptors (mu, delta, or kappa) and their possible interactions, in the depressive effects of RB101 in inflammatory nociceptive conditions induced by intraplantar carrageenin (6 mg/150 microl of saline). We used beta-funaltrexamine (beta-FNA), naltrindole (NTI), and nor-binaltorphimine (BNI) as specific antagonists for mu, delta- and kappa-opioid receptors, respectively. c-Fos protein-immunoreactivity (c-Fos-IR) was evaluated as the number of c-Fos-IR nuclei in the lumbar spinal cord 90 min after carrageenin. c-Fos-IR nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of segments L4-L5 (areas containing numerous neurons responding exclusively, or not, to nociceptive stimuli). RB101(S) (30 mg/kg, i.v.) significantly reduced the total number of carrageenin-evoked c-Fos-IR nuclei (30% reduction, P<0.01). This effect was completely blocked by beta-FNA (10 mg/kg, i.v.), or NTI (1 mg/kg, i.v.). In contrast, BNI (2.5 mg/kg, i.v.) did not reverse the reducing effects of RB101(S) on carrageenin-evoked c-Fos protein expression. These results suggest that functional interactions occur between mu- and delta-opioid receptors in enkephalin-induced antinociceptive effects.
Asunto(s)
Disulfuros/farmacología , Edema/metabolismo , Edema/patología , Encefalinas/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Médula Espinal/metabolismo , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Carragenina , Disulfuros/uso terapéutico , Edema/tratamiento farmacológico , Encefalinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Fenilalanina/uso terapéutico , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacosRESUMEN
In behavioural tests, RB101 (N-[(S)-2-benzyl-3[(S)(2-amino-4-methyl-thio)butyldithio]-1-oxopropyl]-L-phenylalanine benzyl ester), a mixed inhibitor of enkephalin-degrading enzymes, induces antinociceptive effects without producing tolerance, or cross-tolerance with morphine. In the present experiments, the acute or chronic effects of enantiomer RB101(S) were examined on the response of spinal cord neurons to nociceptive inflammatory stimulation (intraplantar injection of carrageenin) using c-Fos studies in awake rats. The number of c-Fos immunoreactive nuclei was evaluated in the lumbar spinal cord 90 min after carrageenin. c-Fos-immunoreactive nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of segments L4-L5 (areas containing numerous neurones responding exclusively, or not, to nociceptive stimuli). In the first experimental series, acute RB101(S) (30 mg/kg, i.v.), morphine (3 mg/kg, i.v.), or respective vehicles were injected in rats chronically treated with RB101(S) (160 mg/kg/day for 4 days, s.c.). In chronically treated RB101(S) rats, both acute RB101(S) and morphine reduced the total number of carrageenin-evoked c-Fos-immunoreactive nuclei. In the second experimental series, acute RB101(S) (30 mg/kg, i.v.) reduced the total number of carrageenin-evoked c-Fos-immunoreactive nuclei with similar magnitude in naive and in morphine-tolerant (100 mg/kg/day for 3 days, s.c.) rats. These data provide further evidence that different cellular mechanisms occurred after chronic stimulation of opioid receptors by morphine or endogenous enkephalins.
Asunto(s)
Disulfuros/farmacología , Encefalinas/metabolismo , Inhibidores Enzimáticos/farmacología , Morfina/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Médula Espinal/enzimología , Analgésicos/farmacología , Animales , Tolerancia a Medicamentos/fisiología , Masculino , Proteínas Proto-Oncogénicas c-fos/análisis , Ratas , Ratas Sprague-Dawley , Médula Espinal/químicaRESUMEN
In a model of mononeuropathic pain (chronic constriction injury of the sciatic nerve, CCI), we have demonstrated that light touch stimuli (stroking) to the paw induced Fos-like immunoreactivity (Fos-LI) in the superficial and deep dorsal horn of the rat spinal cord (Catheline et al., Pain 80 (1999a) 347). The efficacy of opioids in neuropathic pain being controversial, we have tested the effects of morphine (0.3, 1 and 3 mg/kg intravenous, i.v.) on this spinal Fos-LI evoked by light tactile stimuli, which could be related to mechanical allodynia. Morphine did not change the level of spinal Fos-LI observed following light touch stimuli in the CCI rats (43 +/- 3, 38 +/- 7, and 37 +/- 4 Fos-LI neurones/40 microm L4-L5 section, respectively, for the three doses versus 32 +/- 4 in the control group). In contrast, the administration of 3 mg/kg of i.v. morphine reduced by 30% the number of Fos-LI neurones induced by heat stimulation (52 degrees C, 15 s duration) in CCI rats (P < 0.05) as in sham-operated rats. These effects were reversed by the systemic administration of naloxone. The lack of effect of morphine on touch-evoked Fos-LI in the superficial dorsal horn reinforces the assertion that dynamic mechanical allodynia is related to information transmitted by A-beta fibres, since opioid receptors are mainly located on thin primary afferent fibres. Our results provide a basis for a certain form of allodynia that is insensitive to morphine.