RESUMEN
The opioid system in the central nervous system regulates depressive-like behavior in animals. Opioid receptors and their endogenous ligands have been focused on as novel therapeutic targets for depression. We synthesized dermorphin (DRM)-dynorphin (DYN) analogs (DRM-DYN001-004) using the message-address concept concerning interactions with opioid receptors. It has previously been reported that DRM-DYN001, 003, and 004 have shown high affinities for µ- and κ-opioid receptors, whereas all analogs had a lower affinity for the δ-opioid receptor than for other receptors using a receptor binding assay. However, it remains unknown whether these analogs show antidepressant-like effects in mice. We examined the effects of DRM-DYN analogs on the duration of immobile behavior in a tail suspension test. Intracerebroventricular administration of DRM-DYN001 in mice shortened the duration of immobile behavior, but did not affect locomotion. The DRM-DYN001-induced antidepressant-like effect was inhibited by co-administration of naloxone (non-selective opioid receptor antagonist), naloxonazine (selective µ1-opioid receptor antagonist), or nor-BNI (κ-opioid receptor antagonist), but not naltrindole (δ-opioid receptor antagonist). These data suggest that DRM-DYN001 exerts an antidepressant-like effect via activation of the central µ1- and κ-opioid receptors in mice and may represent a new lead peptide for further investigation for the development of novel therapeutic approaches for depression.
Asunto(s)
Antidepresivos , Dinorfinas , Péptidos Opioides , Receptores Opioides kappa , Animales , Antidepresivos/administración & dosificación , Antidepresivos/farmacología , Dinorfinas/administración & dosificación , Dinorfinas/química , Ratones , Antagonistas de Narcóticos/farmacología , Péptidos Opioides/administración & dosificación , Péptidos Opioides/química , Receptores Opioides , Receptores Opioides kappa/metabolismoRESUMEN
The brain mechanism responsible for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) is important for maintaining reproductive function in mammals. Accumulating evidence suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH and subsequent gonadotropin secretion. Dynorphin A (Dyn) and its receptor, kappa-opioid receptor (KOR, encoded by Oprk1), have been shown to be involved in the suppression of pulsatile GnRH/luteinizing hormone (LH) release. On the other hand, it is still unclear whether the inhibitory Dyn signaling affects KNDy neurons or KOR-expressing non-KNDy cells in the ARC or other brain regions. We therefore aimed to clarify the role of ARC-specific Dyn-KOR signaling in the regulation of pulsatile GnRH/LH release by the ARC specific cell deletion of KOR-expressing cells using Dyn-conjugated-saporin (Dyn-SAP). Estrogen-primed ovariectomized female rats were administered Dyn-SAP to the ARC. In situ hybridization of Oprk1 showed that ARC Dyn-SAP administration significantly decreased the number of Oprk1-expressing cells in the ARC, but not in the ventromedial hypothalamic nucleus and paraventricular nucleus. The frequency of LH pulses significantly increased in animals bearing the ARC Dyn-SAP administration. The number of Kiss1-expressing cells in the ARC was not affected by ARC Dyn-SAP treatment. Dyn-KOR signaling within the ARC seems to mediate the suppression of the frequency of pulsatile GnRH/LH release, and ARC non-KNDy KOR neurons may be involved in the mechanism modulating GnRH/LH pulse generation.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Hormona Luteinizante/sangre , Neuronas/metabolismo , Receptores Opioides kappa/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Dinorfinas/administración & dosificación , Femenino , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Saponinas/administración & dosificaciónRESUMEN
The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Péptidos/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Estabilidad de Medicamentos , Dinorfinas/administración & dosificación , Dinorfinas/sangre , Dinorfinas/farmacocinética , Femenino , Glucolípidos/administración & dosificación , Glucolípidos/química , Técnicas In Vitro , Liposomas/administración & dosificación , Liposomas/química , Nanopartículas/química , Antagonistas de Narcóticos/administración & dosificación , Antagonistas de Narcóticos/sangre , Antagonistas de Narcóticos/farmacocinética , Péptidos/sangre , Estabilidad Proteica , Proteolisis , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/antagonistas & inhibidores , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the µ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and ß-funaltrexamine (ß-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP. Antinociception induced by analog 2 through 4 was not reversed by δ opioid receptor (DOP) or κ opioid receptor (KOP) antagonist; antibodies against dynorphin-A (1-17), dynorphin-B (1-13), and Leu5/Met5-enkephalin had no impact on the antinociceptive effects of these analogs. In contrast, antinociceptive effects induced by a spinal injection of the fluorine substituted analog 1 were significantly reversed by KOP antagonism. Furthermore, intrathecal pretreatment with antibodies against dynorphin-B (1-13) attenuated the antinociceptive effect of analog 1. These results indicate that the antinociceptive activity exerted by intrathecally-administered analog 1 is mediated, in part, through KOP with increased release of dynorphin-B (1-13). The chemical modifications used in the present study may serve as a useful tool to gain insight into the mechanisms of endomorphins activity.
Asunto(s)
Analgésicos Opioides/química , Analgésicos Opioides/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos Opioides/química , Péptidos Opioides/farmacología , Analgesia , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/antagonistas & inhibidores , Análisis de Varianza , Animales , Anticuerpos/inmunología , Dinorfinas/administración & dosificación , Dinorfinas/antagonistas & inhibidores , Dinorfinas/química , Dinorfinas/farmacología , Encefalina Leucina/administración & dosificación , Encefalina Leucina/antagonistas & inhibidores , Encefalina Leucina/química , Encefalina Leucina/farmacología , Encefalina Metionina/administración & dosificación , Encefalina Metionina/antagonistas & inhibidores , Encefalina Metionina/química , Encefalina Metionina/farmacología , Flúor/química , Inyecciones Espinales , Masculino , Ratones , Naloxona/administración & dosificación , Naloxona/farmacología , Naltrexona/administración & dosificación , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Oligopéptidos/administración & dosificación , Oligopéptidos/antagonistas & inhibidores , Péptidos Opioides/administración & dosificación , Péptidos Opioides/antagonistas & inhibidores , Dolor/tratamiento farmacológico , Dolor/metabolismo , Dimensión del Dolor , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Receptores sigma/antagonistas & inhibidoresRESUMEN
Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1ß and TNF-α release; inhibiting IL-1ß and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™-hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10µM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.
Asunto(s)
Dinorfinas/administración & dosificación , Inflamación/tratamiento farmacológico , Receptor Toll-Like 4/genética , Factor de Transcripción ReIA/genética , Animales , Línea Celular , Dinorfinas/inmunología , Humanos , Inmunomodulación/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/genética , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , FN-kappa B/genética , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Dynorphin 1-17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1-17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, µ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1-17 and fragments (1-6, 1-7 and 1-9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1-7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund's Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1-7, 1-17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1-7 and 1-9 displayed the greatest activity across all receptor subtypes, while dynorphin 1-7, 1-9 and 1-17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1-7 and U50488H, but not dynorphin 1-17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1-7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1-7 mediates analgesia.
Asunto(s)
Dinorfinas/administración & dosificación , Inflamación/tratamiento farmacológico , Dolor/tratamiento farmacológico , Receptores Opioides delta/genética , Receptores Opioides kappa/genética , Receptores Opioides mu/genética , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/administración & dosificación , Analgesia/métodos , Animales , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Inflamación/genética , Dolor/genética , Dolor/patología , Ratas , TransfecciónRESUMEN
Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5'GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5'GNTI, is presented using this approach.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Dinorfinas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores Opioides kappa/metabolismo , Analgésicos Opioides/farmacología , Animales , Bencenoacetamidas/farmacología , Cuerpo Estriado/metabolismo , Dinorfinas/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas de Narcóticos/farmacología , Unión Proteica , Pirrolidinas/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/genética , Transducción de Señal/efectos de los fármacos , Radioisótopos de AzufreRESUMEN
It has been reported that kappa opioid receptor (KOR) is expressed in the paraventricular nucleus of thalamus (PVT), a brain region associated with arousal, drug reward and stress. Although intra-PVT infusion of KOR agonist was found to inhibit drug-seeking behavior, it is still unclear whether endogenous KOR agonists directly regulate PVT neuron activity. Here, we investigated the effect of the endogenous KOR agonist dynorphin-A (Dyn-A) on the excitability of mouse PVT neurons at different developmental ages. We found Dyn-A strongly inhibited PVT neurons through a direct postsynaptic hyperpolarization. Under voltage-clamp configuration, Dyn-A evoked an obvious outward current in majority of neurons tested in anterior PVT (aPVT) but only in minority of neurons in posterior PVT (pPVT). The Dyn-A current was abolished by KOR antagonist nor-BNI, Ba(2+) and non-hydrolyzable GDP analogue GDP-ß-s, indicating that Dyn-A activates KOR and opens G-protein-coupled inwardly rectifying potassium channels in PVT neurons. More interestingly, by comparing Dyn-A currents in aPVT neurons of mice at various ages, we found Dyn-A evoked significant larger current in aPVT neurons from mice around prepuberty and early puberty stage. In addition, KOR activation by Dyn-A didn't produce obvious desensitization, while mu opioid receptor (MOR) activation induced obvious desensitization of mu receptor itself and also heterologous desensitization of KOR in PVT neurons. Together, our findings indicate that Dyn-A activates KOR and inhibits aPVT neurons in mice at various ages especially around puberty, suggesting a possible role of KOR in regulating aPVT-related brain function including stress response and drug-seeking behavior during adolescence.
Asunto(s)
Dinorfinas/metabolismo , Neuronas/fisiología , Núcleo Hipotalámico Paraventricular/fisiología , Receptores Opioides kappa/metabolismo , Animales , Bario/administración & dosificación , Cationes/administración & dosificación , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Guanosina Difosfato/administración & dosificación , Guanosina Difosfato/análogos & derivados , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BL , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Neuronas/efectos de los fármacos , Neurotransmisores/administración & dosificación , Neurotransmisores/metabolismo , Núcleo Hipotalámico Paraventricular/crecimiento & desarrollo , Técnicas de Placa-Clamp , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inhibidores , Maduración Sexual/fisiología , Tionucleótidos/administración & dosificación , Técnicas de Cultivo de TejidosRESUMEN
It is known that stress changes state and reactivity of humoral systems of stress, particularly the hypothalamic-pituitary-adrenal system (HPA) and the dynorphin-K-opioid system (DKOS) in any age periods, including ones of early postnatal development. Supposedly these changes are underlying some disorders. Difference in state and reactivity of the HPA system is well established. But the role of DKOS is not clear. Further study of this requires summarizing of the literature data on physiology of DKOS activation and ethological features of the activation in different periods of postnatal development. It is possible to conclude that the mode of reaction to stimulation of the DKOS differs in the early development in contrast to adult animals. The mode of reaction can be changed in relation to the periods of development of the system of stress-reactivity and can depend on prior activation of the stress system in a particular period.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Dinorfinas/administración & dosificación , Desarrollo Embrionario/efectos de los fármacos , Receptores Opioides kappa/metabolismo , Estrés Psicológico/tratamiento farmacológico , Animales , Humanos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/fisiopatología , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/patología , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/fisiopatología , Estrés Psicológico/fisiopatologíaRESUMEN
Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.
Asunto(s)
Membrana Celular/metabolismo , Encefalinas/metabolismo , Neuropéptidos/metabolismo , Péptidos Opioides/metabolismo , Precursores de Proteínas/metabolismo , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Dinorfinas/administración & dosificación , Dinorfinas/metabolismo , Endorfinas/administración & dosificación , Endorfinas/metabolismo , Encefalinas/genética , Humanos , Ligandos , Microscopía Confocal , Neuropéptidos/administración & dosificación , Péptidos Opioides/administración & dosificación , Células PC12 , Precursores de Proteínas/genética , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: The N- and C-terminal regions of dynorphin (Dyn) A (1-17) activate opioid and N-methyl-D-aspartate receptors, respectively. Earlier studies demonstrated that Dyn-converting enzyme cleaved Dyn A (1-17) mainly at the Arg(6)-Arg(7) bond, resulting in the production of N- and C-terminal region peptide fragments, and that this enzyme was not inhibited by a mixture of the three peptidase inhibitors (PIs) amastatin (A), captopril (C), and phosphoramidon (P). The purpose of the present study was to evaluate antinociceptive potential and toxicity with intracerebroventricular administration of Dyn A (1-17) or (1-13) under pretreatment with a mixture of A, C, and P and/or Dyn-converting enzyme inhibitor (p-hydroxymercuribenzoate). METHODS: Peptide fragments from Dyn A (1-17) following incubation with membrane preparation under pretreatment with a mixture of the three PIs was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Infusion of drugs and peptides into the third ventricle in rats was performed via indwelling cannulae. Induction of antinociception and toxicity by Dyn A (1-17), Dyn A (1-13), Dyn A (1-6), or Dyn A (7-17) were determined by the tail-flick test and induction of barrel rotation, respectively. The effects of the PIs on antinociception and toxicity were evaluated by a dose-response study and a comparison of differences among various combinations of Dyn A (1-17) or Dyn A (1-13) and the three PIs and p-hydroxymercuribenzoate. RESULTS: MALDI-TOF-MS analysis identified Dyn A (1-6) and Dyn A (1-10) fragments as products following incubation of Dyn A (1-17) with membrane preparation of rat midbrain under pretreatment with a mixture of the three PIs. Pretreatment with a mixture of the three PIs produced an approximately 30-fold augmentation in antinociception induced by low-dose intracerebroventricular administration of Dyn A (1-17) or (1-13) in a µ-, δ- and κ-opioid receptor antagonist-reversible manner, but without signs of toxicity such as barrel rotation in the rat. Dyn A (1-17)-induced antinociception and toxicity was greater than that of Dyn A (1-6), Dyn A (1-13), or Dyn A (7-17) at the same dose. Dyn A (1-17)-induced antinociception and toxicity under pretreatment with various combinations of the three PIs and p-hydroxymercuribenzoate was greater than that with a mixture of the three PIs alone. CONCLUSION: These findings suggest that administration of a mixture of the three PIs increases Dyn A (1-17)- or (1-13)-induced antinociception under physiological conditions without toxicity.
Asunto(s)
Analgésicos Opioides/toxicidad , Analgésicos/efectos adversos , Analgésicos/farmacología , Dinorfinas/toxicidad , Inhibidores de Proteasas/farmacología , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Química Encefálica/efectos de los fármacos , Captopril/administración & dosificación , Captopril/farmacología , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Dinorfinas/farmacología , Glicopéptidos/administración & dosificación , Glicopéptidos/farmacología , Inyecciones Intraventriculares , Masculino , Dimensión del Dolor/efectos de los fármacos , Péptidos/administración & dosificación , Péptidos/farmacología , Ratas , Ratas Wistar , Receptores Opioides/efectos de los fármacosRESUMEN
BACKGROUND: The long term use of opioids for the treatment of pain leads to a group of maladaptations which includes opioid-induced hyperalgesia (OIH). OIH typically resolves within few days after cessation of morphine treatment in mice but is prolonged for weeks if histone deacetylase (HDAC) activity is inhibited during opioid treatment. The present work seeks to identify gene targets supporting the epigenetic effects responsible for OIH prolongation. RESULTS: Mice were treated with morphine according to an ascending dose protocol. Some mice also received the selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) additionally. Chronic morphine treatment with simultaneous HDAC inhibition enhanced OIH, and several spinal cord genes were up-regulated. The expression of Bdnf (Brain-derived neurotrophic factor) and Pdyn (Prodynorphin) were most closely related to the observed behavioral changes. ChIP (Chromatin immuoprecipation) assays demonstrated that promoter regions of Pdyn and Bdnf were strongly associated with aceH3K9 (Acetylated histone H3 Lysine9) after morphine and SAHA treatment. Furthermore, morphine treatment caused an increase in spinal BDNF and dynorphin levels, and these levels were further increased in SAHA treated mice. The selective TrkB (tropomyosin-receptor-kinase) antagonist ANA-12 reduced OIH when given one or seven days after cessation of morphine. Treatment with the selective kappa opioid receptor antagonist nor-BNI also reduced established OIH. The co-administration of either receptor antagonist agent daily with morphine resulted in attenuation of hyperalgesia present one day after cessation of treatment. Additionally, repeated morphine exposure induced a rise in BDNF expression that was associated with an increased number of BDNF+ cells in the spinal cord dorsal horn, showing strong co-localization with aceH3K9 in neuronal cells. Lastly, spinal application of low dose BDNF or Dynorphin A after resolution of OIH produced mechanical hypersensitivity, with no effect in controls. CONCLUSIONS: The present study identified two genes whose expression is regulated by epigenetic mechanisms during morphine exposure. Treatments aimed at preventing the acetylation of histones or blocking BDNF and dynorphin signaling may reduce OIH and improve long-term pain using opioids.
Asunto(s)
Analgésicos Opioides/toxicidad , Epigénesis Genética/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/patología , Morfina/toxicidad , Médula Espinal/metabolismo , Animales , Antineoplásicos/administración & dosificación , Azepinas/administración & dosificación , Benzamidas/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/genética , Modelos Animales de Enfermedad , Dinorfinas/administración & dosificación , Dinorfinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Naltrexona/administración & dosificación , Naltrexona/análogos & derivados , Antagonistas de Narcóticos/administración & dosificación , Dimensión del Dolor/efectos de los fármacos , Médula Espinal/efectos de los fármacos , VorinostatRESUMEN
It has been shown previously that norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (5'-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1,2]. In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss-Webster mice, zyklophin at doses of 0.1, 0.3 and 1mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20mg/kg, i.p.) at 18-20 h before challenge with zyklophin (0.3mg/kg) did not markedly affect scratching. Additionally, KOPR-/- mice given 0.3mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR-/- mice was confirmed with ex vivo radioligand binding using [(3)H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5'-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch.
Asunto(s)
Dinorfinas/efectos adversos , Fragmentos de Péptidos/efectos adversos , Prurito/inducido químicamente , Receptores Opioides kappa/antagonistas & inhibidores , Animales , Bencenoacetamidas/farmacología , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Noqueados , Naltrexona/análogos & derivados , Naltrexona/farmacología , Fragmentos de Péptidos/administración & dosificación , Pirrolidinas/farmacología , Ensayo de Unión Radioligante , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genéticaRESUMEN
A niosomal formulation, functionalized with N-palmitoylglucosamine, was developed as potential brain targeted delivery system of dynorphin-B. In fact, this endogenous neuropeptide, selective agonist of k opioid receptors, is endowed with relevant pharmacological activities on the central nervous system, including a marked antinociceptive effect, but is unable to cross the blood brain barrier (BBB), thus requiring intracerebroventricular administration. Statistical design of experiments was utilized for a systematic evaluation of the influence of variations of the relative amounts of the components of the vesicle membrane (Span 60, cholesterol and SolulanC24) on vesicle mean diameter, polydispersity index and drug entrapment efficiency, chosen as the responses to optimize. A Scheffé simplex-centroid design was used to obtain the coefficients of the postulated mathematical model. The study of the response surface plots revealed that variations of the considered factors had different effects on the selected responses. The desirability function enabled for finding the optimal mixture composition, which represented the best compromise to simultaneously optimize all the three responses. The experimental values obtained with the optimized formulation were very similar to the predicted ones, proving the validity of the proposed regression model. The optimized niosomal formulation of dynorphin-B administered intravenously to mice (100mg/kg) showed a pronounced antinociceptive effect, significantly higher (P<0.05) than that given by i.v. administration of the simple solution of the peptide at the same concentration, proving its effectiveness in enabling the peptide brain delivery. These positive results suggest that the proposed approach could be successfully extended to other neuro-active peptides exerting a strong central action, even at low doses, but unable to cross the BBB.
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Analgésicos/administración & dosificación , Encéfalo/efectos de los fármacos , Portadores de Fármacos/química , Dinorfinas/administración & dosificación , Endorfinas/administración & dosificación , Glucolípidos/química , Analgésicos/farmacocinética , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Dinorfinas/farmacocinética , Dinorfinas/farmacología , Dinorfinas/uso terapéutico , Endorfinas/farmacocinética , Endorfinas/farmacología , Endorfinas/uso terapéutico , Glucolípidos/síntesis química , Inyecciones Intravenosas , Inyecciones Intraventriculares , Liposomas , Masculino , Ratones , Dolor/tratamiento farmacológico , Dolor/metabolismo , Receptores Opioides kappa/agonistasRESUMEN
We characterized pain behavior and cutaneous blood flow response induced by activation of the spinal transient receptor potential ankyrin 1 (TRPA1) channel using intrathecal drug administrations in the rat. Additionally, we assessed whether the pronociceptive actions induced by intrathecally administered dynorphin A, cholecystokinin or prostaglandin F(2α) are mediated by the spinal TRPA1 channel. Cinnamaldehyde, a TRPA1 agonist, produced a dose-related (3-10 µg) cutaneous blood flow increase and mechanical hypersensitivity effect. These effects at the currently used doses were of short duration and attenuated, although not completely, by pretreatment with A-967079, a TRPA1 antagonist. The cinnamaldehyde-induced hypersensitivity was also reduced by pretreatment with minocycline (an inhibitor of microglial activation), but not by carbenoxolone (a gap junction decoupler). In vitro study, however, indicated that minocycline only poorly blocks the TRPA1 channel. The mechanical hypersensitivity effect induced by dynorphin A, but not that by cholecystokinin or prostaglandin F(2α), was attenuated by a TRPA1 antagonist Chembridge-5861528 as well as A-967079. The cinnamaldehyde-induced cutaneous blood flow increase was not suppressed by MK-801, an NMDA receptor antagonist, or bicuculline, a GABA(A) receptor antagonist. The results indicate that spinal TRPA1 channels promote mechanical pain hypersensitivity and due to antidromic activation of nociceptive nerve fibers increase cutaneous blood flow. The attenuation of the cinnamaldehyde-induced hypersensitivity effect by minocycline may be explained by action other than block of the TRPA1 channel. Moreover, the spinal TRPA1 channel is involved in mediating the pronociceptive action of dynorphin A, but not that of the spinal cholecystokinin or prostaglandin F(2α).
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Analgésicos no Narcóticos/uso terapéutico , Dolor de Espalda/tratamiento farmacológico , Dinorfinas/antagonistas & inhibidores , Hiperalgesia/tratamiento farmacológico , Células del Asta Posterior/efectos de los fármacos , Piel/efectos de los fármacos , Canales Catiónicos TRPC/antagonistas & inhibidores , Acroleína/administración & dosificación , Acroleína/efectos adversos , Acroleína/análogos & derivados , Acroleína/antagonistas & inhibidores , Analgésicos no Narcóticos/administración & dosificación , Animales , Dolor de Espalda/etiología , Dolor de Espalda/metabolismo , Conducta Animal/efectos de los fármacos , Colecistoquinina/administración & dosificación , Colecistoquinina/antagonistas & inhibidores , Colecistoquinina/metabolismo , Dinoprost/administración & dosificación , Dinoprost/antagonistas & inhibidores , Dinoprost/metabolismo , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Dinorfinas/metabolismo , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Inyecciones Espinales , Masculino , Minociclina/administración & dosificación , Minociclina/uso terapéutico , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Oximas/administración & dosificación , Oximas/uso terapéutico , Estimulación Física/efectos adversos , Células del Asta Posterior/metabolismo , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/agonistas , Canales Catiónicos TRPC/metabolismoRESUMEN
The effects of various protease inhibitors on naloxone-precipitated withdrawal jumping were examined in morphine-dependent mice. The doses of morphine were subcutaneously given twice daily for 2 days (day 1, 30 mg/kg; day 2, 60 mg/kg). On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after final injection of morphine (60 mg/kg), and the number of jumping was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by the intracerebroventricular administration of N-ethylmaleimide (0.5 nmol) and Boc-Tyr-Gly-NHO-Bz (0.4 nmol), inhibitors of cysteine proteases involved in dynorphin degradation, 5 min before each morphine treatment during the induction phase, with none given on the test day, as well as by dynorphin A (62.5 pmol) and dynorphin B (250 pmol). However, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, caused no changes. The present results suggest that cysteine protease inhibitors suppress naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation.
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Inhibidores de Cisteína Proteinasa/farmacología , Dependencia de Morfina/fisiopatología , Naloxona/farmacología , Síndrome de Abstinencia a Sustancias/fisiopatología , Animales , Dipéptidos/administración & dosificación , Dipéptidos/metabolismo , Dinorfinas/administración & dosificación , Dinorfinas/metabolismo , Etilmaleimida/administración & dosificación , Etilmaleimida/farmacología , Inyecciones Intraventriculares , Masculino , RatonesRESUMEN
BACKGROUND: Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice. METHODS: The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis. RESULTS: Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice. CONCLUSIONS: These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.
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Encéfalo/fisiología , Dinorfinas/fisiología , Inflamación/fisiopatología , Médula Espinal/fisiología , Animales , Conducta Animal , Química Encefálica/genética , Dinorfinas/administración & dosificación , Dinorfinas/farmacología , Encefalinas/genética , Adyuvante de Freund , Inmunohistoquímica , Inflamación/inducido químicamente , Inyecciones Espinales , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Dolor/psicología , Precursores de Proteínas/genética , ARN/genética , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transmisión Sináptica/fisiologíaRESUMEN
Dynorphin (DYN) fragments are the members of the endogenous opioid system and postulated ligands for the opioid receptors. Infusion of DYN(1-17) fragment into the rat dorsal striatum caused a significant increase in acetylcholine and decrease in dopamine overflow. Contrary to this, infusions of DYN(2-17) fragment into the rat dorsal striatum caused a significant increase in dopamine and decrease in acetylcholine overflow. Intrastriatal infusions of different doses of the acetylcholinesterase blocker, neostigmine, augmented acetylcholine and inhibited dopamine overflow in a dose-dependent manner. The opposing responses of the DYN fragments suggest that the N-terminal residue plays a key role in presynaptic neuromodulation.
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Acetilcolina/metabolismo , Química Encefálica/efectos de los fármacos , Dopamina/metabolismo , Dinorfinas/farmacología , Neostriado/fisiología , Animales , Inhibidores de la Colinesterasa/farmacología , Cromatografía Líquida de Alta Presión , Dinorfinas/administración & dosificación , Electroquímica , Masculino , Microdiálisis , Microinyecciones , Neostigmina/farmacología , Neostriado/anatomía & histología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Morphine-3-glucuronide (M3G) administered centrally produces dose-dependent neuro-excitatory behaviours in rodents via a predominantly non-opioid mechanism. The endogenous opioid peptide, dynorphin A (Dyn A) (1-17), is rapidly cleaved in vivo to the relatively more stable fragment Dyn A(2-17) which also produces excitatory behaviours in rodents via a non-opioid mechanism. This study investigated the possible contribution of Dyn A(2-17) to the neuro-excitatory behaviours evoked by supraspinally and spinally administered M3G in male Sprague-Dawley (SD) rats. Marked qualitative differences in behaviours were apparent following administration of M3G and Dyn A(2-17). Administration of 11 nmol i.c.v. doses of M3G produced intermittent myoclonic jerks, tonic-clonic convulsions, and ataxia, as well as postural changes, whereas i.c.v. Dyn A(2-17) at 15 nmol produced effects on body posture alone. Administration of 11 nmol i.t. doses of M3G produced intermittent explosive motor activity, and touch-evoked agitation, as well as postural changes, whereas i.t. Dyn A(2-17) at 15 nmol produced postural changes, touch-evoked agitation, and paralysis. Pre-treatment with Dyn A antiserum (200 microg) markedly attenuated total behavioural excitation following i.c.v. and i.t. administration of Dyn A(2-17) by approximately 94% and 78%, respectively. However, total behavioural excitation following i.c.v. and i.t. administration of M3G was less markedly attenuated (both approximately 27%) by pre-treatment with Dyn A antiserum, with reductions in tonic-clonic convulsions ( approximately 43%), explosive motor behaviour ( approximately 28%), and touch-evoked agitation ( approximately 22%). The present findings discount a major role for Dyn A in mediating the neuro-excitatory effects of M3G, although it may contribute to maintaining some individual neuro-excitatory behaviours.
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Estimulantes del Sistema Nervioso Central/farmacología , Dinorfinas/farmacología , Derivados de la Morfina/farmacología , Fragmentos de Péptidos/farmacología , Animales , Área Bajo la Curva , Estimulantes del Sistema Nervioso Central/administración & dosificación , Dinorfinas/administración & dosificación , Inyecciones Intraventriculares , Inyecciones Espinales , Masculino , Derivados de la Morfina/administración & dosificación , Actividad Motora/efectos de los fármacos , Parálisis/inducido químicamente , Fragmentos de Péptidos/administración & dosificación , Postura , Agitación Psicomotora/psicología , Ratas , Ratas Sprague-DawleyRESUMEN
Dynorphins, the endogenous opioid peptides derived from prodynorphin may participate not only in the inhibition, but also in facilitation of spinal nociceptive transmission. However, the mechanism of pronociceptive dynorphin actions, and the comparative potential of prodynorphin processing products to induce these actions were not fully elucidated. In our studies, we examined pronociceptive effects of prodynorphin fragments dynorphins A and B and big dynorphin consisting of dynorphins A and B, and focused on the mechanisms underlying these effects. Our principal finding was that big dynorphin was the most potent pronociceptive dynorphin; when administered intrathecally into mice at extremely low doses (1-10fmol), big dynorphin produced nociceptive behavior through the activation of the NMDA receptor ion-channel complex by acting on the polyamine recognition site. We next examined whether the endogenous dynorphins participate in the spinal nociceptive transmission using N-ethylmaleimide (NEM) that blocks dynorphin degradation by inhibiting cysteine proteases. Similar to big dynorphin and dynorphin A, NEM produced nociceptive behavior mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site. Our findings support the notion that endogenous dynorphins are critical neurochemical mediators of spinal nociceptive transmission in uninjured animals. This chapter will review above-described phenomena and their mechanism.