RESUMEN
Nucleoside reverse transcriptase inhibitors (NRTIs) are key components of HIV/AIDS treatment to reduce viral load. However, antiretroviral toxic neuropathy has become a common peripheral neuropathy among HIV/AIDS patients leading to discontinuation of antiretroviral therapy, for which the underlying pathogenesis is uncertain. This study examines the role of neurofilament (NF) proteins in the spinal dorsal horn, DRG and sciatic nerve after NRTI neurotoxicity in mice treated with zalcitabine (2',3'-dideoxycitidine; ddC). ddC administration up-regulated NF-M and pNF-H proteins with no effect on NF-L. The increase of pNF-H levels was counteracted by the silencing of HuD, an RNA binding protein involved in neuronal development and differentiation. Sciatic nerve sections of ddC exposed mice showed an increased axonal caliber, concomitantly to a pNF-H up-regulation. Both events were prevented by HuD silencing. pNF-H and HuD colocalize in DRG and spinal dorsal horn axons. However, the capability of HuD to bind NF mRNA was not demonstrated, indicating the presence of an indirect mechanism of control of NF expression by HuD. RNA immunoprecipitation experiments showed the capability of HuD to bind the BDNF mRNA and the administration of an anti-BDNF antibody prevented pNF-H increase. These data indicate the presence of a HuD - BDNF - NF-H pathway activated as a regenerative response to the axonal damage induced by ddC treatment to counteract the antiretroviral neurotoxicity. Since analgesics clinically used to treat neuropathic pain are ineffective on antiretroviral neuropathy, a neuroregenerative strategy might represent a new therapeutic opportunity to counteract neurotoxicity and avoid discontinuation or abandon of NRTI therapy.
Asunto(s)
Antirretrovirales , Proteína 4 Similar a ELAV/metabolismo , Proteínas de Neurofilamentos/metabolismo , Células Receptoras Sensoriales/metabolismo , Zalcitabina , Animales , Anticuerpos/farmacología , Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteína 4 Similar a ELAV/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Silenciador del Gen , Masculino , Ratones , Fármacos Neuroprotectores/farmacología , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nervio Ciático/metabolismo , Nervio Ciático/patología , Neuropatía Ciática/inducido químicamente , Neuropatía Ciática/genética , Neuropatía Ciática/metabolismo , Neuropatía Ciática/prevención & control , Células Receptoras Sensoriales/patología , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Regulación hacia ArribaRESUMEN
The antiretroviral toxic neuropathy, a distal sensory polyneuropathy associated with antiretroviral treatment, is a frequently occurring neurological complication during treatment of patients with AIDS and often leads to discontinuation of antiretroviral therapy. The mechanisms by which antiretroviral drugs contribute to the development of neuropathic pain are not known. Using drugs that reduce intracellular calcium ions (Ca(2+)), we investigated the hypothesis that altered cytosolic Ca(2+) concentration contributes to the 2',3'-dideoxycytidine (ddC)-evoked painful neuropathy. Administration of ddC induced mechanical and cold allodynia, which were abolished by intrathecal administration of TMB-8, a blocker of Ca(2+) release from intracellular stores, and by ryanodine, a RyR antagonist. Treatment with the IP3R antagonist heparin prevented mechanical allodynia with no effect on thermal response. To further clarify the pathway involved, we investigated the role of HuD, a RNA binding protein involved in neuronal function. HuD silencing reverted both mechanical and cold allodynia inducing, a phenotype comparable to that of ryanodine-exposed mice. HuD binding to the RyR2 mRNA, the most abundant RyR isoform in the spinal cord, was demonstrated and RyR2 silencing prevented the ddC-induced neuropathic pain. A positive regulation of gene expression on CaMKIIα by HuD was also observed, but sequestration of CaMKIIα had no effect on ddC-induced allodynia. The present findings identify a spinal RyR2 pathway activated in response to ddC administration, involving the binding activity on RyR2 mRNA by HuD. We propose the modulation of the RyR2 pathway as a therapeutic perspective in the management of antiretroviral painful neuropathy.
Asunto(s)
Proteínas ELAV/metabolismo , Umbral del Dolor/efectos de los fármacos , Dolor/inducido químicamente , Dolor/patología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/patología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Médula Espinal/fisiología , Zalcitabina/toxicidad , Analgésicos no Narcóticos/farmacología , Animales , Fármacos Anti-VIH/toxicidad , Apomorfina/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Agonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Proteínas ELAV/genética , Proteína 4 Similar a ELAV , Conducta Exploratoria/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Hiperalgesia/terapia , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Neuroblastoma/patología , Dolor/complicaciones , Dolor/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológicoRESUMEN
BACKGROUND: Despite sensitivity of MRI to diagnose multiple sclerosis (MS), prognostic biomarkers are still needed for optimized treatment. OBJECTIVE: The objective of this paper is to identify cerebrospinal fluid (CSF) diagnostic biomarkers of MS using quantitative proteomics and to analyze their expression at different disease stages. METHODS: We conducted differential analysis of the CSF proteome from control and relapsing-remitting MS (RRMS) patients followed by verification by ELISA of candidate biomarkers in CSF and serum in control, clinically isolated syndrome (CIS), RRMS and progressive MS (PMS) patients. RESULTS: Twenty-two of the 527 quantified proteins exhibited different abundances in control and RRMS CSF. These include chitinase 3-like protein 1 (CHI3L1) and 2 (CHI3L2), which showed a strong expression in brain of MS patients, especially in astrocytes and microglial cells from white matter plaques. CSF and serum CHI3L1 levels increased with the disease stage and CIS patients with high CSF (>189 ng/ml) and serum (>33 ng/ml) CHI3L1 converted more rapidly to RRMS (log rank test, p < 0.05 and p < 0.001, respectively). In contrast, CSF CHI3L2 levels were lower in PMS than in RRMS patients. Accordingly, CSF CHI3L1/CHI3L2 ratio accurately discriminated PMS from RRMS. CONCLUSIONS: CSF CHI3L1 and CHI3L2 and serum CHI3L1 might help to define MS disease stage and have a prognostic value in CIS.
Asunto(s)
Adipoquinas/sangre , Adipoquinas/líquido cefalorraquídeo , Quitinasas/líquido cefalorraquídeo , Lectinas/sangre , Lectinas/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/diagnóstico , Adulto , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Encéfalo/metabolismo , Proteína 1 Similar a Quitinasa-3 , Quitinasas/sangre , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , ProteómicaRESUMEN
Nucleoside reverse transcriptase inhibitors (NRTIs) are known to produce painful neuropathies and to enhance states of pain hypersensitivity produced by HIV-1 infection in patients with AIDS leading to discontinuation of antiretroviral therapy, thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current study, we tested the hypothesis that HuD, an RNA binding protein known to be an essential promoter of neuronal differentiation and survival, might be involved in the response to NRTI-induced neuropathy. Antiretroviral neuropathy was induced by a single intraperitoneal administration of 2',3'-dideoxycytidine (ddC) in mice. HuD was physiologically expressed in the cytoplasm of the soma and in axons of neurons within DRG and spinal cord and was considerably overexpressed following ddC treatment. ddC up-regulated spinal GAP43 protein, a marker of neuroregeneration, and this increase was counteracted by HuD silencing. GAP43 and HuD colocalize in DRG and spinal dorsal horn (SDH) axons and administration of an anti-GAP43 antibody aggravated the ddC-induced axonal damage. The administration of a protein kinase C (PKC) inhibitor or the PKCγ silencing prevented both HuD and GAP43 increased expression. Conversely, treatment with the PKC activator PDBu potentiated HuD and GAP43 overexpression, demonstrating the presence of a spinal PKC-dependent HuD-GAP43 pathway activated by ddC. These results indicated that HuD recruitment and GAP43 protein increase are mechanistically linked events involved in the response to antiretroviral-induced neurodegenerative processes.
Asunto(s)
Antirretrovirales/toxicidad , Proteínas ELAV/metabolismo , Proteína GAP-43/metabolismo , Dolor/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Médula Espinal/metabolismo , Zalcitabina/toxicidad , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacología , Dolor/fisiopatología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Fosfopiruvato Hidratasa/metabolismo , Proteína Quinasa C/metabolismo , Médula Espinal/efectos de los fármacos , Factores de TiempoRESUMEN
Patients treated with nucleoside reverse transcriptase inhibitors (NRTIs) develop painful neuropathies that lead to discontinuation of antiretroviral therapy thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathy are not known. In order to elucidate the mechanisms underlying this drug-induced neuropathy, we have characterized cellular events in the central nervous system following antiretroviral treatment. Systemic administration of the antiretroviral agent, 2',3'-dideoxycytidine (ddC) considerably increased the expression and phosphorylation of protein kinase C (PKC) γ and É, enzymes highly involved in pain processes, within periaqueductal grey matter (PAG), and, to a lesser extent, within thalamus and prefrontal cortex. These events appeared in coincidence with thermal and mechanical allodynia, but PKC blockade did not prevent the antiretroviral-induced pain hypersensitivity, ruling out a major involvement of PKC in the ddC-induced nociceptive behaviour. An increased expression of GAP43, a marker of neuroregeneration, and decreased levels of ATF3, a marker of neuroregeneration, were detected in all brain areas. ddC treatment also increased the expression of HuD, a RNA-binding protein target of PKC known to stabilize GAP43 mRNA. Pharmacological blockade of PKC prevented HuD and GAP43 overexpression. Silencing of both PKCγ and HuD reduced GAP43 levels in control mice and prevented the ddC-induced GAP43 enhanced expression. Present findings illustrate the presence of a supraspinal PKC-mediated HuD-GAP43 pathway activated by ddC. Based on our results, we speculate that antiretroviral drugs may recruit the HuD-GAP43 pathway, potentially contributing to a response to the antiretroviral neuronal toxicity.
Asunto(s)
Proteínas ELAV/metabolismo , Proteína GAP-43/metabolismo , Dolor/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Proteína Quinasa C/metabolismo , Inhibidores de la Transcriptasa Inversa/efectos adversos , Zalcitabina/efectos adversos , Animales , Fármacos Anti-VIH/efectos adversos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteína 4 Similar a ELAV , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Masculino , Ratones , Dolor/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/inducido químicamenteRESUMEN
Despite a number of antimigraine drugs belonging to different pharmacological classes are available, there is a huge unmet need for better migraine pharmacotherapy. We here demonstrated the capability of Hypericum perforatum, popularly called St. John's wort (SJW), to relieve meningeal nociception in an animal model induced by administration of the nitric oxide (NO) donors glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). GTN and SNP produced a delayed meningeal inflammation, as showed by the upregulation of interleukin (IL)-1ß and inducible NO synthase (iNOS), and a prolonged cold allodynia and heat hyperalgesia with a time-course consistent with NO-induced migraine attacks. A single oral administration of a SJW dried extract (5mg/kg p.o.) counteracted the nociceptive behaviour and the overexpression of IL-1ß and iNOS. To clarify the cellular pathways involved, the expression of protein kinase C (PKC) and downstream effectors was detected. NO donors increased expression and phosphorylation of PKCγ, PKCÉ and transcription factors, such as nuclear factor (NF)-κB, cyclic AMP response element binding protein (CREB), Signal Transducer and Activator of Transcription (STAT)-1. All these molecular events were prevented by SJW and hypericin, a SJW main component. In conclusion, SJW counteracted the NO donor-induced pain hypersensitivity and meningeal activation by blocking PKC-mediated pathways involving NF-κB, CREB, STAT1. These results might suggest SJW as an innovative and safe perspective for migraine pain.
Asunto(s)
Hypericum/química , Meninges/efectos de los fármacos , Trastornos Migrañosos/tratamiento farmacológico , Nocicepción/efectos de los fármacos , Extractos Vegetales/uso terapéutico , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Masculino , Meninges/enzimología , Ratones , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
BACKGROUND: Despite the substantial improvement that antimigraine drugs brought to migraineurs, there is the need for a long-acting and better tolerated migraine treatment than actual pharmacotherapy. St. John's wort (SJW), a medicinal plant endowed with a favourable tolerability profile, showed numerous bioactivities. We here investigated the pain-relieving property of SJW and its main components, hypericin and flavonoids, in a mouse model induced by nitric oxide (NO) donors administration. METHODS: The NO donors nitroglycerin and sodium nitroprusside (SNP) induced allodynia (cold plate test) and hyperalgesia (hot plate test). Western blotting experiments were performed to detect c-Fos and protein kinase C (PKC) expression within periaqueductal grey matter (PAG). RESULTS: A single oral administration of an SJW dried extract (5 mg/kg p.o.) produced a prolonged relief from pain hypersensitivity. Similarly, preventive SJW administration increased the latency to the induction of hyperalgesia and reduced the duration of the painful symptomatology. Among SJW main components, hypericin showed a similar profile of activity, whereas flavonoids were devoid of any antihyperalgesic effect. To clarify the cellular pathways involved in the SJW mechanism of action, we examined the effects induced by the herbal drug on PKC. NO donors' administration upregulated and increased phosphorylation of PKCγ and PKCε isoforms within PAG that was prevented by SJW treatment. The absence of behavioural side effects or altered animals' locomotor activity by SJW was demonstrated. CONCLUSIONS: These results suggest SJW as a safe therapeutic perspective for migraine pain, and indicate PKC as an innovative target for antimigraine therapy.
Asunto(s)
Hypericum/química , Trastornos Migrañosos/tratamiento farmacológico , Dolor/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Conducta Animal/efectos de los fármacos , Western Blotting , Frío , Ergotamina/uso terapéutico , Calor , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/psicología , Indometacina/uso terapéutico , Inyecciones Intraventriculares , Masculino , Ratones , Trastornos Migrañosos/inducido químicamente , Trastornos Migrañosos/complicaciones , Actividad Motora/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Nitroglicerina , Nitroprusiato , Dolor/inducido químicamente , Dolor/etiología , Sustancia Gris Periacueductal/fisiología , Extractos Vegetales/uso terapéutico , Equilibrio Postural/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Vasoconstrictores/uso terapéutico , VasodilatadoresRESUMEN
The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios/química , Antiinflamatorios/farmacología , Pirroles/química , Pirroles/farmacología , Analgésicos/química , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/uso terapéutico , Humanos , Pirroles/uso terapéutico , Relación Estructura-ActividadRESUMEN
Oxaliplatin is a platinum-based chemotherapy drug characterized by the development of a painful peripheral neuropathy which is reproduced in rodent animal models with features observed in humans. Our focus was to explore the alterations of intracellular second messengers at supraspinal level in oxaliplatin-induced mechanical hyperalgesia. In our experiments, chronic administration of oxaliplatin to rats induced mechanical hyperalgesia which lasted for many days. When the hyperalgesic rats were submitted to paw pressure test in the presence of selective PKC inhibitor Calphostin C supraspinally administered, hyperalgesic effect could be reversed showing that PKC activity in supraspinal brain regions is needed. Concurrently, oxaliplatin chronic treatment induced a specific upregulation of gamma isoforms of PKC and increased phosphorylation of gamma/epsilon PKC isoforms within thalamus and PAG. Phosphorylation was reversed when PKC activity was inhibited by Calphostin C. Distinct PKC-activated MAPK pathways, including p38MAPK, ERK1/2 and JNK, were investigated in chronic oxaliplatin rat. A dramatic phosphorylation increase, Calphostin C sensitive, could be observed in thalamus and PAG for p38MAPK. These data show that, in oxaliplatin-induced neuropathy, enhanced mechanical nociception is strictly correlated with increased phosphorylation of specific intracellular mediators in PAG and thalamus brain regions pointing to a role of these supraspinal centers in oxaliplatin-induced neuropathic pain mechanism.
Asunto(s)
Antineoplásicos , Compuestos Organoplatinos , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Proteína Quinasa C/fisiología , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Inyecciones Intraventriculares , Masculino , Naftalenos/farmacología , Oxaliplatino , Dimensión del Dolor , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Fosforilación , Estimulación Física , Equilibrio Postural/efectos de los fármacos , Presión , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
In this study, the optimisation of biopharmaceutical properties of a dried commercial extract of St John's Wort were evaluated employing the in vivo forced swimming test (FST). Three new dosage forms containing beta-cyclodextrin and surfactants (SDS, ASC8) were compared in the FST with the commercial extract. The commercial extract showed antidepressant activity in mice after 60 min at a dosage of 100 mg/kg. The same antidepressant activity appeared in 30 min with a micellar solution of SDS containing the same quantity of extract (100 mg/kg), while with micelles of ASC8 the effect appeared at 15 min and with a dosage of 30 mg/kg. In the case of beta-cyclodextrin the best results were obtained at 30 min, administering 60 mg/kg of the extract. Finally, the influence of the formulations on the water solubility of the constituents of the extract is reported. The tensides dramatically enhanced solubility, in particular that of the more lipophilic compounds, in the case of beta-cyclodextrin this effect was very pronounced for flavonoids and biapigenin, lower for hypericins and practically insignificant for hyperforins.
Asunto(s)
Antidepresivos/farmacocinética , Hypericum/química , Extractos Vegetales/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Excipientes/farmacología , Masculino , Ratones , Micelas , Plantas Medicinales , Dodecil Sulfato de Sodio/farmacología , Solubilidad , Natación , beta-Ciclodextrinas/farmacologíaRESUMEN
Evidence has accumulated for the involvement of Ca(2+) in the pathophysiology of mood disorders. Elevations in both resting and stimulated intracellular Ca(2+) levels in patients with affective disorders have been reported. The role of inositol-1,4,5-trisphosphate receptors (InsP3Rs), which allow mobilization of intracellular Ca(2+) stores, was, then, investigated in the mouse forced swimming test. InsP3R antagonists (heparin, xestospongin C) as well as an inositol monophosphatase inhibitor (LiCl) showed an antidepressant activity of intensity comparable to clinically used antidepressants. InsP3Rl, InsP3R2 and InsP3R3 knockdown mice were obtained to investigate the role of InsP3R isoforms. We generated mice carrying a cerebral knockdown of InsP3Rl, InsP3R2 and InsP3R3 proteins by administering antisense oligonucleotides complementary to the sequence of InsP3Rl, InsP3R2 and InsP3R3. These antisense-treated mice showed a specific InsP3R protein level reduction in the mouse cerebral cortex and hippocampus, demonstrated by immunoblotting, immunoprecipitation and immunocytochemistry experiments. Knockdown mice for each InsP3R isoforms showed an antidepressant behaviour and the induced phenotype was reversible disappearing 7 days after the end of the treatment. The absence of impairment of locomotor activity and spontaneous mobility in InsP3R knockdown mice was revealed. These results indicate the involvement of the InsP3R-mediated pathway in the modulation of depressive conditions and may be useful for the development of new therapeutical strategies for the treatment of mood disorders.
Asunto(s)
Conducta Animal/fisiología , Depresión/genética , Depresión/psicología , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Animales , Western Blotting , Química Encefálica/genética , Técnicas de Silenciamiento del Gen , Inmunoprecipitación , Inyecciones Intraventriculares , Inositol/farmacología , Cloruro de Litio/farmacología , Compuestos Macrocíclicos/farmacología , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Oligonucleótidos Antisentido/farmacología , Oxazoles/farmacología , Equilibrio Postural/efectos de los fármacos , Equilibrio Postural/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Natación/psicologíaRESUMEN
The importance of an intracellular calcium content increase to obtain cholinergic antinociception was demonstrated. The physiological and pathological role of ryanodine receptors (RyRs), receptors involved in the mobilization of intracellular calcium stores, at the CNS level is poorly understood. The aim of the present study was, therefore, to investigate the role of supraspinal endoplasmic type 1, 2 and 3 RyR subtypes in muscarinic antinociception in conditions of acute thermal (hotplate test) and inflammatory (abdominal constriction test) pain. In the absence of isoform selective RyR antagonists, types 1, 2 and 3 RyR knockdown mice were obtained. Western blotting experiments were performed to quantify the RyR isoform protein levels in knockdown mice demonstrating a selective protein level reduction in knockdown animals. I.c.v. pretreatment with an antisense oligonucleotide (aODN) against type 1 or type 3 RyR prevented cholinergic antinociception in the hotplate test shifting to the right of the physostigmine dose-response curve. This antagonistic effect disappeared 7 days after the end of the aODN administration. Conversely, the physostigmine analgesia remained unmodified in type 2 RyR knockdown mice. Similar results were obtained in the abdominal constriction test. Mice undergoing aODN treatments showed neither alteration of animals' gross behavior nor locomotor impairment (rota-rod and hole board tests). These results elucidate the intracellular mechanism underlying muscarinic antinociception. A selective involvement of RyR1 and RyR3 in supraspinal muscarinic analgesia was demonstrated whereas RyR2 appears not to play an essential role in acute thermal and inflammatory pain.
Asunto(s)
Encéfalo/metabolismo , Oligonucleótidos Antisentido/farmacología , Dolor/fisiopatología , Receptores Muscarínicos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Western Blotting , Encéfalo/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Inmunoprecipitación , Masculino , Ratones , Ratones Noqueados , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Fisostigmina/farmacología , Ratas , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacosRESUMEN
The involvement of central endoplasmic inositol 1,4,5-trisphosphate receptors (IP3R) in muscarinic antinociception was investigated in the mouse hot plate test. Selective knockdown of type 1, 2 and 3 IP3R was obtained by means of an antisense oligonucleotide (aODN) strategy. A selective IP3R protein level reduction of approximately 30-50% produced by aODN administration for each receptor subtype investigated was demonstrated by Western blotting experiments. I.c.v. pretreatment with an aODN complementary to the sequence of the type 2 IP3R (0.1-3 nmol per mouse i.c.v.) prevented the antinociception induced by physostigmine (0.15 mg kg(-1) s.c.) and oxotremorine (60 microg kg(-1) s.c.). Similarly, an aODN against type 3 IP3R (0.1-3 nmol per mouse i.c.v.) antagonized cholinergic antinociception. A shift to the right of the physostigmine dose-response curve was obtained after anti-type 2 IP3R2 and anti-type 3 IP3R treatments. Conversely, pretreatment with an aODN complementary to the sequence of type 1 IP3R (0.1-5 nmol per mouse i.c.v.) did not modify the antinociception induced by physostigmine and oxotremorine. Mice undergoing treatment with aODNs did not show any impairment of the locomotor activity, spontaneous motility and exploratory activity as revealed by the rota-rod and hole board tests. These results indicate a selective involvement of type 2 and 3 IP3R in central muscarinic antinociception in mice.
Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Nociceptores/fisiología , Receptores Muscarínicos/fisiología , Animales , Western Blotting , Conducta Exploratoria/efectos de los fármacos , Inyecciones Intraventriculares , Receptores de Inositol 1,4,5-Trifosfato/biosíntesis , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Nociceptores/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Oxotremorina/farmacología , Dimensión del Dolor/efectos de los fármacos , Fisostigmina/farmacología , Equilibrio Postural/efectos de los fármacos , ARN Mensajero/genética , Receptores Muscarínicos/efectos de los fármacosRESUMEN
DM232 (unifiram) and DM235 (sunifiram) are potent cognition-enhancers, which are four order of magnitude more potent than piracetam. These compounds, although not showing affinity in binding studies for the most important central receptors or channels, are able to prevent amnesia induced by modulation of several neurotransmission systems. These compounds are able to increase the release of acetylcholine from rat cerebral cortex, and, as far as unifiram is concerned, to increase the amplitude of fEPSP in rat hippocampal slices. In vitro experiments, performed on hippocampal slices, also supported the hypothesis of a role of the AMPA receptors for the cognition-enhancing properties of unifiram and sunifiram.
Asunto(s)
Cognición/efectos de los fármacos , Nootrópicos/farmacología , Piperazinas/farmacología , Pirroles/farmacología , Animales , Sistema Nervioso Central/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Nootrópicos/uso terapéutico , Piperazinas/uso terapéutico , Pirroles/uso terapéutico , Transmisión Sináptica/efectos de los fármacosRESUMEN
Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts.
Asunto(s)
Gryllidae/fisiología , Gryllidae/parasitología , Helmintos/fisiología , Proteómica , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Proteínas del Helminto/análisis , Proteínas del Helminto/química , Interacciones Huésped-Parásitos , Proteínas de Insectos/análisis , Proteínas de Insectos/química , Masculino , Datos de Secuencia Molecular , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insect's responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshopper's central nervous system (CNS) by producing certain 'effective' molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the host's CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.
Asunto(s)
Conducta Animal/fisiología , Regulación de la Expresión Génica , Saltamontes/fisiología , Saltamontes/parasitología , Helmintos/metabolismo , Proteínas de Insectos/metabolismo , Animales , Encéfalo/metabolismo , Ritmo Circadiano/genética , Electroforesis en Gel Bidimensional , Saltamontes/genética , Saltamontes/metabolismo , Helmintos/genética , Interacciones Huésped-Parásitos , Procesamiento de Imagen Asistido por Computador , Proteínas de Insectos/genética , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The post-receptorial mechanism of the amnesic action of the alpha2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the alpha-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 microg per mouse i.c.v.) reversed the amnesia induced by both alpha2-agonists. Similarly, anti-Gialpha1 (6.25-12.5 microg per mouse i.c.v.), anti-Gialpha3 (3.12-12.5 microg per mouse i.c.v.), anti-Goalpha1 (12.5-25 microg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Gialpha2 (3.12-25 microg per mouse i.c.v.) and anti-Goalpha2 (12.5-25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by the alpha2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Amnesia/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Transducción de Señal/efectos de los fármacos , Agonistas alfa-Adrenérgicos/toxicidad , Amnesia/inducido químicamente , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Clonidina/farmacología , Clonidina/toxicidad , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores Adrenérgicos alfa 2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The effect of the i.c.v. administration of antisense oligodeoxynucleotides directed against the alpha subunit of different Gi-proteins (anti-Gialpha(1), anti-Gialpha(2), anti-Gialpha(3), anti-Goalpha(1), anti-Goalpha(2)) on the amnesia induced by the H(1)-antihistamine diphenhydramine (20 mg kg(-1) s.c.) was evaluated in the mouse passive avoidance test. Pretreatment with anti-Gialpha(1) (12.5-25 microg per mouse i.c.v.) and anti-Gialpha(2) (25 microg per mouse i.c.v.), administered 24 and 18 h before test, prevented antihistamine-induced amnesia. By contrast, pretreatment with an anti-Gialpha(3) (25 microg per mouse i.c.v.), anti-Goalpha(1) (25 microg per mouse i.c.v.) and anti-Goalpha(2) (25 microg per mouse i.c.v.) did not modify the detrimental effect induced by diphenhydramine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by the Gi(1)- and Gi(2)-protein pathway in the transduction mechanism involved in the impairment of memory processes produced by the H(1)-antihistamine diphenhydramine.
Asunto(s)
Amnesia/inducido químicamente , Amnesia/metabolismo , Difenhidramina/toxicidad , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Masculino , Ratones , Oligonucleótidos Antisentido/farmacología , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiologíaRESUMEN
DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.
Asunto(s)
Amnesia/tratamiento farmacológico , Nootrópicos/farmacología , Piperazinas/farmacología , Pirroles/farmacología , Receptores AMPA/fisiología , Amnesia/inducido químicamente , Animales , Reacción de Prevención/efectos de los fármacos , Cognición/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrofisiología/métodos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Quinurénico/farmacología , Masculino , Ratones , N-Metilaspartato/farmacología , Nootrópicos/química , Piperazinas/química , Pirroles/química , Quinoxalinas/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismoRESUMEN
The region from the third external loop to the C terminus of MOR-1 appeared to be critical to the selective binding of MOR-1 ligands as DAMGO and morphine to MOR-1. To study the pharmacological properties of the third extracellular loop an antibody was raised in rabbits against the sequence 304-316 which is unique to MOR-1 and includes the third external loop; the anti-MOR-1 antibody was affinity purified against the immunogen sequence and characterized by [3H]DAMGO and Western blotting; [3H]DPDPE binding assay remained unchanged in the presence of the antibody. Anti-MOR-1 IgG was characterized as a neutral antagonist in Chinese hamster ovary (CHO) cells hyperexpressing constitutively active MOR-1s; in fact, anti-MOR-1 IgG completely reversed the inhibition induced by the MOR-1 agonist endomorphin1, endomorphin2, DAMGO and morphine on forskolin stimulated cyclic AMP (cAMP) accumulation and attenuated both the action of the selective MOR-1 agonist DAMGO to increase [35S]GTPgammaS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [35S]GTPgammaS binding. Radioligand binding assay using membrane suspensions from CHO cells hyperexpressing MOR-1 revealed a significant decreased binding affinity and capacity of all the tested MOR-1 selective ligands after preincubation with anti-MOR-1 IgG. Therefore, the third extracellular loop of MOR-1 appeared to be a key element for the binding of MOR-1 ligands.