RESUMO
OBJECTIVES: Neuropathic pain is characterized by long-lasting, intractable pain. Sciatic nerve ligation is often used as an animal model of neuropathic pain, and the spared nerve injury (SNI) model, in which the common peroneal nerve (CPN) and tibial nerve (TN) are ligated, is widely used. In the present study, we evaluated the analgesic effect of a cholinergic agonist, carbachol, on a neuropathic pain model prepared by sural nerve (SN) ligation in mice. METHODS: The SN was tightly ligated as a branch of the sciatic nerve. Mechanical and thermal allodynia, and hyperalgesia were assessed using von Frey filaments and heat from a hot plate. The analgesic effects of intracerebroventricularly-administered morphine and carbachol were compared. RESULTS: SN ligation resulted in a significant decrease in pain threshold for mechanical stimulation 1 day after ligation. In response to thermal stimulation, allodynia was observed at 50°C and hyperalgesia at 53 and 56°C 3 days after ligation. Content of thiobarbituric acid reactive substances (TBARS) in the spinal cord increased significantly at 6 and 12 h after ligation. Acetylcholine content of the spinal cord also increased at 5 and 7 days after ligation. Intracerebroventricular administration of carbachol at 7 days after ligation produced a marked analgesic effect against mechanical and thermal stimuli, which was stronger and longer-lasting than morphine at all experimental time points. CONCLUSION: These findings suggest that cholinergic nerves are involved in allodynia and hyperalgesia of the SN ligation neuropathic pain model.
Assuntos
Carbacol , Modelos Animais de Doenças , Hiperalgesia , Neuralgia , Nervo Sural , Animais , Hiperalgesia/tratamento farmacológico , Masculino , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Carbacol/farmacologia , Ligadura , Camundongos , Nervo Sural/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Limiar da Dor/efeitos dos fármacos , Morfina/farmacologia , Analgésicos/farmacologia , Medição da Dor , Medula Espinal/efeitos dos fármacos , Acetilcolina/metabolismoRESUMO
1,2,3,4-tetrahydroisoquinoline (TIQ) is endogenously present in the human brain, and some of its derivatives are thought to contribute to the induction of Parkinson's disease (PD)-like signs in rodents and primates. In contrast, the endogenous TIQ derivative 1-methyl-TIQ (1-MeTIQ) is reported to be neuroprotective. In the present study, we compared the effects of artificially modified 1-MeTIQ derivatives (loading an N-propyl, N-propenyl, N-propargyl, or N-butynyl group) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD-like signs in mice. In a behavioral study, MPTP-induced bradykinesia was significantly decreased by all compounds. However, only 1-Me-N-propargyl-TIQ showed an inhibitory effect by blocking the MPTP-induced reduction in striatal dopamine content and the number of nigral tyrosine hydroxylase-positive cells. Western blot analysis showed that 1-Me-N-propargyl-TIQ and 1-Me-N-butynyl-TIQ potently prevented the MPTP-induced decrease in dopamine transporter expression, whereas 1-MeTIQ and 1-Me-N-propyl-TIQ did not. These results suggest that although loading an N-propargyl group on 1-MeTIQ clearly enhanced neuroprotective effects, other N-functional groups showed distinct pharmacological properties characteristic of their functional groups. Thus, the number of bonds and length of the N-functional group may contribute to the observed differences in effect.
Assuntos
Intoxicação por MPTP , Fármacos Neuroprotetores , Transtornos Parkinsonianos , Tetra-Hidroisoquinolinas , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Humanos , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/prevenção & controleRESUMO
Chemotherapy-induced peripheral neuropathy has an important impact on the quality of life of cancer patients. Vincristine-induced neuropathy is a major dose-limiting side effect. Symptoms of peripheral neuropathy are spontaneous pain, allodynia, and hyperalgesia. To analyze the contribution of substance P to the development of vincristine-induced mechanical allodynia/hyperalgesia, substance P levels in the rat spinal dorsal horn were analyzed after vincristine treatment. Mechanical allodynia/hyperalgesia was tested with the von Frey filaments 14 days after intraperitoneal (i.p.) administration of vincristine 0.1 mg/kg/day in rats. Vincristine-induced mechanical allodynia/hyperalgesia after day 14 was significantly inhibited by the neurokinin 1 receptor antagonist, aprepitant (20 mg/kg, s.c.). Immunohistochemistry showed that vincristine treatment significantly increased substance P expression (30.3% ± 2.4%) compared to saline treatment in the superficial layers of the spinal dorsal horn. Moreover, vincristine treatment significantly increased the substance P level in the spinal cord. These results suggest that vincristine treatment increases substance P in the spinal dorsal horn, and that aprepitant attenuates mechanical allodynia/hyperalgesia in vincristine-induced neuropathic rats.
Assuntos
Neuralgia , Substância P , Animais , Modelos Animais de Doenças , Humanos , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Qualidade de Vida , Ratos , Ratos Sprague-Dawley , Medula Espinal , Vincristina/toxicidadeRESUMO
Painful peripheral neuropathy is a common adverse effect of paclitaxel (PTX) treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia, TRPV1 expression in the rat spinal cord was analyzed after intraperitoneal administration of 2 and 4 mg/kg PTX. PTX treatment increased the expression of TRPV1 protein in the spinal cord. Immunohistochemistry showed that PTX (4 mg/kg) treatment increased TRPV1 protein expression in the superficial layers of the spinal dorsal horn 14 days after treatment. Behavioral assessment using the paw withdrawal response showed that PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia after 14 days was significantly inhibited by oral or intrathecal administration of the TRPV1 antagonist AMG9810. We found that intrathecal administration of small interfering RNA (siRNA) to knock down TRPV1 protein expression in the spinal cord significantly decreased PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia. Together, these results demonstrate that TRPV1 receptor expression in spinal cord contributes, at least in part, to the development of PTX-induced painful peripheral neuropathy. TRPV1 receptor antagonists may be useful in the prevention and treatment of PTX-induced peripheral neuropathic pain.
Assuntos
Hiperalgesia/metabolismo , Paclitaxel/efeitos adversos , Medula Espinal/metabolismo , Canais de Cátion TRPV/metabolismo , Acrilamidas/administração & dosagem , Acrilamidas/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Modelos Animais de Doenças , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Injeções Intraperitoneais , Injeções Espinhais , Masculino , Paclitaxel/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Ratos , Canais de Cátion TRPV/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacosRESUMO
Transient receptor potential (TRP) receptors are involved in the development of chemotherapy-induced peripheral neuropathic pain, which is a common side effect of selected chemotherapeutic agents such as oxaliplatin. However, the precise contribution of TRPs to this condition remains unknown. Cold hypersensitivity is the hallmark of oxaliplatin-induced neuropathy, so we used a preclinical model of oxaliplatin-induced cold hypersensitivity in rats to determine the effects of oxaliplatin on TRP channels. To this end, immunohistochemistry was used to examine TRP vanilloid 1 (TRPV1), TRP ankyrin 1 (TRPA1), and TRP melastatin 8 (TRPM8) expression in the rat dorsal root ganglion (DRG) after 4days of oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that oxaliplatin significantly increased acute cold hypersensitivity after 4days of treatment. Double-staining immunohistochemistry showed that 4days after oxaliplatin treatment, there was increased co-expression of TRPA1 and TRPV1 in isolectin B4-positive small-sized DRG neurons, as well as a significant increase in the co-localization of TRPM8 and neurofilament 200 in medium-sized DRG neurons. In addition, in situ hybridization revealed that TRPV1 protein was co-expressed with TRPA1 mRNA on day 4 after oxaliplatin administration. Thus, at an early stage following oxaliplatin treatment there is an increased expression of TRPA1 and TRPV1 in small-sized DRG neurons and of TRPM8 in medium-sized DRG neurons. Collectively, these changes may contribute to the development of oxaliplatin-induced peripheral neuropathic pain.
Assuntos
Antineoplásicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Animais , Temperatura Baixa , Síndromes Periódicas Associadas à Criopirina/fisiopatologia , Gânglios Espinais/metabolismo , Hiperalgesia/induzido quimicamente , Masculino , Neuralgia/induzido quimicamente , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxaliplatina , Ratos Wistar , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Oxaliplatin is a chemotherapeutic agent that is effective against various types of cancer including colorectal cancer. Acute cold hyperalgesia is a serious side effect of oxaliplatin treatment. Although the therapeutic drug pregabalin is beneficial for preventing peripheral neuropathic pain by targeting the voltage-dependent calcium channel α2δ-1 (Cavα2δ-1) subunit, the effect of oxaliplatin-induced acute cold hypersensitivity is uncertain. To analyze the contribution of the Cavα2δ-1 subunit to the development of oxaliplatin-induced acute cold hypersensitivity, Cavα2δ-1 subunit expression in the rat spinal cord was analyzed after oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that 6 mg/kg oxaliplatin-induced cold hypersensitivity 2 and 4 days later. Oxaliplatin-induced acute cold hypersensitivity 4 days after treatment was significantly inhibited by pregabalin (50 mg/kg, p.o.). Oxaliplatin (6 mg/kg, i.p.) treatment increased the expression level of Cavα2δ-1 subunit mRNA and protein in the spinal cord 2 and 4 days after treatment. Immunohistochemistry showed that oxaliplatin increased Cavα2δ-1 subunit protein expression in superficial layers of the spinal dorsal horn 2 and 4 days after treatment. These results suggest that oxaliplatin treatment increases Cavα2δ-1 subunit expression in the superficial layers of the spinal cord and may contribute to functional peripheral acute cold hypersensitivity.
Assuntos
Antineoplásicos/toxicidade , Canais de Cálcio/metabolismo , Expressão Gênica/efeitos dos fármacos , Compostos Organoplatínicos/toxicidade , Medula Espinal/metabolismo , Doença Aguda , Animais , Canais de Cálcio/genética , Síndromes Periódicas Associadas à Criopirina/induzido quimicamente , Síndromes Periódicas Associadas à Criopirina/genética , Síndromes Periódicas Associadas à Criopirina/prevenção & controle , Masculino , Oxaliplatina , Pregabalina/administração & dosagem , Pregabalina/uso terapêutico , Ratos WistarRESUMO
Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4mg/kg) treatment significantly increased substance P expression (37.6±3.7% on day 7, 43.6±4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy.
Assuntos
Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Substância P/metabolismo , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Masculino , Ratos , Ratos Wistar , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismoRESUMO
Guillain-Barré syndrome is a type of acute inflammatory neuropathy that causes ataxia and is associated with the IgG anti-GM1 antibody. However, the pathogenic role of the IgG anti-GM1 antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. Thus, the aim of the present study was to investigate the effects of the IgG anti-GM1 monoclonal antibody (mAb) on spontaneous muscle action potentials (SMAPs), and the effects of calcium channel blockers, in a rat spinal cord-muscle co-culture system. In addition, the binding of IgG anti-GM1 mAb to calcium channels was investigated in the rat hemidiaphragm. The frequency of SMAPs in the innervated muscle cells was acutely inhibited by the IgG anti-GM1 mAb; however, this effect was blocked by the N-type calcium channel blocker, ω-conotoxin GVIA (30 nM). Furthermore, the P/Q-type calcium channel blocker, ω-agatoxin IVA (10 nM), was found to partially block the IgG anti-GM1 mAb-induced inhibitory effect in the spinal cord-muscle co-culture system. Immunohistochemical analysis of the rat hemidiaphragm indicated that IgG anti-GM1 mAb binding overlapped with anti-Cav2.2 (α1B) antibody binding in the nerve terminal. In addition, IgG anti-GM1 mAb binding partially overlapped with anti-Cav2.1 (α1A) antibody binding. Thus, the results demonstrated that the IgG anti-GM1 mAb binds to calcium channels in the nerve terminals of NMJs. Therefore, the inhibitory effect of IgG anti-GM1 mAb on SMAPs may involve N-type and P/Q-type calcium channels in motor nerve terminals at the NMJ.
RESUMO
BACKGROUND: Peripheral cold neuropathic pain is a serious side effect of oxaliplatin treatment. However, the mechanism of oxaliplatin-induced cold hyperalgesia is unknown. In the present study, we investigated the effects of oxaliplatin on transient receptor potential ankyrin 1 (TRPA1) in dorsal root ganglion (DRG) neurons of rats. RESULTS: Behavioral assessment using the acetone spray test showed that 3 and 6 mg/kg oxaliplatin (i.p.) induced acute cold hypersensitivity after 1, 2, 4, and 7 days. Real-time PCR showed that oxaliplatin (6 mg/kg) significantly increased TRPA1 mRNA expression in DRGs at days 1, 2, and 4. Western blotting revealed that oxaliplatin significantly increased TRPA1 protein expression in DRGs at days 2, 4, and 7. Moreover, in situ hybridization histochemistry revealed that most TRPA1 mRNA-labeled neurons in the DRGs were small in size. Oxaliplatin significantly increased co-localization of TRPA1 expression and isolectin B4 binding in DRG neurons. Oxaliplatin induced a significant increase in the percent of TRPA1 mRNA-positive small neurons in DRGs at days 1, 2, and 4. In addition, we found that intrathecal administration of TRPA1 antisense, but not TRPA1 mismatched oligodeoxynucleotides, knocked down TRPA1 expression and decreased oxaliplatin-induced cold hyperalgesia. Double labeling showed that p-p38 mitogen-activated protein kinase (MAPK) was co-expressed in TRPA1 mRNA-labeled neurons at day 2 after oxaliplatin administration. Intrathecal administration of the p38 MAPK inhibitor, SB203580, significantly decreased oxaliplatin-induced acute cold hypersensitivity. CONCLUSIONS: Together, these results demonstrate that TRPA1 expression via activation of p38 MAPK in DRG neurons, at least in part, contributes to the development of oxaliplatin-induced acute cold hyperalgesia.
Assuntos
Síndromes Periódicas Associadas à Criopirina/induzido quimicamente , Gânglios Espinais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Doença Aguda , Animais , Síndromes Periódicas Associadas à Criopirina/enzimologia , Síndromes Periódicas Associadas à Criopirina/fisiopatologia , Gânglios Espinais/enzimologia , Gânglios Espinais/fisiopatologia , Imidazóis/farmacologia , Masculino , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Ratos , Canal de Cátion TRPA1 , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Psilocin (3-[2-(dimethylamino)ethyl]-1H-indol-4-ol) is a hallucinogenic component of the Mexican mushroom Psilocybe mexicana and a skeletal serotonin (5-HT) analogue. Psilocin is the active metabolite of psilocybin (3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate). In the present study, we examined the effects of systemically administered psilocin on extracellular dopamine and 5-HT concentrations in the ventral tegmental area (VTA), nucleus accumbens, and medial prefrontal cortex of the dopaminergic pathway in awake rats using in vivo microdialysis. Intraperitoneal administration of psilocin (5, 10 mg/kg) significantly increased extracellular dopamine levels in the nucleus accumbens. Psilocin did not affect the extracellular 5-HT level in the nucleus accumbens. Conversely, systemic administration of psilocin (10 mg/kg) significantly increased extracellular 5-HT levels in the medial prefrontal cortex of rats, but dopamine was decreased in this region. However, neither extracellular dopamine nor 5-HT levels in the VTA were altered by administration of psilocin. Behaviorally, psilocin significantly increased the number of head twitches. Thus, psilocin affects the dopaminergic system in the nucleus accumbens. In the serotonergic system, psilocin contribute to a crucial effect in the medial prefrontal cortex. The present data suggest that psilocin increased both the extracellular dopamine and 5-HT concentrations in the mesoaccumbens and/or mesocortical pathway.
Assuntos
Dopamina/metabolismo , Alucinógenos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Psilocibina/análogos & derivados , Serotonina/metabolismo , Animais , Masculino , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Psilocibina/farmacologia , Ratos Wistar , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
Neuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the central nervous system. However, the mechanisms by which nNOS activity is regulated are not completely understood. In the present study, the effects of protein kinases on the phosphorylation of nNOS in GH3 rat pituitary tumor cells were evaluated. We show that phosphorylation of nNOS at Ser1412 could be induced by the phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) agonist insulin, the calcium/calmodulin-dependent protein kinase II (CaM-K II) agonist A23187 or the cAMP-dependent protein kinase A (PKA) agonist IBMX, respectively. The phosphorylation levels of nNOS at Ser1412, induced by activation of Akt/PKB or CaM-K II, but not by PKA signaling, were reduced by pre-treatment with the NO donor diethylamine-NONOate. This inhibitory effect could be reversed by addition of a reducing reagent, dithiothreitol. Furthermore, the levels of phosphorylation of nNOS at Ser1412, induced by Akt/PKB or CaM-K II but not by PKA signaling, were enhanced by inhibition of nNOS activity with 7-nitroindazole. These findings suggest that the activation of nNOS can be catalyzed by at least three protein kinases, Akt/PKB, CaM-K II or PKA. NO generated from nNOS feedback prevents the activation of nNOS by inhibiting either Akt/PKB or CaM-K II but not PKA signaling.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Calcimicina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Ditiotreitol/farmacologia , Hidrazinas/farmacologia , Indazóis/farmacologia , Insulina/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Transdução de SinaisRESUMO
We show that Ca(2+)/calmodulin(CaM)-dependent protein kinase I (CaMKI) is directly inhibited by its S-glutathionylation at the Cys(179). In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S-glutathionylation of CaMKI at Cys(179) detected by mass spectrometry. Mutagenesis studies confirmed that S-glutathionylation of Cys(179) is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys(179) is an important mechanism in processing calcium signal transduction in cells.
Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Cisteína/química , Glutationa/metabolismo , Sítios de Ligação/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Diamida/farmacologia , Íons/metabolismo , Espectrometria de Massas , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Animais , Benzilaminas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Cisteína/metabolismo , Ativação Enzimática , Hipocampo/citologia , Hipocampo/metabolismo , Hidrazinas/metabolismo , Indazóis/metabolismo , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Inibidores de Proteínas Quinases/metabolismo , Ratos , S-Nitroso-N-Acetilpenicilamina/metabolismo , Serina/metabolismo , Sulfonamidas/metabolismo , Hormônio Liberador de Tireotropina/metabolismoRESUMO
Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.
Assuntos
Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sequência de Aminoácidos , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Fosforilação , Ratos , Serina/metabolismoRESUMO
We have previously demonstrated that pressure application of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) onto some neurons in the anterior hypothalamic area of rats increases neural activity in vivo and that this PKC activation-induced increase of neural activity is enhanced in spontaneously hypertensive rats (SHR), an animal model for genetic hypertension. Activation of PKC increases expression of the c-fos gene, an important transcription factor and proto-oncogene thought to be a marker of neural activity. To evaluate PKC isoforms responsible for neural activation, we examined which isoforms of PKC are involved in the PKC activation-induced c-fos gene expression in neuronal cultures of Wistar rat and spontaneously hypertensive rat (SHR) brains. PMA increased c-fos gene expression in neuronal cultures of Wistar rat brain and the PMA-induced c-fos gene expression was inhibited by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7). The PKCalpha,beta,gamma activator thymeleatoxin also increased c-fos gene expression, while the PKCdelta,epsilon activator ingenol did not affect it. In addition, the PMA-induced c-fos gene expression was inhibited by PKCbetaantisense oligonucleotides (AON) but not by PKCalpha and PKCgammaAONs. In SHR brain neuronal cultures, the PMA-induced c-fos gene expression was enhanced as compared with that of Wistar Kyoto rats (WKY), while basal c-fos gene expression was almost the same in both neuronal cultures. The enhancement of PMA-induced c-fos gene expression in SHR brain cultures was abolished by PKCbetaAON. These findings suggest that in rat brain neuronal cultures, PMA increases c-fos gene expression via activation of PKC and that PKCbetaisoforms are partly involved in the PMA-induced c-fos gene expression. In neuronal cultures of SHR brain, it appears that the PMA-induced c-fos gene expression is also enhanced via PKCbeta.
Assuntos
Encéfalo/enzimologia , Genes fos/efeitos dos fármacos , Genes fos/fisiologia , Hipertensão/enzimologia , Neurônios/enzimologia , Ésteres de Forbol/farmacologia , Proteína Quinase C/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Neurônios/efeitos dos fármacos , Proteína Quinase C beta , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
1. Angiotensin II AT1A receptors are thought to play an important role in the development of hypertension. The transcriptional factor Sp1 is a ubiquitous transcriptional factor associated with GC-rich promoters and involved in basal promoter activity. 2. To examine basal transcriptional levels regulation of the rat AT1A receptor gene, we determined whether two GC-box-related regions within 100 bp of the rat AT1A receptor gene promoter are involved in the basal expression of the gene in A10 cells, a vascular smooth muscle cell line. 3. The electrophoretic mobility shift assay demonstrated that incubation of the -98/-79 region and -58/-34 region sequence oligonucleotides with nuclear extracts of rat hypothalamus, liver and adrenal formed DNA-protein complexes and that the addition of unlabelled oligonucleotides containing the Sp1 consensus sequence blocked the formation of the DNA-protein complex. The addition of antibody against Sp1 also blocked the formation of the DNA-protein complex. 4. The promoter/luciferase reporter assay demonstrated that the reporter activity of AT1A receptor promoters mutated either within the -98/-79 or the -58/-34 region was lower than that of intact AT1A receptor promoters. 5. The promoter activity of AT1A receptor promoters mutated within those two regions was lower than that of promoters mutated within either the -98/-79 or the -58/-34 region. 6. These findings suggest that GC-box-regulated sequences within the -98/-79 region and the -58/-34 region are additively involved in basal expression level of the AT1A receptor gene in A10 cells.
Assuntos
Sequência Rica em GC/genética , Regulação da Expressão Gênica/fisiologia , Genes Reguladores/genética , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/genética , Animais , Sequência de Bases , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Luciferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Oligonucleotídeos/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Endogâmicos WKY , Fator de Transcrição Sp1/genética , TransfecçãoRESUMO
The transcriptional factor Sp1 is associated with GC-rich promoters and involved in basal promoter activity. A GC-box-related sequence is located within the -58 to -34 base pair region of the angiotensin type 1 receptor gene promoter. We examined whether Sp1 in the hypothalamus was increased in spontaneously hypertensive rats (SHR) and whether inhibition of Sp1 binding sites suppressed angiotensin type 1 receptor expression and thus decreased blood pressure in SHR. Western blot analysis showed that Sp1 protein levels were increased in nuclear extracts of hypothalamus from SHR. Electrophoretic mobility shift assay (EMSA) using oligonucleotides containing Sp1 consensus sequence and -58 to -34 region sequence oligonucleotides showed that DNA-protein complexes were greater in nuclear extracts of hypothalamus from SHR than those of Wistar Kyoto rats (WKY). Sp1 decoy phosphorothioate oligodeoxynucleotides injected into the lateral ventricle produced a decrease in blood pressure in SHR, and decreased angiotensin type 1 receptor mRNA levels and number of angiotensin receptors in the hypothalamus of SHR. Pressor responses to angiotensin II but not to carbachol injected into the lateral ventricle were decreased in the Sp1 decoy-treated SHR. The results of the present study suggest that Sp1 levels in the hypothalamus of SHR are increased, and that inhibition of the binding of Sp1 to its binding sites decreases angiotensin type 1 receptor expression and blood pressure in SHR. The possibility cannot be ruled out that the Sp1 decoy oligodeoxynucleotides (ODN) also suppressed transcriptions of genes other than the angiotensin type 1 receptor gene.
Assuntos
Pressão Sanguínea/fisiologia , Hipotálamo/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Receptores de Angiotensina/biossíntese , Fator de Transcrição Sp1/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Injeções Intraventriculares , Masculino , Oligonucleotídeos Antissenso/administração & dosagem , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Angiotensina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genéticaRESUMO
The angiotensin type 1a (AT1a) receptor gene is overexpressed in the brain and peripheral tissues of spontaneously hypertensive rats (SHR). We examined whether there are mutations responsible for overexpression of the AT1a receptor gene in the SHR AT1a receptor promoter region. Genomic DNA was extracted from the livers of SHR and Wistar Kyoto rats (WKY) of Izumo strain (SHR/Izm and WKY/Izm, respectively). Fragments of the AT1a receptor gene promoter region were amplified by polymerase chain reaction (PCR). Amplified fragments were purified by agarose gel electrophoresis, and the purified fragments were cloned using pTBlue T-Vector. Sequence analysis identified one single base mutation unique to the SHR AT1a receptor gene promoter region when compared to that of WKY. The sequence of the mutation site in SHR was the same as that of Sprague Dawley rats. Using an electrophoretic mobility shift assay, we compared gel patterns formed by DNA-protein complexes using ds-oligonucleotides representing region-1624 to-1595 of the SHR and WKY AT1a receptor promoters. There were 3 major similar DNA-protein complexes against WKY and SHR oligonucleotides. In addition, the oligonucleotide bearing the SHR sequence produced an extra band. Promoter/luciferase reporter assay demonstrated that the promoter activity of SHR AT1a receptor promoters (-2050 to +57) was lower than that of WKY. These results suggest that there is one single mutation unique to the SHR AT1a receptor gene promoter region, but that the mutation is not responsible for overexpression of the AT1 a receptor gene in SHR.
Assuntos
Hipertensão/genética , Ratos Endogâmicos SHR/genética , Receptor Tipo 1 de Angiotensina/genética , Animais , Sequência de Bases , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Genes Reporter , Luciferases/genética , Masculino , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Ratos , Ratos Endogâmicos WKY , Transcrição Gênica/genéticaRESUMO
We previously reported that pressure loading of the vascular wall can activate mitogen-activated protein kinases (MAPKs), enzymes believed to be involved in the pathway for cell proliferation, partly via the vascular angiotensin system in isolated perfused rat aorta. In this study, we examined whether cyclic stretching of vascular smooth muscle cells (VSMC) also produces activation of p42 and p44 MAPKs in cultured rat VSMC and whether stretch-induced MAPK activation is mediated via angiotensin and endothelin systems in VSMC. Cyclic stretching of VSMC produced an elongation-dependent and frequency-dependent increase in p42 and p44 MAPK activity. The stretch-induced p42 and p44 MAPK activation was inhibited by the angiotensin receptor antagonist losartan and by the angiotensin-converting enzyme inhibitor, captopril. The MAPK activation was also inhibited by the endothelin receptor antagonist cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl) (BQ123) and by the endothelin-converting enzyme inhibitor phosphoramidon. Replacement of medium with culture medium of stretched cells caused MAPK activation, which was inhibited by losartan and BQ123. The results of the present study suggest that cyclic stretching of VSMC can activate p42 and p44 MAPKs and that the MAPK activation is mediated via angiotensin and endothelin systems in VSMC.
Assuntos
Angiotensinas/metabolismo , Endotelinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/enzimologia , Pressorreceptores/enzimologia , Angiotensinas/antagonistas & inibidores , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiologia , Células Cultivadas , Endotelinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Músculo Liso Vascular/citologia , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
1. We previously reported that activation function of mitogen-activated protein kinases (MAPK) is enhanced in aorta strips from both prehypertensive and hypertensive spontaneously hypertensive rats (SHR) and that this enhancement of MAPK activation results from enhanced MAPK activation reactivity to angiotensin (Ang) II in SHR aorta strips. 2. The purpose of the present study was to examine whether the enhanced function of the vascular angiotensin system observed in SHR aorta strips results from genetic alterations of vascular smooth muscle cells from SHR. 3. Basal MAPK activity was within normal limits in cells from 4-week-old SHR, whereas enzyme activity was enhanced in 9-week-old SHR compared with age-matched Wistar-Kyoto (WKY) rats. 4. Mitogen-activated protein kinase activation reactivity to AngII and endothelin-1 was enhanced in 9-week-old SHR cells but not in 4-week-old SHR cells. The enhancement of basal MAPK activity in 9-week-old SHR cells was abolished by a combination of the angiotensin AT(1) receptor antagonist losartan and the endothelin receptor antagonist BQ123. 5. These findings suggest that MAPK activation function in 4-week-old SHR cells is not enhanced. Thus, it appears that factors outside vascular smooth muscle cells are needed for the enhanced MAPK activation observed in 4-week-old SHR aorta strips. In 9-week-old SHR, MAPK activation function is enhanced in cells themselves and this function may, at least in part, contribute to the enhanced MAPK activation observed in SHR aorta strips.