RESUMO
Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.
Assuntos
Anafilaxia , Hipersensibilidade a Drogas , Camundongos , Animais , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Ligantes , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Caspofungina/metabolismo , Caspofungina/farmacologia , Peptídeos/farmacologia , Antibacterianos/farmacologia , Mastócitos/metabolismo , Degranulação Celular/fisiologiaRESUMO
Spinal cord injury (SCI) is a devastating neurological disorder that usually damages sensorimotor and autonomic functions. Signaling pathways can play a key role in the repair process of SCI. The plexin-B2 acts as a receptor for angiogenin and mediates ribosomal RNA transcription, influencing cell survival and proliferation. Protein kinase B serine/threonine kinase interacts with angiogenin to form a positive feedback effect. Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor can induce angiogenin nuclear translocation. Moreover, the BDNF can promote the secretion of angiogenin. Interestingly, all of them can activate the angiogenin/plexin-B2 axis. Muscone has anti-inflammatory and proliferative features as it can inhibit nuclear transcription factor kappa-B (NF-κB) and activate the angiogenin/plexin-B2 axis, thus being significant agent in the SCI repair process. Herein, we review the potential mechanism of angiogenin/plexin-B2 axis activation and the role of muscone in SCI treatment. Muscone may attenuate inflammatory responses and promote neuronal regeneration after SCI.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Traumatismos da Medula Espinal , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular , Cicloparafinas , Humanos , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Ribonuclease Pancreático , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cerebral amyloid ß (Aß) proteostasis is compromised under neuronal overexcitation, long-term neuroinflammation and brain aging. Using the animal model of LPS-induced neuroinflammation we demonstrated that treatment with levetiracetam, a specific modulator of synaptic vesicle glycoprotein SV2A, rescues abnormal synaptic vesicle (SV) fusion and neurotransmitter release, decreasing elevated hippocampal APP levels in vivo. Therapy with levetiracetam upregulates the SV2A in hippocampus and restores the level of apolipoprotein E, involved in brain Aß aggregation/clearance and resolution of inflammation. We demonstrated that oligomers of Aß1-42 and Aß1-40 peptides promote SV clustering, which reduces the rate and plateau level of subsequent homo- and heterotypic SNARE-mediated SV fusion. Oligomeric Aß1-42 lowered ΔpH gradient across the vesicular membrane, thus affecting their neurotransmitter storage capacity. In contrast, monomers of Aß1-42 and Aß1-40 had negligible impact on studied processes. Our data suggests that in the course of progression of neuroinflammation oligomeric forms of Aß1-42 and Aß1-40 can compromise the SV fusion machinery and that antiepileptic agent levetiracetam, acting on SV recycling and restricting overexcitation, is able to affect APP processing and Aß generation within the hippocampus in vivo.
Assuntos
Amiloidose/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Hipocampo/efeitos dos fármacos , Levetiracetam/administração & dosagem , Glicoproteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Nootrópicos/administração & dosagem , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Amiloidose/induzido quimicamente , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Células Cultivadas , Hipocampo/metabolismo , Hipocampo/patologia , Lipopolissacarídeos/toxicidade , Masculino , Glicoproteínas de Membrana/agonistas , Proteínas do Tecido Nervoso/agonistas , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Ratos , Ratos WistarRESUMO
CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/agonistas , Receptores de Fator de Crescimento Neural/agonistas , Animais , Apoptose , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Experiments aimed to evaluate the tissue distribution of Mas-related G protein-coupled receptor D (MrgD) revealed the presence of immunoreactivity for the MrgD protein in the rostral insular cortex (rIC), an important area for autonomic and cardiovascular control. To investigate the relevance of this finding, we evaluated the cardiovascular effects produced by the endogenous ligand of MrgD, alamandine, in this brain region. Mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in urethane anesthetized rats. Unilateral microinjection of equimolar doses of alamandine (40 pmol/100 nL), angiotensin-(1-7), angiotensin II, angiotensin A, and Mas/MrgD antagonist d-Pro7-Ang-1-7 (50 pmol/100 nL), Mas antagonist A779 (100 pmol/100 nL), or vehicle (0.9% NaCl) were made in different rats (n = 4-6/group) into rIC. To verify the specificity of the region, a microinjection of alamandine was also performed into intermediate insular cortex (iIC). Microinjection of alamandine in rIC produced an increase in MAP (Δ = 15 ± 2 mmHg), HR (Δ = 36 ± 4 beats/min), and RSNA (Δ = 31 ± 4%), but was without effects at iIC. Strikingly, an equimolar dose of angiotensin-(1-7) at rIC did not produce any change in MAP, HR, and RSNA. Angiotensin II and angiotensin A produced only minor effects. Alamandine effects were not altered by A-779, a Mas antagonist, but were completely blocked by the Mas/MrgD antagonist d-Pro7-Ang-(1-7). Therefore, we have identified a brain region in which alamandine/MrgD receptor but not angiotensin-(1-7)/Mas could be involved in the modulation of cardiovascular-related neuronal activity. This observation also suggests that alamandine might possess unique effects unrelated to angiotensin-(1-7) in the brain.
Assuntos
Angiotensina I/farmacologia , Pressão Arterial/efeitos dos fármacos , Sistema Cardiovascular/inervação , Córtex Cerebral/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Rim/inervação , Proteínas do Tecido Nervoso/agonistas , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Sistema Nervoso Simpático/efeitos dos fármacos , Animais , Córtex Cerebral/fisiologia , Ligantes , Masculino , Microinjeções , Proteínas do Tecido Nervoso/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/metabolismo , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Sistema Nervoso Simpático/fisiologiaRESUMO
Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.
Assuntos
Anafilaxia/induzido quimicamente , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Fragmentos de Peptídeos/efeitos adversos , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Vancomicina/efeitos adversos , Anafilaxia/imunologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/efeitos adversos , Células HEK293 , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/imunologia , Receptores de Neuropeptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Síndrome , Vancomicina/administração & dosagem , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The discovery of a G-protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional nonmetabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch clamp, we found that the activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature EPSC frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists 3,5-dihydroxybenzoic acid, 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1, the basal activity was increased compared with WT. HCAR1 mediates its effect in neurons through a Giα-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 downmodulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and α2A-adrenergic receptors, through a mechanism involving both its Giα and Gißγ subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a downmodulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Giα and Gißγ intracellular pathways to functionally interact with other Gi-coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENT Expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) was recently described in neurons. Here, we describe the physiological role of this G-protein-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCAR1 activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross talk of HCAR1 with other GPCRs, such as GABAB, adenosine A1- and α2A-adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Lactatos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Potenciais de Ação , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , AMP Cíclico/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
Purpose: Pannexin 1 channels are deemed to play important roles in inflammation. However, there is limited information regarding their roles in fungal infection diseases, especially fungal keratitis. This study aimed to investigate the role of pannexin 1 channels in Aspergillus fumigatus (A. fumigatus) keratitis. Materials and Methods: Mouse models or immortalized human corneal epithelial cells (HCECs) were infected with or without A. fumigatus for given time. The expression of pannexin 1 channels was tested by qPCR, western blot and immunofluorescence staining. Mice of A. fumigatus keratitis were pretreated with carbenoxolone (CBX) or 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) to block or activate the opening of pannexin 1 channels respectively. The clinical score was recorded. Cornea tissues were examined for the downstream signals of pannexin 1 channels, including NLRP3, Caspase-1 and IL-1ß, and myeloperoxidase (MPO) by PCR and ELISA. Data were analyzed with commercial data analysis software and a P < 0.05 was considered to be statistically significant. Results: Upon A. fumigatus infection, pannexin 1 expression increased at both the mRNA and the protein levels in mice corneas (P< 0.05, n = 3). Immunofluorescence indicated that pannexin 1 channels were mainly located in the corneal epithelial layer, and they were upregulated after A. fumigatus infection. In vitro, the same tendency was found at the mRNA and the protein levels in HCECs (P< 0.05, n = 8). In mouse model, blockage of pannexin 1 channels by CBX caused more severely keratitis. The downstream signals of pannexin 1 channels (NLRP3/Caspase-1/IL-1ß) and MPO were down-regulated. Whereas activation the opening of pannexin 1 channels by BzATP reduced corneal infection with increased expression of Caspase-1 and IL-1ß. Conclusions: Pannexin 1 channels play important roles in the regulation of progression and leucocytes aggregation during corneal A. fumigatus infection via the NLRP3/Caspase-1/IL-ß pathway.
Assuntos
Aspergilose/metabolismo , Caspase 1/metabolismo , Conexinas/genética , Úlcera da Córnea/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-1beta/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas do Tecido Nervoso/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergillus fumigatus , Western Blotting , Carbenoxolona/farmacologia , Células Cultivadas , Conexinas/agonistas , Conexinas/antagonistas & inibidores , Úlcera da Córnea/tratamento farmacológico , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/metabolismo , Infecções Oculares Fúngicas/tratamento farmacológico , Infecções Oculares Fúngicas/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
AG-30/5C is an angiogenic host defense peptide that activates human mast cells (MC) via an unknown mechanism. Using short hairpin RNA-silenced human MC line LAD2 and stably transfected RBL-2H3 cells, we demonstrate that AG-30/5C induces MC degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2). Most G protein-coupled receptors signal via parallel and independent pathways mediated by G proteins and ß-arrestins. AG-30/5C and compound 48/80 induced similar maximal MC degranulation via MRGPRX2, which was abolished by pertussis toxin. However, compound 48/80 induced a robust ß-arrestin activation as determined by transcriptional activation following arrestin translocation (Tango), but AG-30/5C did not. Overnight culture of MC with compound 48/80 resulted in reduced cell surface MRGPRX2 expression, and this was associated with a significant decrease in subsequent MC degranulation in response to compound 48/80 or AG-30/5C. However, AG-30/5C pretreatment had no effect on cell surface MRGPRX2 expression or degranulation in response to compound 48/80 or AG-30/5C. Icatibant, a bradykinin B2 receptor antagonist, promotes MC degranulation via MRGPRX2 and causes pseudoallergic drug reaction. Icatibant caused MC degranulation via a pertussis toxin-sensitive G protein but did not activate ß-arrestin. A screen of the National Institutes of Health Clinical Collection library led to the identification of resveratrol as an inhibitor of MRGPRX2. Resveratrol inhibited compound 48/80-induced Tango and MC degranulation in response to compound 48/80, AG-30/5C, and Icatibant. This study demonstrates the novel finding that AG-30/5C and Icatibant serve as G protein-biased agonists for MRGPRX2, but compound 48/80 signals via both G protein and ß-arrestin with distinct differences in receptor regulation.
Assuntos
Antagonistas de Receptor B2 da Bradicinina/farmacologia , Bradicinina/análogos & derivados , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Animais , Bradicinina/farmacologia , Células Cultivadas , Células HEK293 , Humanos , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismoRESUMO
Nonylphenol (NP) and Cadmium (Cd) are two common contaminants that can be detected in aquatic environments. Nevertheless, the combined toxicity of NP and Cd at environmentally relevant concentrations in aquatic organisms has not been thoroughly characterized to date. In the present study, the interactions between NP and Cd on male Sebastiscus marmoratus were studied. After 21â¯days of exposure, the brain aromatase activity was observed to be significantly induced by 100â¯ng/L NP and 40⯵g/L Cd, whereas all of the concentrations of co-treatment resulted in an increase in brain aromatase activity. Additionally, NP could also reduce plasma testosterone concentration, while NP, Cd and their mixture could induce plasma 17ß-estradiol (E2) concentration and VTG concentration. The interactions between NP and Cd on the reproductive physiology were antagonism. Our results also support the notion of using these indicators as biomarkers for exposure to EDCs and further extend the boundary of biomonitoring to environmental levels.
Assuntos
Cádmio/toxicidade , Genitália Masculina/efeitos dos fármacos , Infertilidade Masculina/veterinária , Perciformes/fisiologia , Fenóis/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Aromatase/química , Aromatase/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Sinergismo Farmacológico , Disruptores Endócrinos/toxicidade , Biomarcadores Ambientais/efeitos dos fármacos , Estradiol/agonistas , Estradiol/sangue , Doenças dos Peixes/sangue , Doenças dos Peixes/induzido quimicamente , Doenças dos Peixes/metabolismo , Doenças dos Peixes/fisiopatologia , Proteínas de Peixes/agonistas , Proteínas de Peixes/metabolismo , Genitália Masculina/fisiopatologia , Infertilidade Masculina/induzido quimicamente , Infertilidade Masculina/metabolismo , Infertilidade Masculina/fisiopatologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Concentração Osmolar , Perciformes/sangue , Testosterona/antagonistas & inibidores , Testosterona/sangue , Testes de Toxicidade Crônica , Vitelogeninas/sangue , Vitelogeninas/químicaRESUMO
Brain tissue manganese (Mn) accumulation is a cirrhosis-associated complication. Cellular mitochondria are among the potential targets for Mn-induced cytotoxicity. Taurine is one of the most abundant amino acids with high concentrations in human brain tissue. Several pharmacological properties including regulation of mitochondrial function are attributed to taurine. The current investigation was designed to evaluate the effect of taurine on Mn-induced mitochondrial impairment in isolated mice brain mitochondria. The brain mitochondria were exposed to increasing concentrations of Mn (0.1-10 mM). Taurine (0.1, 1, and 10 mM) was added as the protective agent. The severe collapse of mitochondrial membrane potential, decreased mitochondrial dehydrogenases activity, mitochondrial swelling, and depleted mitochondrial adenosine triphosphate (ATP) were evident in Mn-exposed mitochondria. It was found that taurine administration preserved mitochondrial ATP, prevented mitochondrial depolarization and swelling, and increased mitochondrial dehydrogenases activity. These data suggest mitochondrial protection as an underlying mechanism for the protective effects of taurine against Mn toxicity.
Assuntos
Encéfalo/efeitos dos fármacos , Manganês/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Taurina/farmacologia , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatia Hepática/tratamento farmacológico , Encefalopatia Hepática/metabolismo , Encefalopatia Hepática/patologia , Cinética , Masculino , Manganês/efeitos adversos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/metabolismo , Permeabilidade/efeitos dos fármacos , Taurina/uso terapêuticoRESUMO
Bifenthrin (BF) is a synthetic pyrethroid pesticide widely used in several countries to manage insect pests on diverse agricultural crops. Growing evidence indicates that BF exposure is associated with an increased risk of developing neurodegenerative disorders. However, the mechanisms by which BF induces neurological and anxiety alterations in the frontal cortex and striatum are not well known. The present in vivo study was carried out to determine whether reactive oxygen species (ROS)-mediated oxidative stress (OS) and neuroinflammation are involved in such alterations. Thirty-six Wistar rats were thus randomly divided into three groups and were orally administered with BF (0.6 and 2.1â¯mg/kg body weight, respectively) or the vehicle (corn oil), on a daily basis for 60 days. Results revealed that BF exposure in rats enhanced anxiety-like behavior after 60 days of treatment, as assessed with the elevated plus-maze test by decreases in the percentage of time spent in open arms and frequency of entries into these arms. BF-treated rats also exhibited increased oxidation of lipids and carbonylated proteins in the frontal cortex and striatum, and decreased glutathione levels and antioxidant enzyme activities including superoxide dismutase, catalase and glutathione peroxidase. Treatment with BF also increased protein synthesis and mRNA expression of the inflammatory mediators cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and nuclear factor-kappaBp65 (NF-kBp65), as well as the production of tumor necrosis factor-α (TNF-α) and ROS. Moreover, BF exposure significantly decreased protein synthesis and mRNA expression of nuclear factor erythroid-2 (Nrf2) and acetylcholinesterase (AChE), as well as gene expression of muscarinic-cholinergic receptors (mAchR) and choline acetyltransferase (ChAT) in the frontal cortex and striatum. These data suggest that BF induced neurological alterations in the frontal cortex and striatum of rats, and that this may be associated with neuroinflammation and oxidative stress via the activation of Nrf2/NF-kBp65 pathways, which might promote anxiety-like behavior.
Assuntos
Ansiedade/etiologia , Inseticidas/toxicidade , Neurite (Inflamação)/induzido quimicamente , Síndromes Neurotóxicas/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Piretrinas/toxicidade , Tremor/etiologia , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/imunologia , Neurônios Colinérgicos/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/imunologia , Lobo Frontal/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inseticidas/administração & dosagem , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurite (Inflamação)/imunologia , Neurite (Inflamação)/metabolismo , Neurite (Inflamação)/fisiopatologia , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Piretrinas/administração & dosagem , Distribuição Aleatória , Ratos WistarRESUMO
The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system, alamandine actions included vasodilation, antihypertensive, and antifibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: 1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; and 2) to evaluate the ability of this axis to prevent angiotensin II (ANG II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by d-Pro7-ANG-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine-induced NO production was preserved in Mas-/- myocytes and lost in MrgD-/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD-/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with ANG II. Treatment of NRCMs with alamandine prevented ANG II-induced hypertrophy. Moreover, the antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by d-Pro7-ANG-(1-7) or inhibitors of NO synthase or AMPK. ß-Alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate ANG II-induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Angiotensina II/toxicidade , Cardiomegalia/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Óxido Nítrico/metabolismo , Oligopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Células Cultivadas , Ativação Enzimática , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas do Tecido Nervoso/metabolismo , Oligopeptídeos/metabolismo , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
SCOPE: We investigated the role of endoplasmic reticulum (ER) stress in the protective effects of EGCG against the neuronal apoptosis in Aß1-42 -induced SH-SY5Y cells and APP/PS1 transgenic mice. METHODS AND RESULTS: Cell viability (CCK8 assay), flow cytometry, Hoechst 33258 staining, immunohistochemistry, transmission electron microscopy (TEM), and western blotting were used. EGCG prevented Aß1-42-induced toxicity in SH-SY5Y cells, increased cell viability, and decreased apoptosis in a dose-dependent manner. In a subsequent mechanism study, it was found that this effect contributed to the down-regulation of GRP78, CHOP, cleaved-caspase-12 and -3. Moreover, EGCG also reduced the cytotoxicity induced by tunicamycin (TM) and thapsigargin (TG), two ER stress activators. Consistent with the in vitro study, EGCG inhibited neuronal apoptosis in the cortex of APP/PS1 transgenic mice, with the mitigation of ER abnormal ultrastructural swelling and the downregulation of ER-stress-associated proteins. CONCLUSION: These results indicate that EGCG attenuates the neurotoxicity in Alzheimer's disease (AD) via a novel mechanism that involves inhibition of ER-stress-associated neuronal apoptosis in vitro and in vivo, suggesting the tremendous potential of EGCG for use in a nutritional preventive strategy against AD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Apoptose , Catequina/análogos & derivados , Suplementos Nutricionais , Estresse do Retículo Endoplasmático , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Animais , Caspase 12/química , Caspase 12/genética , Caspase 12/metabolismo , Caspase 3/química , Caspase 3/genética , Caspase 3/metabolismo , Catequina/metabolismo , Catequina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Fármacos Neuroprotetores/uso terapêutico , Nootrópicos/metabolismo , Nootrópicos/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Distribuição Aleatória , Fator de Transcrição CHOP/agonistas , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Benzylpiperazine has been designated as Schedule I substance under the Controlled Substances Act by Drug Enforcement Administration. Benzylpiperazine is a piperazine derivative, elevates both dopamine and serotonin extracellular levels producing stimulatory and hallucinogenic effects, respectively, similar to methylenedioxymethamphetamine (MDMA). However, the comparative neurotoxic effects of Piperazine derivatives (benzylpiperazine and benzoylpiperazine) have not been elucidated. Here, piperazine derivatives (benzylpiperazine and benzoylpiperazine) were synthesized in our lab and the mechanisms of cellular-based neurotoxicity were elucidated in a dopaminergic human neuroblastoma cell line (SH-SY5Y). We evaluated the in vitro effects of benzylpiperazine and benzoylpiperazine on the generation of reactive oxygen species, lipid peroxidation, mitochondrial complex-I activity, catalase activity, superoxide dismutase activity, glutathione content, Bax, caspase-3, Bcl-2 and tyrosine hydroxylase expression. Benzylpiperazine and benzoylpiperazine induced oxidative stress, inhibited mitochondrial functions and stimulated apoptosis. This study provides a germinal assessment of the neurotoxic mechanisms induced by piperazine derivatives that lead to neuronal cell death.
Assuntos
Apoptose/efeitos dos fármacos , Agonistas de Dopamina/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Alucinógenos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Piperazinas/toxicidade , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Drogas Desenhadas/química , Drogas Desenhadas/toxicidade , Agonistas de Dopamina/química , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Alucinógenos/química , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Estrutura Molecular , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Piperazinas/química , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismoRESUMO
BNN27, a C17-spiroepoxy derivative of DHEA, was shown to have antiapoptotic properties via mechanisms involving the nerve growth factor receptors (tropomyosin-related kinase A [TrkA]/neurotrophin receptor p75 [p75NTR]). In this study, we examined the effects of BNN27 on neural/glial cell function, apoptosis, and inflammation in the experimental rat streptozotocin (STZ) model of diabetic retinopathy (DR). The ability of BNN27 to activate the TrkA receptor and regulate p75NTR expression was investigated. BNN27 (2,10, and 50 mg/kg i.p. for 7 days) administration 4 weeks post-STZ injection (paradigm A) reversed the diabetes-induced glial activation and loss of function of amacrine cells (brain nitric oxide synthetase/tyrosine hydroxylase expression) and ganglion cell axons via a TrkA receptor (TrkAR)-dependent mechanism. BNN27 activated/phosphorylated the TrkAY490 residue in the absence but not the presence of TrkAR inhibitor and abolished the diabetes-induced increase in p75NTR expression. However, it had no effect on retinal cell death (TUNEL+ cells). A similar result was observed when BNN27 (10 mg/kg i.p.) was administered at the onset of diabetes, every other day for 4 weeks (paradigm B). However, BNN27 decreased the activation of caspase-3 in both paradigms. Finally, BNN27 reduced the proinflammatory (TNFα and IL-1ß) and increased the anti-inflammatory (IL-10 and IL-4) cytokine levels. These findings suggest that BNN27 has the pharmacological profile of a therapeutic for DR, since it targets both the neurodegenerative and inflammatory components of the disease.
Assuntos
Células Amácrinas/efeitos dos fármacos , Anti-Inflamatórios/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Retinopatia Diabética/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Receptor trkA/agonistas , Retina/efeitos dos fármacos , Células Amácrinas/imunologia , Células Amácrinas/metabolismo , Células Amácrinas/patologia , Animais , Anti-Inflamatórios/administração & dosagem , Axônios/efeitos dos fármacos , Axônios/imunologia , Axônios/metabolismo , Axônios/patologia , Desidroepiandrosterona/administração & dosagem , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/imunologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Relação Dose-Resposta a Droga , Proteínas do Olho/agonistas , Proteínas do Olho/metabolismo , Feminino , Gânglios Sensitivos/efeitos dos fármacos , Gânglios Sensitivos/imunologia , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/patologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Fármacos Neuroprotetores/administração & dosagem , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural/agonistas , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Retina/imunologia , Retina/patologia , Retina/fisiopatologia , EstreptozocinaRESUMO
This study aimed at studying the potential neuroprotective effect of Rutin hydrate (RH) alone or in conjugation with α-tocopherol against cadmium chloride (CdCl2)-induced neurotoxicity and cognitive impairment in rats and to investigate the mechanisms of action. Rats intoxicated with CdCl2 were treated with the vehicle, RH, α-tocopherol or combined treatment were examined, and compared to control rats received vehicle or individual doses of either drug. Data confirmed that RH improves spatial memory function by increasing acetylcholine availability, boosting endogenous antioxidant potential, activating cell survival and inhibiting apoptotic pathways, an effect that is more effective when RH was conjugated with α-tocopherol. Mechanism of RH action includes activation of PP2A mediated inhibiting of ERK1/2 and JNK apoptotic pathways and inhibition of PTEN mediated activation of mTOR survival pathway. In conclusion, RH affords a potent neuroprotection against CdCl2-induced brain damage and memory dysfunction and co-administration of α-tocopherol enhances its activity.
Assuntos
Apoptose , Encéfalo/metabolismo , Suplementos Nutricionais , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Síndromes Neurotóxicas/prevenção & controle , Rutina/uso terapêutico , Acetilcolina/agonistas , Acetilcolina/metabolismo , Animais , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Cloreto de Cádmio/antagonistas & inibidores , Cloreto de Cádmio/toxicidade , Intoxicação por Cádmio/fisiopatologia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/prevenção & controle , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Transtornos da Memória/prevenção & controle , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/metabolismo , Ratos Wistar , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismo , alfa-Tocoferol/uso terapêuticoRESUMO
Ghrelin functions as a neuroprotective agent and saves neurons from various insults include ischemic injury. However, it remains to be elucidated whether ghrelin protects neuronal cells against ischemic injury-induced excessive autophagy. Autophagy is required for the maintenance of neural stem cell homeostasis. However, regarding autophagic cell death, it is commonly assumed that excessive autophagy leads to self-elimination of mammalian cells. The purpose of this study was to investigate the potential neuroprotection effects of ghrelin from excessive autophagy in adult rat hippocampal neural stem cells (NSCs). Oxygen-Glucose Deprivation (OGD) strongly induces autophagy in adult rat hippocampal NSCs. Ghrelin treatment inhibited OGD-induced cell death of adult rat hippocampal NSCs assessed by cell-counting-kit-8 assay. Ghrelin also suppressed OGD-induced excessive autophagy activity. The protective effect of ghrelin was accompanied by an increased expression levels of Bcl-2, p-62 and decreased expression level of LC3-II, Beclin-1 by Western blot. Furthermore, ghrelin reduced autophagosome formation and number of GFP-LC3 transfected puncta. In conclusion, our data suggest that ghrelin protects adult rat hippocampal NSCs from excessive autophagy in experimental stroke (oxygen-glucose deprivation) model. Regulating autophagic activity may be a potential optimizing target for promoting adult rat hippocampal NSCs based therapy for stroke.
Assuntos
Autofagia , Grelina/metabolismo , Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neuroproteção , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteína Sequestossoma-1/agonistas , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Células-Tronco Adultas/ultraestrutura , Animais , Apoptose , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/metabolismo , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/patologia , Hipocampo/ultraestrutura , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Sequestossoma-1/metabolismoRESUMO
Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and ß subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4ß2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.
Assuntos
Membrana Celular/efeitos dos fármacos , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Algoritmos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Multimerização Proteica , Transporte Proteico , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagem Individual de MoléculaRESUMO
Chemotherapeutic drugs such as paclitaxel cause painful peripheral neuropathy in many cancer patients and survivors. Although NMDA receptors (NMDARs) at primary afferent terminals are known to be critically involved in chemotherapy-induced chronic pain, the upstream signaling mechanism that leads to presynaptic NMDAR activation is unclear. Group I metabotropic glutamate receptors (mGluRs) play a role in synaptic plasticity and NMDAR regulation. Here we report that the Group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) significantly increased the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of monosynaptic EPSCs evoked from the dorsal root. DHPG also reduced the paired-pulse ratio of evoked EPSCs in spinal dorsal horn neurons. These effects were blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), but not by an mGluR1 antagonist. MPEP normalized the frequency of miniature EPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats but had no effect in vehicle-treated rats. Furthermore, mGluR5 protein levels in the dorsal root ganglion and spinal cord synaptosomes were significantly higher in paclitaxel- than in vehicle-treated rats. Inhibiting protein kinase C (PKC) or blocking NMDARs abolished DHPG-induced increases in the miniature EPSC frequency of spinal dorsal horn neurons in vehicle- and paclitaxel-treated rats. Moreover, intrathecal administration of MPEP reversed pain hypersensitivity caused by paclitaxel treatment. Our findings suggest that paclitaxel-induced painful neuropathy is associated with increased presynaptic mGluR5 activity at the spinal cord level, which serves as upstream signaling for PKC-mediated tonic activation of NMDARs. mGluR5 is therefore a promising target for reducing chemotherapy-induced neuropathic pain.