RESUMO
Predicting lithium-ion battery degradation is worth billions to the global automotive, aviation and energy storage industries, to improve performance and safety and reduce warranty liabilities. However, very few published models of battery degradation explicitly consider the interactions between more than two degradation mechanisms, and none do so within a single electrode. In this paper, the first published attempt to directly couple more than two degradation mechanisms in the negative electrode is reported. The results are used to map different pathways through the complicated path dependent and non-linear degradation space. Four degradation mechanisms are coupled in PyBaMM, an open source modelling environment uniquely developed to allow new physics to be implemented and explored quickly and easily. Crucially it is possible to see 'inside the model and observe the consequences of the different patterns of degradation, such as loss of lithium inventory and loss of active material. For the same cell, five different pathways that can result in end-of-life have already been found, depending on how the cell is used. Such information would enable a product designer to either extend life or predict life based upon the usage pattern. However, parameterization of the degradation models remains as a major challenge, and requires the attention of the international battery community.
RESUMO
A yeast two-hybrid screen, using the carboxyl tail of the human mu opioid receptor as bait and a human brain cDNA library as target, indicated that the carboxyl terminal portion of hlj1, a member of the human heat shock protein 40 family, interacts with the carboxyl tail of the human mu opioid receptor. To determine if direct in vitro binding occurs between these two proteins, we performed overlay experiments. Results from the overlay experiments showed that binding occurs between the His fusion protein of hlj1 and the GST fusion protein of the carboxyl tail of the human mu opioid receptor. In contrast, no binding with the His fusion protein of hlj1 occurred with GST alone or the GST fusion protein of the third cytoplasmic loop of the human mu opioid receptor. Results from co-immunoprecipitation studies, carried out in whole HEK cell lysates, confirmed in vivo binding between these two proteins. Immunofluorescent studies, using laser scanning confocal microscopy, showed significant co-localization between hlj1 and the human mu opioid receptor in the cell membrane. The function of this protein-protein interaction and its physiological relevance in animal and human brain is yet to be determined.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Receptores Opioides mu/metabolismo , Sítios de Ligação/fisiologia , Western Blotting/métodos , Linhagem Celular , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Microscopia Confocal/métodos , Mutagênese/fisiologia , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Receptores Opioides mu/química , Transfecção/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: To assess the resuscitation knowledge and confidence of newly-qualified doctors in New Zealand (NZ) in 2003. DESIGN: Anonymous questionnaires were distributed to all newly-qualified doctors in NZ (n=279). PARTICIPANTS: Two hundred and thirty-three respondents from hospitals throughout NZ (84% response rate). MAIN OUTCOME MEASURES: Resuscitation training received during medical school and use of recommended text (Level 7 of NZ resuscitation manual), confidence in resuscitation skills and core knowledge of basic and advanced resuscitation. RESULTS: 98.3% of doctors received advanced resuscitation training during their final year of medical school, of these 64.6% had received training in the previous 6 months. The mean knowledge score was 56.6% and 45% of doctors made 'fatal errors'. Eighty-four percentage of doctors had read the Level 7 manual and 72.6% found it very or extremely useful. Those who had read the manual had higher scores and were less likely to make a 'fatal error'. Having attended a cardiac arrest and having received training within the last 6 months improved doctor confidence in resuscitation. CONCLUSIONS: The resuscitation knowledge and confidence of newly-qualified doctors in NZ are sub-optimal, with some doctors displaying dangerous deficiencies. Our results suggest mandatory attainment of an advanced resuscitation certificate, six-monthly practical resuscitation sessions and increased exposure to real resuscitation situations should be implemented to improve undergraduate resuscitation training.
Assuntos
Reanimação Cardiopulmonar/educação , Reanimação Cardiopulmonar/estatística & dados numéricos , Competência Clínica/estatística & dados numéricos , Conhecimentos, Atitudes e Prática em Saúde , Estudantes de Medicina/estatística & dados numéricos , Adulto , Atitude do Pessoal de Saúde , Educação Médica/estatística & dados numéricos , Feminino , Pesquisas sobre Atenção à Saúde , Parada Cardíaca/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Nova ZelândiaRESUMO
UNLABELLED: We present a 15-month-old boy who developed fulminant hepatic failure after ingesting 10 ml of clove oil. After 24 h, the ALT level was in excess of 13,000 U/l, with blood urea and creatinine of 11.8 mmol and 134 micromol/l respectively. The hepatic impairment resolved after intravenous administration of N-acetylcysteine so that 6 h later, the ALT level was approximately 10,000 U/l. His liver synthetic function and clinical status improved over the next 4 days. This is the first such case report of its kind in Europe. Analysis of a national database revealed a 14-fold increase in home accidents related to aromatherapy from 1994-1999. Clove oil has important hepatotoxic effects. CONCLUSION: Recent growth in aromatherapy sales has been accompanied by an unfortunate increase in accidental poisoning from these products. Clove oil warrants special attention. Ingesting as little as 10 ml causes hepatotoxicity which can be treated with N-acetylcysteine.
Assuntos
Acetilcisteína/uso terapêutico , Bases de Dados como Assunto , Eugenol/intoxicação , Falência Hepática/induzido quimicamente , Falência Hepática/tratamento farmacológico , Óleos Voláteis/intoxicação , Acidentes Domésticos , Feminino , Humanos , Lactente , Masculino , Resultado do Tratamento , Reino UnidoAssuntos
Dor Abdominal/etiologia , Colonoscopia/efeitos adversos , Ruptura Esplênica/etiologia , Colo/lesões , Diagnóstico Diferencial , Feminino , Hematoma/etiologia , Hemoperitônio/etiologia , Humanos , Perfuração Intestinal/diagnóstico , Pessoa de Meia-Idade , Dor de Ombro/etiologia , Ruptura Esplênica/diagnósticoRESUMO
Analysis of the DNA sequence of the human mu-opioid receptor gene (MOR) revealed that a region overlapping the start codon was substantially homologous to a DNA element named the neurorestrictive suppressor element (NRSE) or restrictive element 1 (RE-1). Transient transfection experiments in the L929 and HEK non-neural cell lines showed that expression of a MOR promoter/reporter gene construct was suppressed in non-neural cell lines by inclusion of this MOR NRSE. Expression from a thymidine kinase promoter was also suppressed when the MOR NRSE was inserted upstream or downstream of the reporter gene. The MOR NRSE did not suppress expression of the reporter gene in neural derived cell lines, IMR-32 and Neuro 2a. The transcription factor REST which binds NRSE thereby enacting the suppression of transcription, was encoded in a plasmid and co-transfected into the IMR-32 cells. The REST co-transfected neuronal derived (IMR-32) cells became sensitive to the MOR NRSE mediated suppression of reporter gene expression. Electrophoretic mobility shift experiments revealed that oligonucleotides containing the MOR NRSE were bound by a factor from nuclear extracts of non-neural cell lines, HeLa and Jurkat. This binding was specifically competed by oligonucleotides containing NRSE sequences previously shown to suppress transcription through REST. Thus an NRSE element overlapping the human MOR start codon suppresses gene expression in non-neural cell lines and may help direct neural tissue specific expression of MOR.
Assuntos
Sequência Consenso , Receptores Opioides mu/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica/fisiologia , Genes Reporter , Células HeLa , Humanos , Células Jurkat , TransfecçãoRESUMO
The putative role of the opioid system in cognitive and memory functions prompted us to search for possible changes in the cohort of the major opioid receptors, mu, delta and kappa, in Alzheimer's disease. The present study examines alterations in opioid receptor levels by quantitative autoradiography. These experiments were carried out on coronal sections of postmortem brains from Alzheimer's disease patients and from aged-matched, dementia-free individuals. Brain sections were labeled with the tritiated forms of mu-, delta- and kappa-opioid ligands; DAMGO ([D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin), DPDPE ([D-Pen2,5]-enkephalin) and bremazocine (in the presence of mu- and delta-ligands), respectively. Nonspecific binding was determined in the presence of naloxone (10 microM). Brain areas analyzed were caudate, putamen, amygdaloid complex, hippocampal formation and various cerebral and cerebellar cortices. Image analyses of autoradiographs show, that in comparison to the same areas in control brain, statistically significant reductions in mu-opioid receptor binding occur in the subiculum and hippocampus of Alzheimer's disease brains. Binding of delta-opioid receptors is also decreased in the amygdaloid complex and ventral putamen of Alzheimer's disease brains. In contrast, large increases of kappa-opioid receptor binding are found in the dorsal and ventral putamen as well as in the cerebellar cortex of Alzheimer's disease brains. Levels of mu- delta- and kappa-opioid receptor binding are unaltered in the caudate, parahippocampal gyrus and occipito-temporal gyrus. These results may suggest an involvement of the endogenous opioid system in some of the multitude of effects that accompany this dementia.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Doença de Alzheimer/patologia , Autorradiografia , Encéfalo/patologia , Densitometria , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , TrítioRESUMO
Opioid receptors are known for their ability to activate diverse second messenger systems. Previously, we showed that selective delta-opioid agonists were able to induce the rapid tyrosine phosphorylation of delta-opioid receptors (delta-ORs) through Src. Src-dependent tyrosine phosphorylation of delta-ORs appears to be important for activation of the mitogen-activated protein kinase cascade and for receptor sequestration into clathrin-coated endosomes, as the Src antagonist, PP1, inhibited both. In an attempt to clarify the role of tyrosine phosphorylation in delta-OR signalling and regulation, we constructed a mutant receptor in which the tyrosine located in the conserved NPXXY motif of the C-terminus was replaced by a phenylalanine (Y318F-delta-OR). Mutation of Y318 resulted in a receptor that was comparable to the wild type in its expression level in HEK-293 cells and in its affinity for opioid ligands. Both receptors showed effective coupling to G proteins and were capable of inhibiting forskolin-stimulated cAMP accumulation with similar potencies. However, the mutant receptor was able to stimulate (35)S-GTPgammaS binding with a lower EC(50) than the wild type receptor. The stimulation of tyrosine phosphorylation in delta-ORs by [D-Thr(2)]-Leu-enkephalin-Thr (DTLET) was significantly less in cells expressing the Y318F-delta-OR than in cells expressing the wild type. In addition, both rapid receptor internalization and down-regulation were markedly attenuated in the mutant. Finally, the mutant receptor was unable to induce a robust activation of the MAPK pathway, suggesting that tyrosine phosphorylation of the delta-OR protein is important for this signalling pathway. These findings implicate tyrosine phosphorylation of Y318 in receptor signalling and agonist-mediated regulation.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Opioides delta/química , Receptores Opioides delta/metabolismo , Tirosina , Sequência de Aminoácidos , Substituição de Aminoácidos , Analgésicos/farmacocinética , Animais , Células CHO , Linhagem Celular , Sequência Conservada , Cricetinae , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacocinética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Mutagênese Sítio-Dirigida , Fenilalanina , Fosforilação , Ensaio Radioligante , Receptores Opioides delta/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , TrítioRESUMO
The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (delta-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for delta-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the delta-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged delta-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the delta-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr(2)]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the delta-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid delta-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the delta-OR protein. Evidence is presented that the delta-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Opioides delta/metabolismo , Tirosina/metabolismo , Animais , Células CHO , Cricetinae , Endossomos/metabolismo , Ativação Enzimática , Oligopeptídeos/farmacologia , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptores Opioides delta/fisiologiaRESUMO
Agonist-promoted internalization (endocytosis) of G-protein-coupled receptors (GPCRs), including all three opioid receptor types (mu, delta and kappa), has been shown to occur via the clathrin endosomal pathway in response to receptor phosphorylation and the actions of the proteins, beta-arrestin and dynamin. Many members of the GPCR family stimulate mitogen-activated protein kinases (MAPK or ERK) activity and, in several cases, it appears that MAPK activation is dependent on receptor internalization. We have reinvestigated the question of whether internalization is obligatory for MAPK activation by opioid receptors, using cell lines expressing the cloned mu or delta receptor. Morphine, which is known to activate both mu and delta receptors, does not induce their rapid internalization into clathrin-coated endosomes. However, morphine produced a robust stimulation of MAPK in both cell lines, as demonstrated by the appearance of phosphorylated MAPK. Moreover, pre-exposure of cells to the internalization inhibitors, concanavalin A or hypertonic sucrose, totally blocked DAMGO mu-selective agonist) and DTLET (delta-selective agonist)-mediated receptor internalization, yet neither treatment affected MAPK phosphorylation induced by these peptides. Our results provide evidence that receptor internalization is not an obligatory requirement for MAPK activation by mu and delta opioid receptors. Hypotheses are presented to explain the seemingly contradictory results obtained from different laboratories.
Assuntos
Endocitose/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Ligação Competitiva , Células CHO , Concanavalina A/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Soluções Hipertônicas/farmacologia , Morfina/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ensaio Radioligante , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Sacarose/farmacologia , Fatores de TempoRESUMO
Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.
Assuntos
Proteínas de Arabidopsis , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Receptores Proteína Tirosina Quinases/genética , Arabidopsis , Genes de Plantas , Proteínas de Homeodomínio/genética , Fenótipo , Coifa/crescimento & desenvolvimento , Proteínas Serina-Treonina QuinasesRESUMO
The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.
Assuntos
Benzenoacetamidas , Receptores Opioides mu/genética , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Animais , Sequência de Bases , Ligação Competitiva/efeitos dos fármacos , Bovinos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clonagem Molecular , Colforsina/farmacologia , Corpo Estriado/química , DNA Complementar/química , DNA Complementar/genética , Diprenorfina/metabolismo , Diprenorfina/farmacologia , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/metabolismo , D-Penicilina (2,5)-Encefalina/farmacologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Dados de Sequência Molecular , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ensaio Radioligante , Receptores Opioides mu/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Radioisótopos de Enxofre , TrítioRESUMO
The regulation of mu-opioid receptor gene expression was investigated using several molecular techniques. Genomic clones containing portions of the human mu-opioid receptor gene were sequenced. 5'-RACE analysis of human brain cDNA confirmed the presence of mRNAs up to -313 from the start codon. As was found for the mouse and rat genes, transcription apparently initiates in the absence of a discernable TATA box. To characterize promoter function, portions of the 5'-flanking region were linked to a reporter gene in transient transfection experiments. Two approximately 50 bp adjacent segments had potent, orientation specific promoter activity. More down-stream segments also had promoter activity. None of the 5'-flanking region constructs showed tissue specificity. The potential role of DNA methylation in preventing ectopic expression was investigated by surveying the methylation state of a CpG rich region straddling the start codon. A neural derived cell line (SH-SY5Y) that expresses the mu-opioid receptor lacked virtually any CpG methylation. In contrast, two neural derived cell lines that do not express the mu-opioid receptor were nearly totally methylated while non-neural cell lines had intermediate levels of CpG methylation. Additional transient transfection experiments revealed that CpG methylation of the 5'-flanking region suppressed reporter gene expression. These results indicate that CpG methylation plays an important role in regulating mu-opioid receptor expression in neural cells; however, no association was found with regulation of expression in non-neural cells.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Receptores Opioides mu/genética , Animais , Carcinoma de Células Escamosas/patologia , Ilhas de CpG , DNA Complementar/genética , Genes Reporter , Humanos , Rim , Células L , Luciferases/biossíntese , Luciferases/genética , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Neuroblastoma/patologia , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Ratos , Receptores Opioides mu/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.
Assuntos
Regulação para Baixo/fisiologia , Proteína Quinase C/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Analgésicos/farmacologia , Analgésicos Opioides/farmacologia , Transporte Biológico/efeitos dos fármacos , Carcinógenos/farmacologia , Diprenorfina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Encefalinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Immunoblotting , Isoenzimas/análise , Isoenzimas/metabolismo , Antagonistas de Entorpecentes/farmacologia , Neuroblastoma , Dibutirato de 12,13-Forbol/farmacologia , Ésteres de Forbol/farmacologia , Proteína Quinase C/análise , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Receptores Opioides mu/antagonistas & inibidores , Sistemas do Segundo Mensageiro/fisiologia , Trítio , Células Tumorais CultivadasRESUMO
Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express mu- and delta-opioid receptors, we investigated whether mu-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by 3H-phorbol ester and [3H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that mu-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [3H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.
Assuntos
Regulação para Baixo/fisiologia , Proteína Quinase C/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Analgésicos/farmacologia , Analgésicos Opioides/farmacologia , Ligação Competitiva/fisiologia , Transporte Biológico/efeitos dos fármacos , Carcinógenos/farmacologia , Diprenorfina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Encefalinas/farmacologia , Humanos , Cinética , Proteínas de Membrana/metabolismo , Morfina/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Neuroblastoma , Dibutirato de 12,13-Forbol/farmacologia , Receptores Opioides mu/antagonistas & inibidores , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Fatores de Tempo , Trítio , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
We have investigated the role of cysteine residues in a highly purified mu opioid receptor protein (muORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of muORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding muORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the mu opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.
Assuntos
Benzomorfanos/metabolismo , Corpo Estriado/metabolismo , Cisteína , Encefalinas/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Reagentes de Sulfidrila/farmacologia , Animais , Bovinos , Membrana Celular/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Etilmaleimida/farmacologia , Cinética , Ligantes , Naloxona/farmacologia , Ensaio Radioligante , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , TrítioRESUMO
Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore, these results support the idea that the striking effects of sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.
Assuntos
Cisteína/fisiologia , Receptores Opioides delta/fisiologia , Alanina/fisiologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Cricetinae , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Serina/fisiologiaRESUMO
Norepinephrine (NE) is known to activate a number of immediate-early genes (IEGs) in the brain which may be involved in prolonged changes in neuronal function. To investigate the function of these genes it would be useful to have a model system in which they are induced in specific populations of cells in specific brain regions without systemic drug administration which can affect multiple sites. In the present paper we have shown that local infusions of NE or of the alpha2-adrenoceptor antagonist, atipamezole, in the mouse amygdala produces localized expression of fos. The expression of fos was blocked by a cocktail of an alpha1-(prazosin) and beta1-adrenoceptor (betaxolol) blocker but not by a selective 5-HT1A blocker (WAY100135). Prazosin and betaxolol did not have a nonspecific reducing action on fos expression. It is concluded that localized expression of fos after NE infusion in the mouse amygdala represents a model system for further studies of the role of IEG expression in central noradrenergic function.
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Tonsila do Cerebelo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Imidazóis/farmacologia , Norepinefrina/farmacologia , Animais , Masculino , CamundongosRESUMO
We compared two types of automatic non-invasive blood pressure measuring device with sphygmomanometey in 47 normotensive and 38 hypertensive women in the third trimester of pregnancy. An automatic oscillometric device (Accutor) and a volume-clamp device (Finapres) significantly underestimated the diastolic pressure as measured by the fourth Korotkoff sound using a Hawksley random zero sphygmomanometer. The mean difference between the sphygmomanometer and Accutor measurement of diastolic blood pressure was +3.1 mmHg in the normotensive women and +8.3 mmHg in the hypertensive women (P = 0.001). The mean difference between the sphygmomanometer and Finapres measurement of diastolic blood pressure was +6.1 mmHg in the normotensive women and +11.5 mmHg in hypertensive women (P = 0.003). The increased use of continuous non-invasive devices to monitor blood pressure in women with hypertension should be accompanied by sound knowledge of their limitations in this group of patients.