RESUMO
Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies.
Assuntos
Autoanticorpos/imunologia , Citocromo P-450 CYP2D6/metabolismo , Hepatite C Crônica/patologia , Adulto , Idoso , Estudos de Coortes , Citocromo P-450 CYP2D6/genética , Dextrometorfano/metabolismo , Dextrorfano/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , SuíçaRESUMO
BACKGROUND AND PURPOSE: CSF loss in spontaneous intracranial hypotension disrupts a well-regulated equilibrium. We aimed to evaluate the volume shift between intracranial compartments in patients with spontaneous intracranial hypotension before and after surgical closure of the underlying spinal dural breach. MATERIALS AND METHODS: In total, 19 patients with spontaneous intracranial hypotension with a proved spinal CSF leak investigated at our institution between July 2014 and March 2017 (mean age, 41.8 years; 13 women) were included. Brain MR imaging-based volumetry at baseline and after surgery was performed with FreeSurfer. In addition, the spontaneous intracranial hypotension score, ranging from 0 to 9, with 0 indicating very low and 9 very high probability of spinal CSF loss, was calculated. RESULTS: Total mean ventricular CSF volume significantly increased from baseline (15.3 mL) to posttreatment MR imaging (18.0 mL), resulting in a mean absolute and relative difference, +2.7 mL and +18.8% (95% CI, +1.2 to +3.9 mL; P < .001). The change was apparent in the early follow-up (mean, 4 days). No significant change in mean total brain volume was observed (1136.9 versus 1133.1 mL, P = .58). The mean spontaneous intracranial hypotension score decreased from 6.9 ± 1.5 at baseline to 2.9 ± 1.5 postoperatively. CONCLUSIONS: Our study demonstrated a substantial increase in ventricular CSF volume in the early follow-up after surgical closure of the underlying spinal dural breach and may provide a causal link between spinal CSF loss and spontaneous intracranial hypotension. The concomitant decrease in the spontaneous intracranial hypotension score postoperatively implies the restoration of an equilibrium within the CSF compartment.
Assuntos
Vazamento de Líquido Cefalorraquidiano/complicações , Vazamento de Líquido Cefalorraquidiano/diagnóstico por imagem , Líquido Cefalorraquidiano/diagnóstico por imagem , Hipotensão Intracraniana/diagnóstico por imagem , Hipotensão Intracraniana/etiologia , Adulto , Idoso , Vazamento de Líquido Cefalorraquidiano/cirurgia , Feminino , Humanos , Hipotensão Intracraniana/cirurgia , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Neuroimagem/métodosRESUMO
The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/metabolismo , Caspases/metabolismo , Processamento de Proteína Pós-Traducional , Células HeLa , Humanos , Fatores Reguladores de Interferon/metabolismo , Modelos Biológicos , Poliovirus/fisiologiaRESUMO
Polymorphisms of the cytochrome P450 2D6 (CYP2D6) gene affecting enzyme activity are involved in interindividual variability in drug efficiency/toxicity. Four phenotypic groups are found in the general population: ultra rapid (UM), extensive (EM), intermediate (IM) and poor (PM) metabolizers. The AmpliChip CYP450 test is the first genotyping array allowing simultaneous analysis of 33 CYP2D6 alleles. The main aim of this study was to evaluate the performance of this test in CYP2D6 phenotype prediction. We first verified the AmpliChip CYP450 test genotyping accuracy for five CYP2D6 alleles routinely analysed in our laboratory (alleles 3,4,5,6, x N; n=100). Results confirmed those obtained by real-time PCR. Major improvements using the array are the detection of CYP2D6 intermediate alleles and identification of the duplicated alleles. CYP2D6 phenotype was determined by assessing urinary elimination of dextromethorphan and its metabolite dextrorphan and compared to the array prediction (n=165). Although a low sensitivity of UM prediction by genotyping was observed, phenotype prediction was optimal for PM and satisfying for EM and IM.
Assuntos
Citocromo P-450 CYP2D6/genética , Análise de Sequência com Séries de Oligonucleotídeos , Farmacogenética/métodos , Dextrometorfano/farmacocinética , Dextrometorfano/urina , Dextrorfano/farmacocinética , Dextrorfano/urina , Frequência do Gene , Genótipo , Humanos , Taxa de Depuração Metabólica/genética , Fenótipo , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-ß-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.
Assuntos
Imidazóis/farmacologia , Necrose/tratamento farmacológico , Piridazinas/farmacologia , Pirimidinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Sulfonamidas/farmacologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína de Domínio de Morte Associada a Fas/genética , Células HEK293 , Células HT29 , Humanos , Indazóis , Células Jurkat , Células L , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/sangue , Sistema Enzimático do Citocromo P-450/sangue , Teste em Amostras de Sangue Seco , Preparações Farmacêuticas/sangue , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Administração Oral , Adulto , Bupropiona/administração & dosagem , Bupropiona/sangue , Bupropiona/farmacocinética , Cafeína/administração & dosagem , Cafeína/sangue , Cafeína/farmacocinética , Cápsulas , Bebidas Gaseificadas , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Café , Inibidores das Enzimas do Citocromo P-450 , Dextrometorfano/administração & dosagem , Dextrometorfano/sangue , Dextrometorfano/farmacocinética , Inibidores Enzimáticos/administração & dosagem , Estudos de Viabilidade , Flurbiprofeno/administração & dosagem , Flurbiprofeno/sangue , Flurbiprofeno/farmacocinética , Humanos , Isoenzimas , Masculino , Midazolam/administração & dosagem , Midazolam/sangue , Midazolam/farmacocinética , Omeprazol/administração & dosagem , Omeprazol/sangue , Omeprazol/farmacocinética , Preparações Farmacêuticas/administração & dosagem , Fenótipo , Projetos Piloto , Valor Preditivo dos Testes , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Espectrometria de Massas em Tandem , Terfenadina/administração & dosagem , Terfenadina/análogos & derivados , Terfenadina/sangue , Terfenadina/farmacocinética , Adulto JovemRESUMO
The family of death domain (DD)-containing proteins are involved in many cellular processes, including apoptosis, inflammation and development. One of these molecules, the adapter protein MyD88, is a key factor in innate and adaptive immunity that integrates signals from the Toll-like receptor/interleukin (IL)-1 receptor (TLR/IL-1R) superfamily by providing an activation platform for IL-1R-associated kinases (IRAKs). Here we show that the DD-containing protein Unc5CL (also known as ZUD) is involved in a novel MyD88-independent mode of IRAK signaling that culminates in the activation of the transcription factor nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase. Unc5CL required IRAK1, IRAK4 and TNF receptor-associated factor 6 but not MyD88 for its ability to activate these pathways. Interestingly, the protein is constitutively autoproteolytically processed, and is anchored by its N-terminus specifically to the apical face of mucosal epithelial cells. Transcriptional profiling identified mainly chemokines, including IL-8, CXCL1 and CCL20 as Unc5CL target genes. Its prominent expression in mucosal tissues, as well as its ability to induce a pro-inflammatory program in cells, suggests that Unc5CL is a factor in epithelial inflammation and immunity as well as a candidate gene involved in mucosal diseases such as inflammatory bowel disease.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas de Transporte/genética , Quimiocinas/biossíntese , Quimiocinas/genética , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Flurbiprofeno/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2C9 , Fluconazol/administração & dosagem , Flurbiprofeno/sangue , Flurbiprofeno/urina , Genótipo , Humanos , Hidrólise , Masculino , Rifampina/administração & dosagem , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug-drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored. EXPERIMENTAL APPROACH: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg x kg(-1) and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session. KEY RESULTS: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C(max) (-0.71 < Spearman correlation coefficient rhos < -0.92). Oxymorphone C(max) was 62% and 75% lower in PM than EM and UM. Noroxymorphone C(max) reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C(max) by 40% and 80%, and increased noroxycodone AUC(infinity) by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC(infinity) and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition. CONCLUSIONS AND IMPLICATIONS: Drug-drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.
Assuntos
Analgésicos Opioides/farmacocinética , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Oxicodona/farmacocinética , Polimorfismo Genético , Adulto , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/sangue , Analgésicos Opioides/química , Estudos Cross-Over , Inibidores do Citocromo P-450 CYP2D6 , Inibidores do Citocromo P-450 CYP3A , Método Duplo-Cego , Interações Medicamentosas/genética , Inibidores Enzimáticos/farmacologia , Genótipo , Humanos , Cetoconazol/farmacologia , Masculino , Desintoxicação Metabólica Fase I/genética , Oxicodona/administração & dosagem , Oxicodona/sangue , Oxicodona/química , Fenótipo , Quinidina/farmacologia , Receptores Opioides mu/metabolismo , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. EXPERIMENTAL APPROACH: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. KEY RESULTS: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. CONCLUSIONS AND IMPLICATIONS: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.
Assuntos
Analgésicos Opioides/uso terapêutico , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Oxicodona/efeitos adversos , Oxicodona/uso terapêutico , Polimorfismo Genético , Adulto , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/farmacocinética , Analgésicos Opioides/farmacologia , Estudos Cross-Over , Inibidores do Citocromo P-450 CYP2D6 , Inibidores do Citocromo P-450 CYP3A , Método Duplo-Cego , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Genótipo , Humanos , Masculino , Desintoxicação Metabólica Fase I/genética , Pessoa de Meia-Idade , Oxicodona/farmacocinética , Oxicodona/farmacologia , Oximorfona/sangue , Limiar da Dor/efeitos dos fármacos , Fenótipo , Desempenho Psicomotor/efeitos dos fármacos , Reflexo Pupilar/efeitos dos fármacos , Adulto JovemAssuntos
Arildialquilfosfatase/genética , Resistência a Medicamentos , Variação Genética , Isquemia Miocárdica/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Ticlopidina/análogos & derivados , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Arildialquilfosfatase/metabolismo , Clopidogrel , Citocromo P-450 CYP2C19 , Resistência a Medicamentos/genética , Genótipo , Humanos , Isquemia Miocárdica/sangue , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/genética , Fenótipo , Agregação Plaquetária/genética , Testes de Função Plaquetária , Ticlopidina/uso terapêuticoRESUMO
We investigated the involvement of mitogen-activated protein kinases (MAPKs) in the maturation of CD83(-) dendritic cells (DC) derived from human blood monocytes. Maturating agents such as LPS and TNF-alpha induced the phosphorylation of members of the three families of MAPK (extracellular signal-regulated kinase l/2, p46/54 c-Jun N-terminal kinase, and p38 MAPK). SB203580, an inhibitor of the p38 MAPK, but not the extracellular signal-regulated kinase l/2 pathway blocker PD98059, inhibited the up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and the DC maturation marker CD83 induced by LPS and TNF-alpha. In addition, SB203580 inhibited the enhancement of the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake induced by LPS and TNF-alpha. Likewise, SB203580 partially prevented the up-regulation of IL-1alpha, IL-1beta, IL-lRa, and TNF-alpha mRNA upon stimulation with LPS and TNF-alpha, as well as the release of bioactive TNF-alpha induced by LPS. DC maturation induced by the contact sensitizers 2,4-dinitrofluorobenzene and NiSO(4), as seen by the up-regulation of CD80, CD86, and CD83, was also coupled to the phosphorylation of p38 MAPK, and was inhibited by SB203580. The irritants SDS and benzalkonium chloride that do not induce DC maturation did not trigger p38 MAPK phosphorylation. Together, these data indicate that phosphorylation of p38 MAPK is critical for the maturation of immature DC. These results also suggest that p38 MAPK phosphorylation in DC may become useful for the identification of potential skin contact sensitizers.
Assuntos
Alérgenos/imunologia , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Irritantes/imunologia , Lipopolissacarídeos/imunologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fator de Necrose Tumoral alfa/imunologia , Antígenos CD/biossíntese , Antígenos CD/imunologia , Compostos de Benzalcônio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Citocinas/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Dinitrofluorbenzeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Ativação Linfocitária/efeitos dos fármacos , Proteína Quinase 10 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Níquel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Dodecilsulfato de Sódio/farmacologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.
Assuntos
Fator Natriurético Atrial/biossíntese , Cálcio/metabolismo , AMP Cíclico/metabolismo , Endotelina-1/farmacologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Alcaloides , Animais , Animais Recém-Nascidos , Benzofenantridinas , Cálcio/farmacologia , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Epoprostenol/biossíntese , Ventrículos do Coração , Cinética , Miocárdio/citologia , Naftalenos/farmacologia , Nifedipino/farmacologia , Fenantridinas/farmacologia , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/biossíntese , Receptores de Endotelina/fisiologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Tionucleotídeos/farmacologiaRESUMO
We studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in epidermal growth factor (EGF)-induced prostacyclin (PGI2) production in cultured, spontaneously-beating neonatal ventricular rat cardiomyocytes. To this purpose, the effect of EGF on cardiomyocyte MAPK phosphorylation, MAPK activity and PGI2-production were investigated, and compared to those induced by the PKC activator 4 beta phorbol 12-myristate 13-acetate (PMA). Both EGF (0.1 microM) and PMA (0.1 microM) induced the rapid and reversible phosphorylation of 42 KDa-MAPK in ventricular cardiomyocytes, responses that were accompanied by transient increases in MAPK activity (190-230% of control values within 5 min), and two- to three-fold increases in PGI2 formation. The tyrosine kinase inhibitors lavendustin (1 microM) and genistein (10 microM) strongly inhibited EGF-induced MAPK activation and PGI2-formation, but had no effect on PMA-stimulated responses. Experiments with the PKC inhibitor CGP 41251 (1 microM) or with PKC-downregulated cells demonstrated that in contrast to the PMA-stimulated responses, EGF-induced MAPK activation and PGI2-production were PKC-independent processes. Investigating the role of MAPK in EGF- and in PMA-promoted PGI2-formation, we found that the MAPK-inhibitor 6-thioguanine (500 microM), as well as the MAPK-kinase-inhibitor PD98059 (50 microM) abolished both EGF- and PMA-stimulated PGI2-production in cardiomyocytes. Our results indicate that MAPK-activation is at the basis of both growth factor receptor and PKC-dependent eicosanoid-formation in ventricular cardiomyocytes, where EGF-induced prostaglandin-production takes place via a PKC-independent pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Epoprostenol/biossíntese , Ventrículos do Coração/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Análise de Variância , Animais , Células Cultivadas , Ativação Enzimática , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Fosforilação , Ratos , Ratos WistarRESUMO
Epidermal growth factor (EGF) was tested for its ability to promote hypertrophic responses in neonatal rat ventricular cardiomyocytes. Exposure of these cells to 100 n m EGF for 2-18 h resulted in a time-dependent increase in protein synthesis reaching 174+/-18% of control values at 18 h. After 30 min stimulation, the mRNA levels of c-jun and c-fos were also increased 20- and 36-fold, respectively. We also investigated EGF-induced activation of Stat (signal transducers and activators of transcription) proteins as well as the possible interactions of this signaling pathway with the p38 and p42/44 MAP kinases cascades. EGF did not activate Stat1 and Stat3, but did induce a rapid and transient activation of Stat5, which corresponded mainly to Stat5b DNA-binding. The EGF-promoted Stat5 DNA-binding was decreased in a concentration-dependent manner by the p38 MAPK inhibitor SB 203580 (IC(50)=1.2 microm), whereas it was tripled by 50 micro m PD 98059, an inhibitor of the p42/44 MAPK cascade. This is the first demonstration that EGF increases protein synthesis and early response gene expression in cardiomyocytes, responses considered as markers of hypertrophy in these cells. The results further show that EGF activates Stat5, that this response requires p38 MAPK stimulation, and it is negatively modulated by p42/44 MAPK.
Assuntos
Cardiomegalia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Ventrículos do Coração/metabolismo , Proteínas do Leite , Transativadores/metabolismo , Animais , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Ventrículos do Coração/citologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosforilação , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Ratos , Ratos Wistar , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Incubation of spontaneously beating ventricular cardiomyocytes from neonatal rats with prostaglandin E(2) (0.1 microM) or forskolin (0.1 microM) simultaneously increased the rate of cellular contraction and atrial natriuretic peptide (ANP) secretion. Both responses were maximal within 10-20 min of application and were accompanied by three- to fourfold increases in cAMP formation. By contrast, a higher regimen of forskolin (10 microM) promoted a 20- to 30-fold increase in basal cAMP production, which was accompanied by the abolition of contractile activity and ANP release. Low regimens of forskolin (0.1 microM) doubled the occurrence of cytosolic Ca(2+) transients associated with monolayer contraction, whereas higher regimens of forskolin (10 microM) completely suppressed Ca(2+) transients. Moreover, in quiescent cultures that were pretreated with ryanodine, tetrodotoxin, nifedipine, or butanedione monoxime, prostaglandin E(2) (0.1 microM) and forskolin (0.1 microM) failed to elicit significant ANP secretion, suggesting that cAMP-elevating agents promote ANP secretion to a great extent via an increase in cellular contraction frequency in ventricular cardiomyocytes.
Assuntos
Fator Natriurético Atrial/metabolismo , AMP Cíclico/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/biossíntese , Citosol/metabolismo , Dinoprostona/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Concentração Osmolar , Ratos , Ratos WistarRESUMO
Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 +/- 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 +/- 65% of control levels at 5 min, and 316 +/- 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 +/- 17% of control values at 5 min and 125 +/- 7% at 15 min). Okadaic acid (1 microM ) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity.