RESUMO
BACKGROUND: Remote ischemic preconditioning (RIPC) protects the heart from ischemia and reperfusion (I/R) injury. The underlying molecular mechanisms are unclear. It has been demonstrated that Connexin 43 (Cx43) is critically involved in cardioprotective interventions including classical ischemic preconditioning. In the present study we investigated the influence of RIPC on the expression patterns of Cx43 after I/R in the rat heart in vivo. METHODS: Male Wistar rats were subjected to 35 min regional myocardial ischemia followed by 2 h reperfusion with or without 4 cycles of 5 minutes bilateral hind limb ischemia and reperfusion (RIPC), to RIPC without ischemia or underwent no intervention (Sham). Infarct size was measured by TTC staining. The myocardium was divided into area at risk (AAR) and area not at risk (non AAR). Expression of Cx43-mRNA and protein was analyzed by qPCR and Western Blot analysis, respectively. Localization of Cx43 was visualized by confocal immunofluorescence staining. RESULTS: RIPC reduced the infarct size (I/R: 73 ± 5% vs. RIPC I/R: 34 ± 14%, p < 0.05). Expression of Cx43 mRNA did not differ between groups. I/R caused a strong decrease of relative Cx43 protein expression in the AAR that was partly abolished by RIPC. Furthermore, RIPC decreased the level of ischemia-induced dephosphorylation of Cx43. Confocal immunofluorescence staining showed that I/R caused a loss of the Cx43 signal at the intercalated discs, while the Cx43 signal at the intercalated discs was partly sustained after RIPC. CONCLUSION: Preservation of Cx43 protein expression and phosphorylation after RIPC might protect the rat heart in vivo.
Assuntos
Conexina 43/metabolismo , Precondicionamento Isquêmico Miocárdico/métodos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Proteínas Quinases/metabolismo , Animais , Extremidades , Coração/fisiopatologia , Hemodinâmica , Masculino , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/patologia , Fosforilação , Ratos , Ratos WistarRESUMO
We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h (p<0.05) and biliary taurocholate excretion from 45.9±13.81 µmol/3 h to 1.87±0.46 µmol/3 h (p<0.05). Mrp2, Bsep and Ntcp peak immunofluorescence in pericentral hepatocytes decreased to 79.0±2.6% (p<0.001), 80.6±8.4% (p<0.05) and 65.8±5.0% (p<0.01), respectively. Pre-induction of HO-1 by HbG200 was largely confined to pericentral hepatocytes. HO-1 induction attenuated the decreased bile flow (0.91±0.16 ml/3 h, p<0.05) and canalicular taurocholate secretion (4.33±1.71 µmol/3 h, p<0.05). Bsep and Mrp2 peak immunofluorescence in pericentral hepatocytes was largely restored. Activation of JNK and Fyn by cold I/R was significantly attenuated by HO-1. Inhibiting HO activity by tin protoporphyrin IX after HbG200 administration reversed the effect on bile flow and canalicular transporter expression. In conclusion, pericentral downregulation of Bsep and Mrp2 following cold I/R is ameliorated by inducing HO-1 and was associated with diminished hepatocellular JNK and Fyn signaling. HO-1 may serve as a therapeutic target to attenuate hepatocellular cholestasis following I/R injury.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bile/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hemoglobinas/metabolismo , Isquemia/metabolismo , Traumatismo por Reperfusão/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Modelos Animais de Doenças , Regulação para Baixo , Isquemia/enzimologia , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologiaRESUMO
BACKGROUND: Pre- and postconditioning describe mechanisms whereby short ischemic periods protect an organ against a longer period of ischemia. Interestingly, short ischemic periods of a limb, in itself harmless, may increase the ischemia tolerance of remote organs, e.g. the heart (remote conditioning, RC). Although several studies have shown reduced biomarker release by RC, a reduction of complications and improvement of patient outcome still has to be demonstrated. Atrial fibrillation (AF) is one of the most common complications after coronary artery bypass graft surgery (CABG), affecting 27-46% of patients. It is associated with increased mortality, adverse cardiovascular events, and prolonged in-hospital stay. We hypothesize that remote ischemic pre- and/or post-conditioning reduce the incidence of AF following CABG, and improve patient outcome. METHODS/DESIGN: This study is a randomized, controlled, patient and investigator blinded multicenter trial. Elective CABG patients are randomized to one of the following four groups: 1) control, 2) remote ischemic preconditioning, 3) remote ischemic postconditioning, or 4) remote ischemic pre- and postconditioning. Remote conditioning is applied at the arm by 3 cycles of 5 minutes of ischemia and reperfusion. Primary endpoint is the incidence AF in the first 72 hours after surgery, detected using a Holter-monitor. Secondary endpoints include length-of-stay on the intensive care unit and in-hospital, and the occurrence of major adverse cardiovascular events at 30 days, 3 months and 1 year.Based on an expected incidence in the control group of 27%, 195 patients per group are needed to detect with 80% power a reduction by 45% following either pre- or postconditioning, while allowing for a 10% dropout and at an alpha of 0.05. With the combined intervention expected to be stronger, we need 75 patients in this group to detect a reduction in incidence of AF of 60%. DISCUSSION: The RICO-trial (the effect of Remote Ischemic Conditioning on atrial fibrillation and Outcome) is a randomized controlled multicenter trial, designed to investigate whether remote ischemic pre- and/or post-conditioning of the arm reduce the incidence of AF following CABG surgery. TRIAL REGISTRATION: ClinicalTrials.gov under NCT01107184.
RESUMO
Calcium-dependent cell death occurs in neurodegenerative diseases and ischemic or traumatic brain injury. We analyzed whether thioureylenes can act in a neuroprotective manner by pharmacological suppression of calcium-dependent pathological pathways. In human neuroblastoma (SK-N-SH) cells, thioureylenes (thiopental, carbimazole) inhibited the calcium-dependent neuronal protein phosphatase (PP)-2B, the activation of the proapoptotic transcription factor nuclear factor of activated T-cells, BAD-induced initiation of caspase-3, and poly-(ADP-ribose)-polymerase cleavage. Caspase-3-independent cell death was attenuated by carbimazole and the protein kinase C (PKC) delta inhibitor rottlerin by a PP-2B-independent mechanism. Neuroprotective effects were mediated by the redox-active sulfur of thioureylenes. Furthermore, we observed that the route of calcium mobilization was differentially linked to caspase-dependent or independent cell death and that BAD dephosphorylation did not necessarily induce intrinsic caspase activation. In addition, a new 30- to 35-kDa caspase-3 fragment with an unknown function was identified. In organotypic hippocampal slice cultures, thioureylenes inhibited caspase-3 activation or reduced N-methyl-d-aspartate and kainic acid receptor-mediated cell death that was independent of caspase-3. Because prolonged inhibition of caspase-3 resulted in caspase-independent cellular damage, different types of cell death must be taken under therapeutic consideration. Here we show that thioureylenes in combination with PKCdelta inhibitors might represent a promising therapeutic approach to attenuate neuronal damage.
Assuntos
Cálcio/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Tiouracila/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cálcio/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Camundongos , Neurônios/patologia , Tiouracila/análogos & derivadosRESUMO
BACKGROUND: The NO--cGMP system plays a key role in the regulation of sinusoidal tonus and liver blood flow with phosphodiesterase-5 (PDE-5) terminating the dilatory action of cGMP. We, therefore, investigated the effects of PDE-5 inhibitors on hepatic and systemic hemodynamics in rats. METHODS: Hemodynamic parameters were monitored for 60 min. after intravenous injection of sildenafil and vardenafil [1, 10 and 100 microg/kg (sil1, sil10, sil100, var1, var10, var100)] in anesthetized rats. RESULTS: Cardiac output and heart rate remained constant. After a short dip, mean arterial blood pressure again increased. Systemic vascular resistance transiently decreased slightly. Changes in hepatic hemodynamic parameters started after few minutes and continued for at least 60 min. Portal (var10 -31%, sil10 -34%) and hepatic arterial resistance (var10 -30%, sil10 -32%) decreased significantly (p < 0.05). At the same time portal venous (var10 +29%, sil10 +24%), hepatic arterial (var10 +34%, sil10 +48%), and hepatic parenchymal blood flow (var10 +15%, sil10 +15%) increased significantly (p < 0.05). The fractional liver blood flow (total liver flow/cardiac output) increased significantly (var10 26%, sil10 23%). Portal pressure remained constant or tended to decrease. 10 microg/kg was the most effective dose for both PDE-5 inhibitors. CONCLUSION: Low doses of phosphodiesterase-5 inhibitors have distinct effects on hepatic hemodynamic parameters. Their therapeutic use in portal hypertension should therefore be evaluated.
Assuntos
Inibidores Enzimáticos/farmacologia , Hemodinâmica/efeitos dos fármacos , Imidazóis/farmacologia , Fígado/irrigação sanguínea , Inibidores da Fosfodiesterase 5 , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Modelos Animais , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Citrato de Sildenafila , Triazinas/farmacologia , Dicloridrato de Vardenafila , Resistência Vascular/efeitos dos fármacosRESUMO
Carbon monoxide (CO) is generated during incomplete combustion of carbon-containing compounds and leads to acute and chronic toxicity in animals and humans depending on the concentration and exposure time. In addition to exogenous sources, CO is also produced endogenously by the activity of heme oxygenases (HOs) and the physiological significance of HO-derived CO has only recently emerged. CO exerts vasoactive, anti-proliferative, anti-oxidant, anti-inflammatory and anti-apoptotic effects and contributes substantially to the important role of the inducible isoform HO-1 as a mediator of tissue protection and host defense. Exogenous application of low doses of gaseous CO might provide a powerful tool to protect organs and tissues under various stress conditions. Experimental evidence strongly suggests a beneficial effect under pathophysiological conditions such as organ transplantation, ischemia/reperfusion, inflammation, sepsis, or shock states. The cellular and molecular mechanisms mediating CO effects are only partially characterized. So far, only a few studies in humans are available, which, however, do not support the promising results observed in experimental studies. The protective effects of exogenous CO may strongly depend on the pathological condition, the mode, time point and duration of application, the administered concentration, and on the target tissue and cell. Differences in bioavailability of endogenous CO production and exogenous CO supplementation might also provide an explanation for the lack of protective effects observed in some experimental and clinical studies. Further randomized, controlled clinical studies are needed to clarify whether exogenous application of CO may turn into a safe and effective preventive and therapeutic strategy to treat pathophysiological conditions associated with inflammatory or oxidative stress.
Assuntos
Antimetabólitos/uso terapêutico , Intoxicação por Monóxido de Carbono/genética , Monóxido de Carbono/uso terapêutico , Antimetabólitos/administração & dosagem , Antimetabólitos/metabolismo , Antimetabólitos/farmacologia , Monóxido de Carbono/administração & dosagem , Monóxido de Carbono/metabolismo , Monóxido de Carbono/farmacologia , HumanosRESUMO
Thionamides, inhibitors of the thyroid peroxidase-mediated iodination, are clinically used in the treatment of hyperthyroidism. However, the use of antithyroid drugs is associated with immunomodulatory effects, and recent studies with thionamide-related heterocyclic thioderivates demonstrated direct anti-inflammatory and immunosuppressive properties. Using primary human T-lymphocytes, we show that the heterocyclic thionamides carbimazole and propylthiouracil inhibit synthesis of the proinflammatory cytokines tumor necrosis factor (TNF)alpha and interferon (IFN)gamma. In addition, DNA binding of nuclear factor (NF)-kappaB, a proinflammatory transcription factor that regulates both TNFalpha and IFNgamma synthesis, and NF-kappaB-dependent reporter gene expression were reduced. Abrogation of NF-kappaB activity was accompanied by reduced phosphorylation and proteolytic degradation of inhibitor of kappaB (IkappaB)alpha, the inhibitory subunit of the NF-kappaB complex. Carbimazole inhibited NF-kappaB via the small GTPase Rac-1, whereas propylthiouracil inhibited the phosphorylation of IkappaBalpha by its kinase inhibitor of kappaB kinase alpha. Methimazole had no effect on NF-kappaB induction, demonstrating that drug potency correlated with the chemical reactivity of the thionamide-associated sulfur group. Taken together, our data demonstrate that thioureylenes with a common, heterocyclic structure inhibit inflammation and immune function via the NF-kappaB pathway. Our results may explain the observed remission of proinflammatory diseases upon antithyroid therapy in hyperthyroid patients. The use of related thioureylenes may provide a new therapeutic basis for the development and application of anti-inflammatory compounds.
Assuntos
Antitireóideos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Antitireóideos/química , Humanos , Quinase I-kappa B/metabolismo , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Various pharmacologic inhibitors of the NF-kappaB pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3(+) T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Linfócitos T/efeitos dos fármacos , Tiopental/farmacologia , Complexo CD3 , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/análise , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Substâncias Protetoras/farmacologia , RNA Mensageiro/análise , Linfócitos T/citologiaRESUMO
Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Pâncreas/citologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Curcumina/farmacologia , Heme Oxigenase-1/genética , Heme Oxigenase-1/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Modulation of the inflammatory stress response by anesthesia may be responsible for an increased susceptibility to infectious complications, such as wound infection or pneumonia. Sevoflurane, a specific inhibitor of activator protein-1, an immediate early transcription factor, induces apoptosis in T-cells. Because p38 can be involved either in pro- or antiapoptotic processes, we examined whether the sevoflurane-induced apoptosis is mediated by p38 activation in Jurkat T-cells. METHODS: Jurkat T-cells were exposed to different concentrations of sevoflurane, isoflurane, or desflurane in vitro. Phosphorylation of mitogen-activated protein (MAP) kinases, upstream kinases, downstream activating transcription factor 2 ATF-2, and caspase-3 processing were evaluated by Western blot. p38 kinase activity was evaluated after immunoprecipitation and phosphorylation of the substrate ATF-2 using Western blot. Apoptosis was assessed using flow cytometry after staining with green fluorescent protein-annexin V. RESULTS: While desflurane had no effect, sevoflurane and isoflurane induced p38 phosphorylation with sevoflurane inducing p38 kinase activity. Sevoflurane did not affect the MAP kinases ERK and JNK. Sevoflurane exposure also induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1), MAP kinase kinases 3 and 6 (MKK3/MKK6), and ATF-2. Pretreatment of cells with the general caspase inhibitor Z-VAD.fmk did not prevent the sevoflurane-induced phosphorylation of p38. Isoflurane- and sevoflurane-mediated caspase-3 processing and apoptosis could not be abolished by pretreatment with the specific p38 inhibitors SB202190 and SB203580. CONCLUSIONS: Sevoflurane is a specific activator of the apoptosis signal-regulating kinase-1-, MKK3/MKK6-p38 MAP kinase cascade in Jurkat T-cells. Our data suggest that sevoflurane-induced p38 activation is not affected by caspase activation. Furthermore, sevoflurane-induced apoptosis is not dependent on p38 MAP kinase activation.
Assuntos
Anestésicos Inalatórios/farmacologia , Ativação Enzimática/efeitos dos fármacos , Éteres Metílicos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Desflurano , Humanos , Inflamação/fisiopatologia , Isoflurano/análogos & derivados , Isoflurano/farmacologia , Células Jurkat , Proteínas de Neoplasias/metabolismo , SevofluranoRESUMO
Microcirculatory dysfunction, reduced oxygen supply and thus impaired hepatosplanchnic organ function, play a pivotal role in the clinical manifestation of sepsis and septic shock. Early correction of the perfusion mismatch is essential to maintain end-organ and gut mu-cosa barrier function and thus to prevent bacterial translocation, bacteraemia and possible multiple organ failure. This review will outline the clinical tightrope-walk optimizing hepa-tosplanchnic oxygen supply while maintaining adequate end-organ perfusion pressure and highlight the diagnostic challenges and limitations faced in clinical practice.
Assuntos
Infecções Bacterianas/terapia , Circulação Hepática , Oxigênio/uso terapêutico , Garantia da Qualidade dos Cuidados de Saúde/métodos , Choque Séptico/terapia , Circulação Esplâncnica , Vasculite/terapia , Infecções Bacterianas/fisiopatologia , Alemanha , Humanos , Insuficiência de Múltiplos Órgãos/prevenção & controle , Guias de Prática Clínica como Assunto , Padrões de Prática Médica , Choque Séptico/fisiopatologia , Vasculite/fisiopatologiaRESUMO
Treatment of hyperthyroidism by thionamides is associated with immunomodulatory effects, but the mechanism of thionamide-induced immunosuppression is unclear. Here we show that thionamides directly inhibit interleukin-2 cytokine expression, proliferation, and the activation (CD69 expression) of primary human T lymphocytes. Inhibition of immune function was associated with a repression of DNA binding of the cooperatively acting immunoregulatory transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT). Likewise, thionamides block the GTPase p21Ras, the mitogen-activated protein kinases, and impair the calcineurin/calmodulin-dependent NFAT dephosphorylation and nuclear translocation. The potency of inhibition correlated with the chemical reactivity of the thionamide-associated sulfur group. Taken together, our data demonstrate that thio-derivates with a common heterocyclic thioureylene-structure mediate a direct suppression of immune functions in T-cells via inhibition of the AP-1/NFAT pathway. Our observations may also explain the clinical and pathological resolution of some secondary, calcineurin, and mitogen-activated protein kinase-associated diseases upon thionamide treatment in hyperthyroid patients. This offers a new therapeutic basis for the development and application of heterocyclic thio-derivates.
Assuntos
Imunossupressores/farmacologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Tiobarbitúricos/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/fisiologia , Proliferação de Células/efeitos dos fármacos , Humanos , Imunossupressores/química , Células Jurkat , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Tiobarbitúricos/químicaRESUMO
The involvement of p38 in fundamental physiological processes and the fact that deregulation often leads to disease indicates the potential impact of p38 dependent mechanisms. Here we demonstrate a new pathway that includes the induction of the mitogen activated protein kinase p38 by protein kinase C and results in a specific phosphorylation of c-Jun in T-lymphocytes. P38 directly phosphorylates c-Jun within its transactivation domain at serine 63 and serine 73 and thus posttranscriptionally affects the presence of DNA-bound phosphorylated c-Jun, a prerequisite for activator protein 1 dependent gene transcription. Moreover, DNA-binding activity of c-Fos, FosB, and JunB were also dependent on the p38 protein kinase activity, whereas JunD, Fra-1 and Fra-2 were not affected. Although we show that stress induced mitogen activated protein kinases share c-Jun as a substrate for phosphorylation, p38 mediated effects could not be rescued by the c-Jun N-terminal kinases. This demonstrates that the protein kinase p38 plays a unique and non-redundant role in posttranslational c-Jun regulation. The induction of a p38 dependent c-Jun phosphorylation was comparable in both CD4(+) and CD8(+) T-cells, proposing a ubiquitous pathway that is not linked to T-cell subtype and effector function. In contrast, ATF-2 was predominantly phosphorylated in CD8(+) T-cells. Different cell lines show p38-dependent c-Jun phosphorylation upon phorbol ester induction but there is evidence that the simian virus 40 large T-antigen may interfere with this pathway.
Assuntos
Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anticorpos/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Dactinomicina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Serina/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Preconditioning with noble gases serves as an effective strategy to diminish tissue injury in different organs. The aim of this study was to investigate the influence of pretreatment with the nonanesthetic noble gas helium on hepatic injury after warm ischemia and reperfusion (IR) in comparison to ischemic preconditioning (IPC). Anesthetized and ventilated rats were randomized into six groups (n = 8/group): sham: after laparotomy, the portal triad was exposed without clamping; IPC was performed with 10 min of partial liver ischemia and 10 min of reperfusion; HePC: three cycles of 5 min with inhalation of helium 70 vol% and intermittent washout; IR: 45 min of ischemia followed by 240 min of reperfusion; IPC-IR: IPC followed by hepatic IR; HePC-IR: pretreatment with helium 70 vol% followed by hepatic IR. Hepatic injury was evaluated by measurement of serum enzymes aspartate aminotransferase and alanine aminotransferase. Hepatic mRNA expression and serum levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) were measured with real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Myeloperoxidase in liver tissue was assessed spectrophotometrically as a marker of neutrophil accumulation. mRNA levels of heme oxygenase 1 in liver tissue were assessed to investigate a protein of the most abundant protective system in the liver. Aspartate aminotransferase and alanine aminotransferase serum activities increased after hepatic IR (sham vs. IR; P < 0.05). The serum levels of liver enzymes after IR were significantly diminished with IPC (P < 0.05), whereas helium pretreatment had no effect. mRNA expression of TNF-α increased in all groups except IPC-IR compared with sham, whereas mRNA expression of IL-10 increased only after helium pretreatment. Serum levels of IL-10 were not affected by any intervention, whereas serum levels of TNF-α and liver myeloperoxidase were increased after IR, but not after HePC-IR. In conclusion, pretreatment with inhaled helium does not attenuate hepatic injury after warm IR of the liver, although there is evidence for a modulation of the inflammatory response.
Assuntos
Hélio/uso terapêutico , Precondicionamento Isquêmico , Fígado/lesões , Traumatismo por Reperfusão/prevenção & controle , Animais , Ensaio de Imunoadsorção Enzimática , Interleucina-10/sangue , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/sangue , Isquemia QuenteRESUMO
STUDY OBJECTIVE: To test the feasibility and efficacy of a new approach to paravertebral catheter placement in patients undergoing major surgery of the breast. DESIGN: Single-group, single-center observational study. SETTING: Operating room, postoperative recovery area, and normal ward of a university hospital. PATIENTS: 25 ASA physical status 1, 2, 3, and 4 patients undergoing major unilateral surgery of the breast. INTERVENTIONS: Paravertebral catheters for intraoperative and postoperative anesthesia and analgesia were applied using the recently described lamina technique. This technique is performed at a more medial puncture site, avoiding the pleura. MEASUREMENTS: Additional opioid requirements were recorded to assess effectiveness of regional anesthesia. At the time of catheter withdrawal, patients, staff nurses, and anesthesiologists who provided postoperative pain management were asked to rate their satisfaction with paravertebral catheter effectiveness. MAIN RESULTS: All patients successfully received a paravertebral catheter using the lamina technique. During the surgical procedure, 84% of patients received no additional opioids after intubation. No patient required opioids as rescue medication postoperatively (visual analog scale rating > 30 mm) or during the rest of the hospital stay. Postoperative analgesia provided with paravertebral catheters was rated very high by patients, staff nurses, and anesthesiologists involved in postoperative care. CONCLUSIONS: The lamina technique for placement of a paravertebral catheter is a feasible and effective technique for intraoperative and postoperative analgesia in patients scheduled for major breast surgery with or without axillary lymph node resection.
Assuntos
Raquianestesia/métodos , Mama/cirurgia , Coluna Vertebral , Adulto , Idoso , Idoso de 80 Anos ou mais , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Período de Recuperação da Anestesia , Anestesia Geral , Neoplasias da Mama/cirurgia , Cateterismo , Feminino , Humanos , Injeções Espinhais , Pessoa de Meia-Idade , Dor Pós-Operatória/tratamento farmacológico , Projetos Piloto , Complicações Pós-Operatórias/epidemiologiaRESUMO
Remote ischemic preconditioning (RIPC) and local ischemic preconditioning (IPC) protect the myocardium from subsequent ischemia/reperfusion (I/R) injury. In this study, the protective effects of early RIPC, IPC, and the combination of both (RIPC-IPC) were characterized. Furthermore, the hypothesis was tested that protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs), important mediators of IPC, are activated in RIPC. Infarct size, serum troponin T, and creatine kinase levels were assessed after 4 × 5-min noninvasive RIPC, local IPC, or a combination of both and 35 min of regional ischemia and 120 min of reperfusion. Protein kinase C ε and the MAPKs extracellular signal-regulated MAPK (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK were analyzed by Western blot analysis and activity assays in the myocardium and skeletal muscle immediately after the preconditioning protocol. Remote ischemic preconditioning, IPC, and RIPC-IPC significantly reduced myocardial infarct size (RIPC-I/R: 54% ± 15%; IPC-I/R: 33% ± 15%; RIPC-IPC-I/R: 33% ± 15%; P < 0.05 vs. I/R [76% ± 14%]) and troponin T release (RIPC-I/R: 15.4 ± 6.4 ng/mL; IPC-I/R: 10.9 ± 7.0 ng/mL; RIPC-IPC-I/R: 9.8 ± 5.6 ng/mL; P < 0.05 vs. I/R [27.1 ± 12.0 ng/mL]) after myocardial I/R. Ischemic preconditioning led to an activation of PKCε and ERK 1/2, whereas RIPC did not lead to a translocation of PKCε to the mitochondria or phosphorylation of the MAPKs ERK 1/2, JNK 1/2, and p38 MAPK. Remote ischemic preconditioning did not induce translocation of PKCε to the mitochondria or phosphorylation of MAPKs in the preconditioned muscle tissue. Remote ischemic preconditioning, IPC, and RIPC-IPC exert early protection against myocardial I/R injury. Remote ischemic preconditioning and local IPC exhibit different activation dynamics of signal transducers in the myocardium. The studied PKC-MAPK pathway is likely not involved in the protective effects of RIPC.
Assuntos
Precondicionamento Isquêmico , Infarto do Miocárdio/metabolismo , Animais , Western Blotting , Creatina Quinase/sangue , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hemodinâmica/efeitos dos fármacos , Precondicionamento Isquêmico Miocárdico , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Infarto do Miocárdio/sangue , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Troponina T/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVES: Microcirculatory derangements caused by ischemia and reperfusion (I/R) play a pivotal role in acute and graft pancreatitis. The inducible enzyme heme oxygenase 1 (HO-1) has been shown to decrease I/R injury by modulation of capillary perfusion in other organs. It was the aim of this study to evaluate the effect of HO-1 induction on pancreatic microcirculation after I/R. METHODS: Rats were randomized into 4 groups: (1) sham controls; (2) 1-hour ischemia and 2-hour reperfusion (I/R); (3) I/R + cobalt protoporphyrin (CoPP), an HO-1 inducer; and (4) I/R + CoPP + tin protoporphyrin, an HO inhibitor. Functional capillary density (FCD) and leukocyte endothelium interaction were analyzed using intravital microscopy during reperfusion. Expression of HO-1 mRNA, HO-1 protein, and HO activity were assessed by Northern blot, Western blot, and an HO activity assay. RESULTS: Functional capillary density decreased significantly in the I/R group as compared with sham controls. Cobalt protoporphyrin treatment increased FCD to control values. In contrast, HO inhibition in CoPP-pretreated animals lowered FCD and increased leukocyte endothelium interaction significantly. Cobalt protoporphyrin administration increased HO-1 mRNA, protein, and HO activity, whereas activity of the enzyme was reduced after injection of tin protoporphyrin. CONCLUSIONS: Heme oxygenase 1 plays a beneficial role in pancreatic microcirculatory derangements after I/R. This could be of therapeutic relevance after pancreas transplantation and other forms of postischemic pancreatitis.
Assuntos
Heme Oxigenase-1/metabolismo , Microcirculação/fisiologia , Pâncreas/irrigação sanguínea , Pancreatite/enzimologia , Traumatismo por Reperfusão/enzimologia , Animais , Capilares/enzimologia , Endotélio Vascular/fisiologia , Endotélio Vascular/fisiopatologia , Leucócitos/fisiologia , Masculino , Microcirculação/efeitos dos fármacos , Pancreatite/induzido quimicamente , Protoporfirinas/toxicidade , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Sprague-DawleyRESUMO
Nitric oxide plays a crucial role in the maintenance of liver function and integrity. During stress, the inducible heme oxygenase-1 protein and its reaction products, including carbon monoxide, also exert potent hepatoprotective effects. We investigated a potential relationship between endogenous nitric oxide synthesis and the hepatic regulation of heme oxygenase-1. Inhibition of nitric oxide synthesis in vivo by injection of l-NAME led to a dose-dependent induction of heme oxygenase-1 mRNA, protein and activity in the rat liver, whereas did not affect the expression of other heat shock proteins. The effect of l-NAME was demonstrated by hemodynamic changes within the liver circulation as measured by ultrasonic flow probes. Inhibition of nitric oxide synthase led to a decline in hepatic arterial and portal venous blood flow, and subsequently caused liver cell damage. In contrast, the combined administration of l-NAME and the nitric oxide-independent intestinal vasodilator dihydralazine completely restored portal venous flow, abolished the liver cell damage, and prevented the upregulation of heme oxygenase-1, despite inhibition of nitric oxide production. In conclusion, nitric oxide deficiency upregulates hepatic heme oxygenase-1, which is reversible by maintaining hepatic blood flow. This interdependence has important implications for the development of therapeutic strategies aimed at modulating the activity of these hepatoprotective mediator systems.
Assuntos
Eritrócitos/metabolismo , Heme Oxigenase-1/metabolismo , Fígado/enzimologia , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/efeitos dos fármacos , Fígado/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico/deficiência , Nitritos/sangue , Nitroarginina/farmacologia , Oxirredução , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. SUMMARY BACKGROUND DATA: Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. METHODS: Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. RESULTS: Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and alpha-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. CONCLUSIONS: This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.
Assuntos
Anestésicos/farmacologia , Heme Oxigenase-1/metabolismo , Isoflurano/farmacologia , Fígado/irrigação sanguínea , Fígado/enzimologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Análise de Variância , Animais , Velocidade do Fluxo Sanguíneo , Northern Blotting , Western Blotting , Técnicas Imunoenzimáticas , Masculino , Malondialdeído/metabolismo , Microcirculação , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Impaired hepatic perfusion after hemorrhagic shock frequently results in hepatocellular dysfunction associated with increased mortality. This study characterizes the effect of the vasodilators dihydralazine and urapidil on hepatocellular perfusion and integrity after hemorrhagic shock and resuscitation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University experimental laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: To register systemic and regional hepatic hemodynamics, rats (n=6 per group) were instrumented and randomly assigned to the following groups: shock+vehicle; shock+dihydralazine (1.5 mg/kg); or shock+urapidil (3 mg/kg). After 1 hr of hemorrhagic shock, animals were resuscitated for 5 hrs and mean arterial pressure was maintained at 70+/-5 mm Hg by administration of dihydralazine or urapidil. To evaluate hepatic heme oxygenase-1 expression and liver injury (determination of levels of alanine and aspartate aminotransferase [ALT, AST] and histology), an additional series of experiments with six animals per group was performed. At the end of each experiment, animals were killed and blood and liver tissue was obtained for subsequent analyses. MEASUREMENTS AND MAIN RESULTS: Dihydralazine increased cardiac output and portal and hepatic microvascular flow (p<.05) and reduced liver injury after shock (lower ALT and AST levels [p<.05]; improvement of histopathological changes). In contrast, urapidil had no effect on portal flow or liver injury. Hepatic heme oxygenase-1 mRNA expression was upregulated in animals subjected to hemorrhagic shock but did not differ among experimental groups. CONCLUSIONS: Dihydralazine increases nutritive portal and hepatic microvascular flow and limits liver injury after hemorrhagic shock. This protective effect appears to be the result of increased cardiac output and increased portal flow. These findings may offer a new strategy for hepatic protection after hemorrhagic shock.