RESUMO
Sepsis-induced cardiogenic shock in combination with severe acute respiratory failure represents a life-threatening combination that is often refractory to the conventional methods of treatment. We describe the case of a 33-year-old patient who developed acute cardiovascular collapse and ARDS secondary to superinfection of Panton-Valentine leukocidin-positive Staphylococcus aureus and H1N1 pneumonia who underwent successful combination therapy for severe sepsis-related cardiomyopathy and respiratory failure using extracorporeal membrane oxygenation and cytokine adsorption therapy.
Assuntos
Citocinas/isolamento & purificação , Oxigenação por Membrana Extracorpórea , Hemoperfusão , Influenza Humana/complicações , Pneumonia Estafilocócica/complicações , Sepse/terapia , Adsorção , Adulto , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Leucocidinas/metabolismo , Pneumonia Estafilocócica/virologia , Síndrome do Desconforto Respiratório/terapia , Síndrome do Desconforto Respiratório/virologia , Insuficiência Respiratória/etiologia , Sepse/virologia , Choque Cardiogênico/terapia , Choque Cardiogênico/virologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismoAssuntos
Lesão Pulmonar Aguda/fisiopatologia , Esofagectomia/efeitos adversos , Síndrome do Desconforto Respiratório/fisiopatologia , Lesão Pulmonar Aguda/etiologia , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/fisiopatologia , Pulmão/cirurgia , Síndrome do Desconforto Respiratório/etiologia , Fatores de RiscoRESUMO
BACKGROUND: Postoperative pulmonary complications (PPC) may result in longer duration of in-hospital stay and even mortality. Both thoracic surgery and intraoperative mechanical ventilation settings add considerably to the risk of PPC. It is unclear if one-lung ventilation (OLV) for thoracic surgery with a strategy of intraoperative high positive end-expiratory pressure (PEEP) and recruitment maneuvers (RM) reduces PPC, compared to low PEEP without RM. METHODS: PROTHOR is an international, multicenter, randomized, controlled, assessor-blinded, two-arm trial initiated by investigators of the PROtective VEntilation NETwork. In total, 2378 patients will be randomly assigned to one of two different intraoperative mechanical ventilation strategies. Investigators screen patients aged 18 years or older, scheduled for open thoracic or video-assisted thoracoscopic surgery under general anesthesia requiring OLV, with a maximal body mass index of 35 kg/m2, and a planned duration of surgery of more than 60 min. Further, the expected duration of OLV shall be longer than two-lung ventilation, and lung separation is planned with a double lumen tube. Patients will be randomly assigned to PEEP of 10 cmH2O with lung RM, or PEEP of 5 cmH2O without RM. During two-lung ventilation tidal volume is set at 7 mL/kg predicted body weight and, during OLV, it will be decreased to 5 mL/kg. The occurrence of PPC will be recorded as a collapsed composite of single adverse pulmonary events and represents the primary endpoint. DISCUSSION: PROTHOR is the first randomized controlled trial in patients undergoing thoracic surgery with OLV that is adequately powered to compare the effects of intraoperative high PEEP with RM versus low PEEP without RM on PPC. The results of the PROTHOR trial will support anesthesiologists in their decision to set intraoperative PEEP during protective ventilation for OLV in thoracic surgery. TRIAL REGISTRATION: The trial was registered in clinicaltrials.gov ( NCT02963025 ) on 15 November 2016.
Assuntos
Ventilação Monopulmonar/métodos , Respiração com Pressão Positiva/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Procedimentos Cirúrgicos Torácicos/métodos , Humanos , Complicações Intraoperatórias/terapia , Projetos de Pesquisa , Tamanho da AmostraRESUMO
After publication of the original article [1], the authors have notified us that two of the collaborator first and last names have been inverted in the "PROTHOR Investigators" table.
RESUMO
The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively. Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40%. In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells. The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter. STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels. Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells. Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake. In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake. The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment. Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side. The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected. It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E. coli.
Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Enterotoxinas/farmacologia , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Hormônios Gastrointestinais , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Peptídeos/farmacologia , Taurina/metabolismo , Alcaloides/farmacologia , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/genética , Células Cultivadas , Toxina da Cólera/farmacologia , AMP Cíclico/análise , GMP Cíclico/análogos & derivados , GMP Cíclico/análise , GMP Cíclico/farmacologia , Humanos , Glicoproteínas de Membrana/genética , Peptídeos Natriuréticos , RNA Mensageiro/análise , EstaurosporinaRESUMO
The inflammatory influence and biological markers of prolonged mechanical-ventilation in uninjured human lungs remains controversial. We investigated exhaled nitric oxide (NO) and carbon monoxide (CO) in mechanically-ventilated, brain-injured patients in the absence of lung injury or sepsis at two different levels of positive end-expiratory pressure (PEEP). Exhaled NO and CO were assessed in 27 patients, without lung injury or sepsis, who were ventilated with 8 ml kg(-1) tidal volumes under zero end-expiratory pressure (ZEEP group, n = 12) or 8 cm H2O PEEP (PEEP group, n = 15). Exhaled NO and CO was analysed on days 1, 3 and 5 of mechanical ventilation and correlated with previously reported markers of inflammation and gas exchange. Exhaled NO was higher on day 3 and 5 in both patient groups compared to day 1: (PEEP group: 5.8 (4.4-9.7) versus 11.7 (6.9-13.9) versus 10.7 (5.6-16.6) ppb (p < 0.05); ZEEP group: 5.3 (3.8-8.8) versus 9.8 (5.3-12.4) versus 9.6 (6.2-13.5) ppb NO peak levels for days 1, 3 and 5, respectively, p < 0.05). Exhaled CO remained stable on day 3 but significantly decreased by day 5 in the ZEEP group only (6.3 (4.3-9.0) versus 8.1 (5.8-12.1) ppm CO peak levels for day 5 versus 1, p < 0.05). The change scores for peak exhaled CO over day 1 and 5 showed significant correlations with arterial blood pH and plasma TNF levels (r s = 0.49, p = 0.02 and r s = -0.51 p = 0.02, respectively). Exhaled NO correlated with blood pH in the ZEEP group and with plasma levels of IL-6 in the PEEP group. We observed differential changes in exhaled NO and CO in mechanically-ventilated patients even in the absence of manifest lung injury or sepsis. These may suggest subtle pulmonary inflammation and support application of real time breath analysis for molecular monitoring in critically ill patients.
Assuntos
Lesões Encefálicas/fisiopatologia , Testes Respiratórios , Monóxido de Carbono/análise , Óxido Nítrico/análise , Respiração Artificial , Adolescente , Adulto , Idoso , Lesões Encefálicas/sangue , Lesões Encefálicas/terapia , Estado Terminal , Expiração , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Pneumonia/diagnóstico , Pneumonia/fisiopatologia , Respiração com Pressão Positiva , Volume de Ventilação Pulmonar , Adulto JovemAssuntos
Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Óxido Nítrico/metabolismo , Vasodilatadores/metabolismo , Adulto , Idoso , Área Sob a Curva , Feminino , Humanos , Inflamação , Transplante de Pulmão/métodos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/química , Isoformas de Proteínas , Testes de Função RespiratóriaRESUMO
Endothelium-derived relaxing factor and exogenous nitrovasodilators are thought to produce smooth muscle relaxation by activation of soluble guanylate cyclase. To investigate whether diminished cyclic GMP (cGMP) accumulation underlies the differences in vascular reactivity to nitrovasodilators between Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), we determined cGMP formation in aortic smooth muscle cells from the two strains. Both cultured cells and aortic rings from 12- to 14-week-old SHR accumulated greater amounts of cGMP on stimulation with exogenous nitrovasodilators (ie, sodium nitroprusside) than those from WKY rats, whereas there was no difference observed in cells from prehypertensive animals (5- to 6-week old) between the two strains. Responsiveness of smooth muscle cells to endothelium-derived relaxing factor was investigated in cocultures of bovine aortic endothelial cells (BAE) and smooth muscle cells from SHR and WKY rats. cGMP accumulation elicited by endothelium-derived relaxing factor released either basally or in response to bradykinin and the calcium ionophore A23187 was greater in smooth muscle from 12- to 14-week-old SHR than from age-matched WKY rats (80 +/- 17 versus 11 +/- 2 for basal; 152 +/- 12 versus 80 +/- 26 for A23187; 163 +/- 21 versus 40 +/- 12 pmol/mg protein per 15 minutes for bradykinin) in SHR/BAE and WKY/BAE cocultures, respectively. Northern blot analysis of steady-state messenger RNA levels for the beta 1 subunit of soluble guanylate cyclase revealed higher levels of the message in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hipertensão/fisiopatologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Sequência de Bases , Northern Blotting , Células Cultivadas , GMP Cíclico/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Guanilato Ciclase/biossíntese , Guanilato Ciclase/genética , Hipertensão/patologia , Masculino , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Óxido Nítrico/antagonistas & inibidores , Sondas de Oligonucleotídeos/química , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatação/fisiologia , ômega-N-MetilargininaRESUMO
Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 mumol/L isobutylmethyl-xanthine or 0.01 to 10 mumol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase-polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both alpha1- and beta1-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC alpha1 subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both.
Assuntos
AMP Cíclico/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Músculo Liso Vascular/enzimologia , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Cromatografia Líquida de Alta Pressão , GMP Cíclico/metabolismo , Immunoblotting , Membranas Intracelulares/metabolismo , Sondas Moleculares/genética , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Solubilidade , Transcrição GênicaRESUMO
We investigated the role of microtubules in the induction of nitric oxide synthase in cultured vascular smooth muscle cells. We found that like interleukin-1 alpha, lipopolysaccharide elicited a time and concentration-dependent accumulation of cyclic GMP via induction of nitric oxide synthase. Nocodazole and colchicine, two chemically distinct microtubule depolymerizing agents, completely prevented lipopolysaccharide- and interleukin-induced (and nitric oxide-mediated) cyclic GMP generation. In contrast to lipopolysaccharide and interleukin-1 alpha, cyclic GMP accumulation in response to sodium nitroprusside, an exogenous nitrovasodilator, was not altered by either nocodazole or colchicine. Our findings demonstrate that microtubule depolymerizing agents inhibit nitric oxide synthase induction and suggest a prominent role for microtubules in mediating the activation of the inducible nitric oxide pathway in smooth muscle cells.
Assuntos
Aminoácido Oxirredutases/biossíntese , Colchicina/farmacologia , Microtúbulos/metabolismo , Músculo Liso Vascular/enzimologia , Nocodazol/farmacologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , GMP Cíclico/biossíntese , Citoesqueleto/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/enzimologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase , Nitroprussiato/farmacologia , Ratos , Ratos Wistar , ômega-N-MetilargininaRESUMO
1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.
Assuntos
Citoesqueleto/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Western Blotting , Células Cultivadas , Colchicina/farmacologia , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/metabolismo , Citocalasinas/farmacologia , Citoesqueleto/enzimologia , Indução Enzimática/efeitos dos fármacos , Supressores da Gota/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Nocodazol/farmacologia , Paclitaxel/farmacologia , RNA/análise , RNA/isolamento & purificação , Ratos , Ratos WistarRESUMO
1. The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC). 2. As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (N(G)-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h. 3. Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 microM and inhibition at higher concentrations. 4. Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells. 5. CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor. 6. RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone. 7. These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins.
Assuntos
Anisomicina/farmacologia , Aorta/efeitos dos fármacos , Cicloeximida/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase/biossíntese , Inibidores da Síntese de Proteínas/farmacologia , Animais , Aorta/citologia , Aorta/enzimologia , Células Cultivadas , GMP Cíclico/metabolismo , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos WistarRESUMO
1. The current study explored potential redox mechanisms of nitric oxide (NO)-induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells. 2. Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration-dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO. 3. Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO. 4. Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation. 5. Study of extracellula interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release. 6. Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells. 7. These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors.
Assuntos
DNA/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Acetilcisteína/farmacologia , Animais , Ácido Ascórbico/farmacologia , Células Cultivadas , DNA/biossíntese , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/farmacologia , Fase G1 , Glutationa/análogos & derivados , Glutationa/farmacologia , Humanos , Hidroxiureia/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Oxirredução , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Ratos , Fase S , S-NitrosoglutationaRESUMO
1. Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO-induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin-1 beta (IL-1 beta). 2. Exposure of rat aortic smooth muscle cells to SNP (10 microM) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30-70% in IL-1 beta- or LPS-pretreated cells, in a pretreatment time-and IL-1 beta- or LPS-concentration-dependent manner. When, however, cells were exposed to IL-1 beta or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3. Pretreatment of rats with LPS (5 mg kg-1, i.v.) for 6 h led to a decrease in aortic ring SNP-induced cyclic GMP accumulation. 4. The IL-1 beta-induced reduction in SNP-stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5. Reverse-transcriptase-polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the alpha, subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS- or IL-1 beta-treated cells, as compared to vehicle-treated cells. 6. Protein levels of the alpha 1 sGC subunit remained unaltered upon exposure to LPS or IL-1 beta, suggesting that the early decreased cyclic GMP accumulation in IL-1 beta- or LPS-pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.
Assuntos
GMP Cíclico/biossíntese , Endotoxinas/farmacologia , Interleucina-1/farmacologia , Nitroprussiato/farmacologia , Vasodilatadores/antagonistas & inibidores , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Immunoblotting , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Reação em Cadeia da Polimerase , Radioimunoensaio , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Vasodilatadores/farmacologiaRESUMO
Lipopolysaccharide (LPS)-induced hypotension and impaired aortic contraction to norepinephrine (NE) are thought to be consequent to induction of nitric oxide synthase (iNOS). Anesthesia is often employed in studies of the mechanisms mediating LPS-induced cardiovascular dysfunction in rats. Since sympathetic nervous system activity and compensatory mechanisms can be altered by anesthesia, this study was designed to determine a) if the cardiovascular dysfunction associated with LPS (5 mg/kg, i.v.)-induced endotoxin shock is enhanced in anesthetized compared with conscious male Wistar rats, and b) the potential role of iNOS in these responses to LPS. Arterial pressure and heart rate were continuously measured via a femoral arterial cannula. Six hours after LPS, conscious rats had a stable mean arterial pressure (MAP) and were tachycardic, while anesthetized rats showed a significant decrease in MAP without tachycardia. Small mesenteric arterioles (200-300 microns) were isolated, and the endothelium was removed six h after LPS. Intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mmHg. Norepinephrine-induced contraction and oscillations/min were impaired to a greater extent in arterioles from LPS-treated anesthetized rats than in those from conscious rats. Calcium-dependent and -independent nitric oxide formation, reflected as cGMP accumulation, were also determined in aortic rings treated with a chelator of Ca2+, EGTA, or the inhibitor of nitric oxide synthase activity, L-NAME. In rings from saline-treated conscious and anesthetized rats, cGMP accumulation was significantly reduced by EGTA and L-NAME, indicating calcium-dependent constitutive (cNOS) activity. However, in aortic rings from LPS-treated conscious and anesthetized rats, cGMP accumulation was not affected by EGTA and was significantly greater in rings from anesthetized vs. conscious rats. These results suggest that cardiovascular dysfunction is more prominent in LPS-treated anesthetized vs. conscious rats. This effect may be related to increased induction of iNOS in the presence of anesthesia.
Assuntos
Adjuvantes Anestésicos/efeitos adversos , Anestesia/efeitos adversos , Sistema Cardiovascular/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Pentobarbital/efeitos adversos , Anestesia/métodos , Animais , Antídotos/farmacologia , Arteríolas/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotoxemia/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Hemodinâmica/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Norepinefrina/farmacologia , Ratos , Ratos Wistar , Resistência Vascular/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
It is still unclear whether cross-tolerance develops between endogenously produced nitric oxide and exogenous nitric oxide donors. Thus, cGMP accumulation was determined in cultured aortic smooth muscle cells exposed to a nitric oxide source. Exposure of human, rat, rabbit, porcine or bovine smooth muscle cells to sodium nitroprusside led to a time- and concentration-dependent development of tolerance. In rat aortic smooth muscle cells, cross-tolerance developed between the sodium nitroprusside and S-nitroso-N-acetylpenicillamine, but not between sodium nitroprusside and atriopeptin. In addition, when rat aortic smooth muscle cells were treated with endotoxin or interleukin-1beta, they displayed lower sodium nitroprusside-induced cGMP accumulation as compared to control cells. When rat aortic smooth muscle cells were exposed to sodium nitroprusside for 12 h they displayed a decreased ability to accumulate cGMP in response to endothelium-derived nitric oxide released from bovine aortic endothelial cells. In addition, co-cultures of rat aortic smooth muscle cells with bovine aortic endothelial cells showed an L-nitroarginine methylester-sensitive decrease in sodium nitroprusside-induced cGMP accumulation compared to single rat aortic smooth muscle cell cultures. We conclude that cross-tolerance between endothelium-derived nitric oxide and exogenously applied nitric oxide donors occurs in vitro.
Assuntos
GMP Cíclico/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Penicilamina/análogos & derivados , Animais , Aorta/efeitos dos fármacos , Bovinos , Células Cultivadas , Tolerância a Medicamentos , Músculo Liso Vascular/metabolismo , Penicilamina/farmacologia , Coelhos , Ratos , SuínosRESUMO
Taurine uptake into cultures of human retinal pigment epithelial (HRPE) cells was monitored for 7 days after seeding. A culture medium containing 16% fetal bovine serum (FBS) was used for 2 days and switched to one with 8% FBS. Uptake of taurine (25 nM) was approximately 1.5 pmol/mg protein/15 min for 3 days, then decreased by 45% and was maintained at a decreased level till the 7th day. When the 16% FBS medium was used for the entire culture period, a similar profile of taurine uptake was observed but decrease of the uptake started on the 3rd day. Treatment of cells with 100 ng/ml cholera toxin (CT) for 24 h between the 6th and 7th days returned taurine uptake to its high level observed at the beginning of the cell culture. A similar CT treatment of cells between the 2nd and 3rd days enhanced taurine uptake significantly but this enhancement was much smaller. CT increased taurine uptake in treatment-time and dose dependent manners. Forskolin (FSK) (10 mM) and 8-Bromocyclic adenosine 3',5'-monophosphate (1 mM) also increased taurine uptake. KT5720 at 1 microM, a selective inhibitor of cAMP-dependent protein kinase (PKA), partially blocked CT-induced enhancement of taurine uptake. The level of cAMP was higher on the 3rd day than the 7th day but its response to 3-isobutyl-1-methyl-xanthine, FSK and CT was similar on both days. A kinetic analysis revealed that CT treatment decreases the apparent Michaelis-Menten constant of the taurine transporter while the drastic reduction of taurine uptake during the cell culture period is due to a decrease in the maximal velocity. The results show that cAMP elevated by CT treatment enhances taurine uptake via an increase in the affinity of the transporter. The decrease of taurine uptake during the culture period seems to be related to a decrease in the amount of the transporter.
Assuntos
Carbazóis , Toxina da Cólera/farmacologia , Proteínas de Membrana Transportadoras , Epitélio Pigmentado Ocular/metabolismo , Taurina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Cinética , Glicoproteínas de Membrana/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Pirróis/farmacologiaRESUMO
Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H(2)O(2) (group 2), I/R (group 3), I/R and EA (group 4), H(2)O(2) coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H(2)O(2) treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H(2)O(2). GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury.
RESUMO
The effects of hydrogen peroxide (H2O2) on the action of basally produced endothelium-derived relaxing factor (EDRF) were investigated by measuring cGMP accumulation in single and cocultures of calf pulmonary artery endothelial cells (CPAEs) and rabbit pulmonary artery smooth muscle cells (RPASMs) as a model for determining the contribution of EDRF dysfunction to altered vascular tone and reactivity frequently associated with oxidant-induced vascular injury. Higher cGMP levels in long-term cocultures (20.4 +/- 1.8 pmol/mg protein/15 min) than in single-cell cultures (CPAE, 9.6 +/- 0.9 pmol/mg protein/15 min; RPASM, 3.7 +/- 0.2 pmol/mg protein/15 min), and CPAE-induced increases (fivefold) in intracellular RPASM cGMP content in short-term cocultures suggest basal release of EDRF. Basal generation and release of an L-arginine-derived endothelial labile factor accounted for the increases in cGMP, since the response was completely blocked by pretreatment of CPAEs with NG-monomethyl L-arginine. Pretreatment of long-term cocultures with H2O2 for 30 minutes resulted in a dose-dependent (0.5-2 mM) decrease in cGMP formation (49-79%). To determine the effects of H2O2 on EDRF synthesis, transport, and RPASM responsiveness, CPAEs or RPASMs were selectively pretreated with H2O2 before establishment of short-term cocultures. In cocultures of H2O2-pretreated CPAEs with untreated RPASMs, RPASM cGMP levels were reduced, suggesting a decrease in EDRF production rather than deterioration of EDRF during transport, because cGMP levels were unaffected by posttreatment with oxygen radical scavengers during coculture. Pretreatment of RPASMs with H2O2 attenuated the untreated CPAE-induced, the putative EDRF S-nitroso-L-cysteine-induced, or the nitroprusside-induced increases in RPASM cGMP levels. This attenuation was prevented by pretreatment with either dimethylthiourea, deferoxamine, or dithiothreitol, suggesting a mechanism of H2O2 action involving iron-catalyzed formation of intracellular hydroxyl radicals and their attack on cellular thiols. H2O2 diminution of cGMP accumulation was not associated with lytic cell injury in the experimental time frame, because morphology and 51Cr release from prelabeled RPASMs and CPAEs were unchanged.