RESUMO
Besides the health benefits of regular exercise, high-level training-above an optimal level-may have adverse effects. In this study, we investigated the effects of long-term vigorous training and its potentially detrimental structural-functional changes in a small animal athlete's heart model. Thirty-eight 4-month-old male guinea pigs were randomized into sedentary and exercised groups. The latter underwent a 15-week-long endurance-training program. To investigate the effects of the intense long-term exercise, in vivo (echocardiography, electrocardiography), ex vivo, and in vitro (histopathology, patch-clamp) measurements were performed. Following the training protocol, the exercised animals exhibited structural left ventricular enlargement and a significantly higher degree of myocardial fibrosis. Furthermore, resting bradycardia accompanied by elevated heart rate variability occurred, representing increased parasympathetic activity in the exercised hearts. The observed prolonged QTc intervals and increased repolarization variability parameters may raise the risk of electrical instability in exercised animals. Complex arrhythmias did not occur in either group, and there were no differences between the groups in ex vivo or cellular electrophysiological experiments. Accordingly, the high parasympathetic activity may promote impaired repolarization in conscious exercised animals. The detected structural-functional changes share similarities with the human athlete's heart; therefore, this model might be useful for investigations on cardiac remodeling.
Assuntos
Cardiomegalia Induzida por Exercícios , Treino Aeróbico , Animais , Cobaias , Masculino , Eletrocardiografia , Coração , Resistência Física , Remodelação VentricularRESUMO
BACKGROUND: Activation of nicotinic receptors with nicotine has been shown to reduce post-surgical pain in clinical and preclinical studies. Choline is a selective agonist at α7-type nicotinic receptors that does not have addictive or sympathetic activating properties. It is anti-nociceptive in animal studies. We conducted a double-blind randomized trial of oral choline supplementation with lecithin to aid in the treatment of pain after gynaecological surgery. METHODS: Sixty women having open gynaecological surgery were randomly assigned to receive 20 g of lecithin before surgery or placebo. Plasma choline concentration and tumour necrosis factor (TNF) were measured. Pain report was the primary outcome measure. RESULTS: We achieved a small but statistically significant increase in choline after surgery with oral supplementation. Plasma TNF was not decreased and pain report was not different between groups at rest or with movement. There were no adverse effects of treatment. CONCLUSIONS: Oral supplementation with lecithin during the perioperative period resulted in very slow absorption and thus only a small increase in plasma choline was achieved. This concentration was inadequate to reduce TNF as has been shown in other studies. The absence of an anti-inflammatory effect was likely related to our failure to demonstrate efficacy in pain reduction.
Assuntos
Colina/uso terapêutico , Suplementos Nutricionais , Procedimentos Cirúrgicos em Ginecologia , Lipotrópicos/uso terapêutico , Dor Pós-Operatória/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Colina/sangue , Método Duplo-Cego , Feminino , Humanos , Lecitinas/administração & dosagem , Lipotrópicos/sangue , Pessoa de Meia-Idade , Medição da Dor/métodos , Projetos Piloto , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Adulto JovemRESUMO
Most computational models of abdominal aortic aneurysms address either the hemodynamics within the lesion or the mechanics of the wall. More recently, however, some models have appropriately begun to account for the evolving mechanics of the wall in response to the changing hemodynamic loads. Collectively, this large body of work has provided tremendous insight into this life-threatening condition and has provided important guidance for current research. Nevertheless, there has yet to be a comprehensive model that addresses the mechanobiology, biochemistry, and biomechanics of thrombus-laden abdominal aortic aneurysms. That is, there is a pressing need to include effects of the hemodynamics on both the development of the nearly ubiquitous intraluminal thrombus and the evolving mechanics of the wall, which depends in part on biochemical effects of the adjacent thrombus. Indeed, there is increasing evidence that intraluminal thrombus in abdominal aortic aneurysms is biologically active and should not be treated as homogeneous inert material. In this review paper, we bring together diverse findings from the literature to encourage next generation models that account for the biochemomechanics of growth and remodeling in patient-specific, thrombus-laden abdominal aortic aneurysms.
Assuntos
Aneurisma da Aorta Abdominal/complicações , Fenômenos Mecânicos , Trombose/complicações , Animais , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/fisiopatologia , Fenômenos Biomecânicos , Humanos , Modelos BiológicosRESUMO
We present a rare case of combined fungal infection in a critically ill 47 year-old patient with chronic hepatitis C at the stage of liver cirrhosis. The patient was admitted for signs of decompensated cirrhosis caused by hepatitis C and increased alcohol consumption. After 2 week hospital stay, his condition was complicated by a pulmonary infiltrate and rapid deterioration followed. Despite intensive care, the patient died. Autopsy findings showed invasive pulmonary aspergillosis. The aim of this case report is to point to a broad differential diagnosis of jaundice and pulmonary infiltrates, thus stressing the value of interdisciplinary cooperation and the need to consider the possibility of invasive fungal infections when caring for liver cirrhosis patients. In addition, several risk factors contributing to the development of fungal diseases in these patients are discussed in the article.
Assuntos
Candidíase/complicações , Doenças do Esôfago/complicações , Hepatite C Crônica/complicações , Aspergilose Pulmonar Invasiva/complicações , Cirrose Hepática Alcoólica/complicações , Infecções Oportunistas/complicações , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/fisiopatologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Diabetes Mellitus Experimental/enzimologia , Endotélio Vascular/enzimologia , Ativação Enzimática , Glucose/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , NF-kappa B/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0151461.].
Assuntos
Asma/imunologia , Asma/fisiopatologia , Poli(ADP-Ribose) Polimerases/metabolismo , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/fisiopatologia , Alérgenos/imunologia , Animais , Asma/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Interleucina-5/genética , Interleucina-5/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerase-1 , Hipersensibilidade Respiratória/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
Beat-to-beat variability in repolarization (BVR) has been proposed as an arrhythmic risk marker for disease and pharmacological action. The mechanisms are unclear but BVR is thought to be a cell level manifestation of ion channel stochasticity, modulated by cell-to-cell differences in ionic conductances. In this study, we describe the construction of an experimentally-calibrated set of stochastic cardiac cell models that captures both BVR and cell-to-cell differences in BVR displayed in isolated canine action potential measurements using pharmacological agents. Simulated and experimental ranges of BVR are compared in control and under pharmacological inhibition, and the key ionic currents determining BVR under physiological and pharmacological conditions are identified. Results show that the 4-aminopyridine-sensitive transient outward potassium current, Ito1, is a fundamental driver of BVR in control and upon complete inhibition of the slow delayed rectifier potassium current, IKs. In contrast, IKs and the L-type calcium current, ICaL, become the major contributors to BVR upon inhibition of the fast delayed rectifier potassium current, IKr. This highlights both IKs and Ito1 as key contributors to repolarization reserve. Partial correlation analysis identifies the distribution of Ito1 channel numbers as an important independent determinant of the magnitude of BVR and drug-induced change in BVR in control and under pharmacological inhibition of ionic currents. Distributions in the number of IKs and ICaL channels only become independent determinants of the magnitude of BVR upon complete inhibition of IKr. These findings provide quantitative insights into the ionic causes of BVR as a marker for repolarization reserve, both under control condition and pharmacological inhibition.
Assuntos
Potenciais de Ação/fisiologia , Biologia Computacional/métodos , Ativação do Canal Iônico/fisiologia , Miócitos Cardíacos/fisiologia , Canais de Potássio/fisiologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Algoritmos , Animais , Canais de Cálcio Tipo L/fisiologia , Células Cultivadas , Simulação por Computador , Cães , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Processos EstocásticosRESUMO
Peroxynitrite and hydroxyl radicals are potent initiators of DNA single-strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). In response to high glucose incubation medium in vitro, or diabetes and hyperglycemia in vivo, reactive nitrogen and oxygen species generation occurs. These reactive species trigger DNA single-strand breakage, which induces rapid activation of PARP. PARP in turn depletes the intracellular concentration of its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation. This process results in acute endothelial dysfunction in diabetic blood vessels. Accordingly, inhibitors of PARP protect against endothelial injury under these conditions. In addition to the direct cytotoxic pathway regulated by DNA injury and PARP activation, PARP also appears to modulate the course of inflammation by regulating the activation of nuclear factor kappaB, and the expression of a number of genes, including the gene for intercellular adhesion molecule 1 and the inducible nitric oxide synthase. The research into the role of PARP in diabetic vascular injury is now supported by novel tools, such as new classes of potent inhibitors of PARP and genetically engineered animals lacking the gene for PARP. Pharmacological inhibition of PARP emerges as a potential approach for the experimental therapy of diabetic vascular dysfunction.
Assuntos
Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Endotélio Vascular/fisiopatologia , Nitrogênio/metabolismo , Oxigênio/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Diabetes Mellitus/enzimologia , Diabetes Mellitus/patologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Radicais Livres/metabolismo , Humanos , Inflamação/metabolismo , Óxido Nítrico/metabolismo , Transdução de SinaisRESUMO
The cellular electrophysiological effects of 1 microM tedisamil (KC 8857) were studied in human atrial and ventricular fibers. Conventional microelectrode technique was applied to record the transmembrane action potentials at stimulation frequency of 100 per min and 37 degrees C. Tedisamil lengthened action potential duration (APD) more in atrial than in ventricular muscle fibers; prolongation of APD90 was 28.9 +/- 3.3% (n = 6; p < 0.05) for atrial and 13.3 +/- 5.2% (n = 6; P < 0.05) for ventricular tissue. The maximal rate of depolarization was depressed slightly, but significantly by 1 microM tedisamil only in ventricular fibers (12.9 +/- 6.5%, n = 6, P < 0.05). From these cellular electrophysiological data it is concluded that the bradycardic/antiischemic agent tedisamil possesses marked Class III properties not only in cardiac tissues of experimental animals but also those of man.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ciclopropanos/farmacologia , Coração/efeitos dos fármacos , Adolescente , Adulto , Criança , Pré-Escolar , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C. RESULTS: An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8). CONCLUSIONS: In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.
Assuntos
Miocárdio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Adulto , Antiarrítmicos/farmacologia , Compostos de Bário/farmacologia , Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cloretos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/citologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Piridinas/farmacologia , Processamento de Sinais Assistido por Computador , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
OBJECTIVE: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)). RESULTS: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier. CONCLUSION: In undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.
Assuntos
Benzodiazepinas/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Miocárdio/metabolismo , Canais de Potássio/efeitos dos fármacos , Adulto , Bloqueadores dos Canais de Cálcio/farmacologia , Separação Celular/métodos , Cromanos/farmacologia , Colforsina/farmacologia , Feminino , Humanos , Síndrome do QT Longo/metabolismo , Masculino , Nisoldipino/farmacologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Canais de Potássio/metabolismo , Piridinas/farmacologia , Sulfonamidas/farmacologiaRESUMO
Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.
Assuntos
Dano ao DNA/fisiologia , Dermatite de Contato/metabolismo , Nitratos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Adjuvantes Imunológicos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Fragmentação do DNA/fisiologia , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Feminino , Marcação In Situ das Extremidades Cortadas , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-8/imunologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos , Necrose , Oxazolona , Pele/imunologia , Pele/metabolismo , Pele/patologia , Tirosina/metabolismoRESUMO
Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naive mice (n = 7 +/+, n = 6 -/-) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naive PARP +/+ and -/- mice. Both groups showed nitrotyrosine staining in the contusion, suggest ing that peroxynitrite is produced in contused brain. In protoco 2, mice (PARP +/+, n = 8; PARP -/-, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP -/- mice demonstrated improved motor and memory function after CC versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detri mental effect of PARP on functional outcome after TBI.
Assuntos
Lesões Encefálicas/fisiopatologia , Lesões Encefálicas/psicologia , Encéfalo/fisiopatologia , Cognição/fisiologia , Aprendizagem em Labirinto , Memória/fisiologia , Atividade Motora/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Lesões Encefálicas/genética , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Sinais (Psicologia) , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/deficiência , Percepção EspacialRESUMO
In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.
Assuntos
Apoptose/efeitos dos fármacos , Nitratos/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Apoptose/fisiologia , Caspases/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Genes bcl-2 , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/genética , Linfócitos T/citologia , Transglutaminases/metabolismoRESUMO
Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3-4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 microM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 microM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Células HL-60/metabolismo , Nitratos/farmacologia , Caspase 3 , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosfatidilserinas/metabolismo , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
Nitroxyl (NO(-)/HNO), has been proposed to be one of the NO(*)-derived cytotoxic species. Although the biological effect of nitroxyl is largely unknown, it has been reported to cause DNA breakage and cytotoxicity. We have therefore investigated whether NO(-)/HNO-induced DNA single-strand breakage activates the nuclear nick sensor enzyme poly(ADP-ribose) polymerase (PARP) and whether PARP activation affects the mode of NO(-)/HNO- induced cell death. NO(-)/HNO generated from Angeli's salt (AS, sodium trioxodinitrate) (0-300 microM) induced DNA single-strand breakage, PARP activation, and a concentration-dependent cytotoxicity in murine thymocytes. AS-induced cell death was also accompanied by decreased mitochondrial membrane potential and increased secondary superoxide production. The cytotoxicity of AS, as measured by propidium iodide uptake, was abolished by electron acceptors potassium ferricyanide, TEMPOL, the intracellular calcium chelator BAPTA-AM, and by PARP inhibitors 3-aminobenzamide (3-AB) and PJ-34. The cytoprotective effect of 3-AB was paralleled by increased output of AS-induced apoptotic parameters such as phosphatidylserine exposure, caspase activation, and DNA fragmentation. No significant increase in tyrosine nitration could be observed in AS-treated thymocytes as opposed to peroxynitrite-treated cells, indicating that tyrosine nitration is not likely to contribute to NO(-)/HNO-induced cytotoxicity. Our results demonstrate that NO(-)/HNO-induced PARP activation shifts the default apoptotic cell death toward necrosis in thymocytes. However, as total PARP inhibition resulted only in 30% cytoprotection, PARP-independent mechanisms dominate NO(-)/HNO-induced cytotoxicity in thymocytes.
Assuntos
Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Óxidos de Nitrogênio/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Substâncias Protetoras/farmacologia , Timo/efeitos dos fármacos , Animais , Apoptose , Caspases/metabolismo , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitratos , Nitritos/toxicidade , Poli(ADP-Ribose) Polimerases/metabolismo , Timo/citologia , TirosinaRESUMO
The cellular mechanism of action of tedisamil (KC-8857) (TED), a novel antiarrhythmic/antifibrillatory compound, was studied on transmembrane currents in guinea pig, rabbit and dog ventricular myocytes by applying the patch-clamp and the conventional microelectrode technique. In guinea pig myocytes the rapid component of the delayed rectifier potassium current (IKr) was largely diminished by 1 microM TED (from 0.88+/-0.17 to 0.23+/-0.07 pA/pF, n=5, p<0.05), while its slow component (IKs) was reduced only by 5 microM TED (from 8.1+/-0.3 to 4.23+/-0.07 pA/pF, n=5, p<0.05). TED did not significantly change the IKr and IKs kinetics. In rabbit myocytes 1 microM TED decreased the amplitude of the transient outward current (I(to)) from 20.3+/-4.9 to 13.9+/-2.8 pA/pF (n=5, p<0.05), accelerated its fast inactivation time constant from 8.3+/-0.6 to 3.5+/-0.5 ms (n=5, p<0.05) and reduced the ATP-activated potassium current (IKATP) from 38.2+/-11.8 to 18.4+/-4.7 pA/pF (activator: 50 microM cromakalim; n=5, p<0.05). In dog myocytes 2 microM TED blocked the fast sodium current (INa) with rapid onset and moderately slow offset kinetics, while the inward rectifier potassium (IK1), the inward calcium (ICa) and even the I(to) currents were not affected by TED in concentration as high as 10 microM. The differences in I(to) responsiveness between dog and rabbit are probably due to the different alpha-subunits of I(to) in these species. It is concluded that inhibition of several transmembrane currents, including IKr, IKs, I(to), IKATP and even INa, can contribute to the high antiarrhythmic/antifibrillatory potency of TED, underlying predominant Class III combined with I A/B type antiarrhythmic characteristics.
Assuntos
Antiarrítmicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ciclopropanos/farmacologia , Canais Iônicos/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antiarrítmicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Ciclopropanos/química , Cães , Relação Dose-Resposta a Droga , Cobaias , Ventrículos do Coração/efeitos dos fármacos , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Coelhos , Função VentricularRESUMO
The cellular electrophysiologic effect of GYKI 16638, a new antiarrhythmic compound was studied and compared with that of sotalol and mexiletine in undiseased human right ventricular muscle preparation by applying the conventional microelectrode technique. GYKI 16638 (5 microM), at stimulation cycle length of 1000 ms, lengthened action potential duration (APD(90)) from 338.9 +/- 28.6 ms to 385.4 +/- 24 ms (n = 9, p > 0.05). This APD lengthening effect, unlike that of sotalol (30 microM), was rate-independent. GYKI 16638, contrary to sotalol and like mexiletine (10 microM), exerted a use-dependent depression of the maximal rate of depolarization (V(max)) which amounted to 36.4 +/- 11.7% at cycle length of 400 ms (n = 5, p < 0.05) and was characterised with an offset kinetical time constant of 298.6 +/- 70.2 ms. It was concluded that GYKI 16638 in human ventricular muscle shows combined Class IB and Class III antiarrhythmic properties, resembling the electrophysiological manifestation seen after chronic amiodarone treatment.
Assuntos
Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Coração/efeitos dos fármacos , Mexiletina/farmacologia , Fenetilaminas/farmacologia , Sotalol/farmacologia , Sulfonamidas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Adulto , Estimulação Elétrica , Eletrofisiologia , Feminino , Coração/fisiologia , Humanos , Técnicas In Vitro , Cinética , Masculino , Músculos Papilares/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacosRESUMO
A fluorimetric method using 4-methylumbelliferyl heptanoate (MUH) has been developed for detecting cell-mediated cytotoxicity and cell proliferation. The assay is based on the hydrolysis of the fluorochrome (MUH) by intracellular esterases of viable cells resulting in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter. Because of a similarity to the principle of the widely used colorimetric MTT assay, a comparison was made between the two assays when measuring cell proliferation and LAK cell cytotoxicity to different target cell types. The results have shown that the MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is completely comparable to the MTT method. The rapidity of the new cytotoxicity assay, 5 h in contrast to 9 h for the MTT assay, its applicability to both adherently and nonadherently growing target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious contender for replacing conventional radioactive techniques.