RESUMO
Ultrasound, a non-invasive therapy method, is a potential tool for medical applications, but its biological effects on vascular smooth muscle cells (VSMCs) have not been characterized. The aim of this study was to explore the effect and possible apoptotic mechanism of VSMCs that were induced by low-frequency ultrasound (LFU). Cell viability and apoptosis of A7r5 cells were evaluated after treating A7r5 cells with a continuous 45-kHz 1.0-W/cm(2) ultrasound (exposure time of 0, 10, 20, 30, and 35 s) by MTT assay and flow cytometry. At the optimum ultrasound exposure condition (30 s), gene chip analysis was performed, and the apoptotic signaling pathway was confirmed by reverse transcription-polymerase chain reaction and Western blot. As measured by flow cytometry, LFU significantly induced A7r5 cell apoptosis. Comparing the ultrasound group with the control group, the protein expression of caspase-9 and caspase-3 was increased by 50 and 57%, respectively; the caspase-3 mRNA level was increased by 37.5%. These findings indicate that an intrinsic pathway plays a major role in apoptosis that is induced by LFU and that LFU can induce A7r5 cell apoptosis via caspase-9- and caspase-3-dependent pathways.
Assuntos
Aorta/metabolismo , Apoptose/efeitos da radiação , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/citologia , Aorta/efeitos da radiação , Caspase 3/biossíntese , Caspase 9/biossíntese , Linhagem Celular , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Miócitos de Músculo Liso/efeitos da radiação , RNA Mensageiro/biossíntese , Ratos , SomRESUMO
PURPOSE: γ-rays (IR) cause an increase in intracellular calcium [Ca(2+)], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis. MATERIALS AND METHODS: Intestinal smooth muscle cells were exposed to 10-50 Gy from a (60)Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence. RESULTS: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E. CONCLUSION: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.
Assuntos
Apoptose/fisiologia , Sinalização do Cálcio/fisiologia , Mitocôndrias Musculares/fisiologia , Contração Muscular/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos da radiação , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta à Radiação , Raios gama , Cobaias , Mitocôndrias Musculares/efeitos da radiação , Contração Muscular/efeitos da radiação , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/efeitos da radiação , Doses de RadiaçãoRESUMO
The objective of this study was to investigate whether low level laser therapy (LLLT) could reduce bronchial hyper-responsiveness (BHR) induced by tumour necrosis factor-alpha (TNF-alpha) modulating the metabolism of inositol phosphate (IP) in bronchial smooth muscle cells (BSMCs). The study was on 28 Wistar rats, randomly divided into four groups. Irradiation (1.3 J/cm(2)) was administered 5 min and 4 h after bronchial smooth muscle (BSM) had been suspended in TNF-alpha baths, and the contractile response-induced calcium ion (Ca(2+)) sensitization was measured. The BSMCs were isolated, and the IP accumulation was measured before and after TNF-alpha immersion in the groups that had been irradiated or not irradiated. BSM segments significantly increased contraction 24 h after TNF-alpha immersion when exposed to carbachol (CCh) as Ca(2+), but it was significantly reduced by 64% and 30%, respectively, after laser treatment. The increase in IP accumulation induced by CCh after TNF-alpha immersion was reduced in the BSMCs by LLLT. The dose of 2.6 J/cm(2) reduced BHR and IP accumulation in the rats' inflammatory BSMCs.
Assuntos
Hiper-Reatividade Brônquica/radioterapia , Terapia com Luz de Baixa Intensidade , Animais , Sequência de Bases , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/fisiopatologia , Cálcio/metabolismo , Carbacol/farmacologia , Primers do DNA/genética , Expressão Gênica/efeitos da radiação , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Compostos Macrocíclicos/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/efeitos da radiação , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/efeitos da radiação , Oxazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We investigated the effects of gamma-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca(2+) handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca(2+), reduced amount of intrareticular Ca(2+), and reduced capacitive Ca(2+) entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca(2+), 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca(2+), amount of intrareticular Ca(2+), capacitative Ca(2+) entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca(2+) handling, and apoptosis appear due to a toxic action of intracellular Ca(2+). Ca(2+)-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca(2+) handling and apoptosis induced by gamma-radiation.
Assuntos
Apoptose/efeitos da radiação , Raios gama , Íleo/efeitos da radiação , Miócitos de Músculo Liso/efeitos da radiação , Retículo Sarcoplasmático/efeitos da radiação , Animais , Cálcio/metabolismo , Caspases/fisiologia , Células Cultivadas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Cobaias , Íleo/fisiologia , Contração Muscular/efeitos da radiação , Miócitos de Músculo Liso/fisiologia , Retículo Sarcoplasmático/fisiologiaRESUMO
The use of gamma-radiation in treatment of pelvic cancer is associated with injury of healthy surrounding tissues and disorders of intestinal motility; however, the cellular mechanisms involved are unclear. We tested the hypothesis that exposure of visceral smooth muscle cells (SMCs) to gamma-radiation induces apoptosis via activation of specific protein kinase C (PKC) isoforms. Cultured SMCs and slices from guinea pig ileum smooth muscle longitudinal layer (GPISMLL) were exposed to 10 to 50 Gy. Flow cytometry in gamma-radiated SMCs showed increased percentage of cells in the sub-G(0)/G(1) phase, a hallmark of apoptosis. gamma-Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of gamma-irradiated GPISMLL showed DNA fragmentation and apoptotic bodies analyzed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling method, whereas the plasma and nuclear membranes were preserved. Confocal microscopy in gamma-radiated SMCs labeled with annexin V-fluorescein showed an increase in apoptotic cells and phosphatidylserine externalization. Contraction of GPISMLL strips in response to KCl and acetylcholine was reduced in tissues exposed to 30 and 50 Gy. gamma-Radiation of GPISMLL caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and increased particulate/cytosolic PKC activity ratio. Western blot analysis revealed significant amounts of alpha- and epsilon-PKC in the cytosolic fraction of control GPISMLL. gamma-Radiation caused an increase in the amount of alpha- and epsilon-PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that gamma-radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of alpha- and epsilon-PKC isoforms.