γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells Sandra Claro a, ⁎, Maria Etsuko Miyamoto Oshiro a , Edna Freymuller b , Eduardo Katchburian c , Esper George Kallas d , Paulo Sérgio Cerri e , Alice Teixeira Ferreira a a Department of Biophysics, Federal University of São Paulo (UNIFESP-EPM), São Paulo, SP, Brazil b Centre of Electron Microscopy, Federal University of São Paulo (UNIFESP-EPM), São Paulo, SP, Brazil c Department of Morphology, Federal University of São Paulo (UNIFESP-EPM), São Paulo, SP, Brazil d Department of Gene Therapy, Federal University of São Paulo (UNIFESP-EPM), São Paulo, SP, Brazil e Department of Morphology, School of Dentistry, São Paulo State University (UNESP), Araraquara, SP, Brazil ABSTRACT ARTICLE INFO Article history: Received 20 August 2007 Received in revised form 25 April 2008 Accepted 19 May 2008 Available online 26 June 2008 Keywords: Apoptosis Calcium Caspase 12 Ileum γ-Radiation Sarco/endoplasmic reticulum We investigated the effects of γ-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 γ-radiation. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Use of high doses of γ-radiation in small volumes of tissue is becoming increasingly common (Pettersen et al., 2007; Foote et al., 1995; Fowler, 1989). Radiation may not only kill the tissue of interest, but also affect surrounding tissue. With intestinal radiotherapy, radiation toxicity is largely explained by injury to the epithelial and submucosal layers, but little is known regarding the effects on intestinal smooth muscle cells. Knowledge of response of these cells to γ-radiation aids the selection of treatments that maximize cancer cell killing with minimal damage to normal cells and tissues. Differentiated cells are more radioresistant than non-differentiated cells because they are able to repair the radiation-induced damage (Hall, 2000). Therefore, the effects of radiation on non-differentiated cells have been more studied (Soloviev et al., 2005; Hallahan et al., 1994; Szumiel et al., 1990). In addition, apoptosis is a cell single death with peculiar feature (Kerr et al., 1972) and radiation-induced signal transduction is an event that can be regulated by a balance between pro- and anti-apoptotic signals in many cell types, (Belka et al., 2004). Gamma radiation causes damage to membrane lipids and proteins such as ion channels, leading to increased permeability to Ca 2+ , which in turn can cause further damage in the cell (Eder et al., 2004; Yu et al., 2001; Cohen-Jonathan, 1999). The sarco/endoplasmic reticulum is a key player in the regulation of intracellular Ca 2+ because of its ability to take up and to release Ca 2+ . This release causes the opening of store-operated Ca 2+ channels (Parekh and Putney, 2005) in the cell membrane, allowing the influx of Ca 2+ , which can lead to SER stress (Boyce and Yuan, 2006; Xu et al., 2005). The sarco/endoplasmic reticulum-released Ca 2+ promotes apoptosis (Rao et al., 2004; Ferrari et al., 2002; Chami et al., 2001; Lam et al., 1994), and other cellular death mechanisms (Chin et al., 2007; Trump and Berzesky, 1996). The stressed sarco/endoplasmic reticulum can activate caspase 12 (Nakagawa et al., 2000), which is released from the sarco/endoplasmic reticulum to the cytosol, where it European Journal of Pharmacology 590 (2008) 20–28 ⁎ Corresponding author. Departamento de Biofísica, Universidade Federal de São Paulo-EPM, Rua Botucatu, 862- ECB-2° andar, Vila Clementino-CEP 04023-062, São Paulo, Brazil. Tel.: +55 11 5572 4583; fax: +55 11 5571 5780. E-mail address: sandra@biofis.epm.br (S. Claro). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.05.038 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar