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CELLULAR AND MOLECULAR

- and -Protein Kinase C Activity during Smooth Muscle Cell Apoptosis in Response to -Radiation

Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil (S.C., M.E.M.O., A.T.F.); Department of Physiology, University of Medical Sciences of Alagoas, Alagoas, Brazil (C.A.K.); and Division of Vascular Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts (R.A.K.)

The use of -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 -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 -radiated SMCs showed increased percentage of cells in the sub-G₀/G₁ phase, a hallmark of apoptosis. -Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of -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 -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. -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 - and -PKC in the cytosolic fraction of control GPISMLL. -Radiation caused an increase in the amount of - and -PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that -radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of - and -PKC isoforms.

Address correspondence to: Sandra Claro, Departamento de Biofísica, Universidade Federal de São Paulo, Escola Paulisa de Medicina, Rua Botucatu 862, Vila Clementino, CEP 04023-062 São Paulo, Brazil. E-mail: sandra{at}biofis.epm.br