Abstract
The use of γ-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 γ-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 γ-radiated SMCs showed increased percentage of cells in the sub-G0/G1 phase, a hallmark of apoptosis. γ-Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of γ-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 γ-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. γ-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 α- and ϵ-PKC in the cytosolic fraction of control GPISMLL. γ-Radiation caused an increase in the amount of α- and ϵ-PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that γ-radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of α- and ϵ-PKC isoforms.
Footnotes
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This study was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo and Sociedade Paulista para o Desenvolvimento da Medicina, Brazil and by the National Institutes of Health (Grants HL-65998 and HL-70659 to R.A.K.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.125930.
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ABBREVIATIONS: LET, linear energy transfer; PS, phosphatidylserine; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; SMC, smooth muscle cell; GPISMLL, guinea pig ileum smooth muscle longitudinal layer; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Gö, Gö-6976, indocarbazole; GF-109203X, bisindolylmaleimide;ϵ-PKC V1–2, ϵ-PKC V1–2 inhibitor peptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr); TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; TdT, terminal deoxynucleotidyl transferase; Ach, acetylcholine.
- Received May 21, 2007.
- Accepted June 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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