alpha- and epsilon-protein kinase C activity during smooth muscle cell apoptosis in response to gamma-radiation.
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.
Claro
Sandra
S
Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil. sandra@biofis.epm.br
Kanashiro
Celia Akemi
CA
Oshiro
Maria Etsuko Miyamoto
ME
Ferreira
Alice Teixeira
AT
Khalil
Raouf Ali
RA
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D005720:Gamma Rays; D006168:Guinea Pigs; D009119:Muscle Contraction / Q000528:radiation effects; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000528:radiation effects*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D021381:Protein Transport; D016192:Resting Phase, Cell Cycle / Q000528:radiation effects
D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon