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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-G 0 /G 1 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.

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Simultaneous recordings of the tension and intracellular Ca2+ concentration of guinea-pig ileum longitudinal smooth muscle strips, as well as 24Na+ and 45Ca2+ influx measurements in cultured myocytes from the same tissue, were used to investigate the mechanisms underlying angiotensin-induced desensitization and tachyphylaxis. Angiotensin II and [2-lysine]-angiotensin II (Lys2All), incubated for prolonged periods (10 min) with muscle strips, induced fading of the contractile response (desensitization) and reappearance of the intracellular Ca2+ concentration oscillations, which were inhibited during the initial increase in cytosolic Ca2+. The desensitization was paralleled, in cultured myocytes, by inhibition of the 45Ca2+ but not of the 24Na+ influxes which were initially stimulated by the peptides. On the other hand, repeated administrations of angiotensin II (but not of Lys2All) caused gradual reduction of the contractile response and of the 24Na+ influx stimulation evoked by the agonist (tachyphylaxis). Treatment with phorbol 12-13 dibutyrate accelerated the desensitization induced by both angiotensin II and by Lys2All and aggravated the tachyphylaxis to angiotensin II. The results support the hypothesis that activation of protein kinase C is responsible for the desensitization and that tachyphylaxis is due to the slow dissociation of angiotensin II from a postulated Na(+)-dependent regulatory site on the receptor.