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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.)

Received for publication May 21, 2007
Accepted June 26, 2007.

	Abstract

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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-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.

Although precautions are usually taken during radiation therapy to limit the exposure of healthy surrounding tissues, potential injury could lead to various normal tissue complications. The form and extent of radiation injury vary according to the specific cell type and its susceptibility to injury. For example, radiotherapy of pelvic cancer is frequently associated with early and late intestinal and rectal toxicity (Tucker et al., 2006). In the early phases, the proliferating intestinal epithelial cells are mainly affected leading to localized inflammation, nausea, vomiting, cramping abdominal pain, and diarrhea. In later phases, progressive vasculitis affects the microvasculature of submucosa leading to thickening, fibrosis, and ulceration of the intestinal wall. Progressive ischemia may result in strictures, intestinal perforation, and possible abscess or fistula formation (Kodner et al., 1999).

Radiation-induced cell death is characterized by loss of cell division capacity and preservation of the plasma membrane leading to the formation of membrane-bound apoptotic bodies (Dewey et al., 1995). Other morphological hallmarks of apoptosis include loss of cell contact, cell shrinkage, chromatin condensation (Kerr et al., 1972; Wyllie et al., 1980), and phosphatidylserine (PS) externalization (Fadok et al., 2000). The apoptotic nucleus is resulted of DNA fragmentation into 180- to 200-bp fragments (Allen et al., 1997).

Radiation-induced apoptosis could also be manifested as reduction in cell growth and in proliferation. The G₂/M checkpoint is the most radiosensitive phase of the cell cycle (Steel, 1997; Hall, 2000d). Cell arrest in G₁/S checkpoint prevents replication of damaged DNA (Jonathan et al., 1999), and the presence of a sub-G₀/G₁ peak is an important hallmark of apoptosis (Evan et al., 1995; McGahon et al., 1995). Radiation-induced apoptosis of human gastric tumor cells could be a late event, with arrested G₂ phase after 12 h and maximal effect in G₁ phase after 72 to 96 h (Yanagihara et al., 1995).

Several signaling mechanisms are implicated in radiation-induced cell death including reactive oxygen species, c-Jun NH₂-terminal kinase, p53, bax, and various protein kinases (England and Cotter, 2005; Liu and Lin, 2005; Shen and Liu, 2006; Przemeck et al., 2007). Protein kinase C (PKC), a mediator of cell growth, may also affect the balance between pro- and antiapoptotic signals (Griner and Kazanietz, 2007; Selvatici et al., 2007). PKC is a multigene family of protein kinases activated by PS, other lipids, Ca²⁺, and phorbol esters (PDBu or PMA) (Nishizuka, 1992; Kanashiro et al., 1998a) and can also be regulated by ionizing radiation (Woloschak et al., 1990; Hallahan et al., 1991; Spitaler et al., 1999; Belka et al., 2004). The net effect of PKC activation on cell apoptosis may be variable because PKC subtypes have distinct functions in the apoptotic process (Nakajima, 2006).

Although the intestinal toxicity associated with radiotherapy is largely explained by injury to the epithelial and submucosal layers, little is known regarding the effects on intestinal smooth muscle cells (SMCs). In SMCs, the apoptotic process could be manifested not only as reduction in cell growth and proliferation but also as contractile dysfunction. In addition, in intestinal smooth muscle, activation of muscarinic receptors by acetylcholine causes contraction by stimulating Ca²⁺ influx and PKC (Murthy, 2006). Furthermore, although PKC activation has been implicated in regulation of SMC growth and contraction, the role of PKC subtypes in SMC apoptosis in response to -radiation is unclear.

The purpose of this study was to test the hypothesis that exposure of guinea pig ileum SMCs to low-LET -radiation with single dose rate induces apoptosis via activation of specific PKC isoforms. Experiments were designed to investigate: 1) whether low-LET radiation of guinea pig ileum SMCs causes apoptosis, growth arrest, and contractile dysfunction; 2) whether radiation-induced SMC apoptosis and growth arrest are prevented during blockade of PKC-mediated signaling; and 3) whether radiation-induced SMC apoptosis and contractile dysfunction are associated with changes in the activity of specific PKC isoforms.

	Materials and Methods

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Guinea Pig Ileum Tissue Slices. Slices from guinea pig ileum smooth muscle longitudinal layer (GPISMLL) were prepared as described previously and following the guidelines of the Institutional Committee for Animal Safety (CP-484/98-UNIFESP-EPM) (Shimuta et al., 1990). Tissue slices were irradiated and placed in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, New York, NY) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM glutamine (Sigma, St. Louis, MO) and maintained in 5% CO₂ atmosphere at 37°C for 72 h postirradiation.

SMC Culture. Primary culture of SMCs was prepared from GPISMLL as described previously (Shimuta et al., 1990). Cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM glutamine. The medium was changed every other day until the cells reached confluence. Purity of SMCs was determined by their positive labeling with monoclonal antibody to myosin (MHC-G-4, 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as detected by fluorescence microscopy. When cells reached confluence, they were irradiated, then placed in supplemented DMEM in 5% CO₂ atmosphere for 24 to 72 h.

-Irradiation. GPISMLL slices and cultured SMCs were exposed to -rays using a low-LET ⁶⁰Co -source (Eldorado 78-Atomic Energy; Canada Ltd., Montreal, QC, Canada). Samples were exposed to absorption doses of 10 to 50 Gy. A 0.5-cm-thick Lucite layer was placed over the samples to attain electronic equilibrium condition (Attix, 1986). The -rays dose rate was 1.80 Gy/min, and the distance between the ⁶⁰Co -source and samples was 80 cm. Control (0 Gy) tissues and cultured SMCs were not exposed to radiation.

Detection of Cell Death. Quantitative fluorescence analysis of apoptotic bodies was determined using the DNA intercalating propidium iodide dye (Roche Diagnostics, Mannheim, Germany). Suspensions of cells were fixed in 50% ethanol and PBS (140 mM NaCl, 3.9 mM Na₂HPO₄·H₂O, 1.7 mM NaH₂PO₄·H₂O) at 4°C for 1 h. The samples were centrifuged, and the pellet was resuspended in PBS, stained with 25 µg/ml propidium iodide, and analyzed by FACS Calibur flow cytometry using Cell Quest software (BD Biosciences, San Jose, CA). Cells were exposed to a focused argon laser beam excitation (488 nm), and red fluorescence from propidium iodide was collected through a band-pass filter (585/42 nm) (Krishan, 1975). Population of cells was distributed according to their allocation in the sub-G₀/G₁ phase determined by the M₂ region, and the G₁, S, and G₂/M phases were determined by the M₁ region of the cell cycle (Philippé et al., 1997). The PKC activator phorbol ester, 1 µM PDBu (Sigma), was added to SMCs 1 h before exposure to -rays. In other experiments, SMCs were treated for 72 h with PKC inhibitors: 1 µM Gö-6976 (Gö), which mainly inhibits Ca²⁺-dependent and I isoforms (Martiny-Baron et al., 1993); 1 µM GF-109203X, inhibitor of PKC, > I > II > > > (Calbiochem, La Jolla, CA) (Toullec et al., 1991); 1 µM benzophenanthridine alkaloid chelerythrine (Sigma), inhibitor of the PKC catalytic domain (Herbert et al., 1990); and 250 nM myristoylated membrane-permeable -PKC V1–2 peptide (-PKC V1–2) (BIOMOL Research Laboratories, Plymouth Meeting, PA), selective inhibitor of -PKC. This selective inhibitor of -PKC has eight amino acids derived from the first unique region (V1) of -PKC, amino acids 14 to 21(Gray et al., 1997).

Light Microscopy. The TUNEL method, as defined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling, was used to assess fragmented DNA (ApopTag Plus Peroxidase In Situ Apoptosis Detections kit; Oncor, Gaithersburg, MD) (Gavrieli et al., 1992). GPISMLL slices were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections (7 µm thick) were mounted on 5% silanized slides, deparaffinized, then predigested with 20 µg/ml proteinase K for 15 min at room temperature, 25°C. Endogenous peroxidase was inactivated by treating the tissue sections with 3% H₂O₂ for 1 h. Residues of digoxigenin-nucleotide were catalytically added to the DNA using TdT in a humid atmosphere at 37° for 1 h. Tissue sections were treated with anti-digoxigenin-peroxidase for 30 min, then stained with diaminobenzidine and methyl green. In other experiments, tissue slices from GPISMLL were embedded in araldite resin, cut into 7-µm-thick sections, and stained with toluidine blue for 5 min. Tissue sections were observed with an Axiovert-135 microscope (Carl Zeiss GmbH, Jena, Germany) with a UPlan-Apochromat 100x/1.35 oil objective.

Confocal Microscopy. SMCs from GPISMLL were grown on cover slips sealed to the bottom of 35-mm Petri dishes, exposed to -radiation, and maintained in 5% CO₂ atmosphere at 37°C for 24 h postirradiation. Cells were fixed in buffered 3.5% formaldehyde for 1 h, then stained with annexin V-fluorescein (1:100; Roche Diagnostics) and 4,6-diamidino-2-phenylindole (1:2000; Molecular Probes, Eugene, OR) for 5 min in buffered solution containing 10 mM Hepes/NaOH, 150 mM NaCl, 1.8 mM CaCl₂, 5 mM KCl, and 1 mM MgCl₂. Annexin V is a Ca²⁺-dependent protein with a strong affinity for PS, which is externalized in the early stages of apoptosis (Koopman et al., 1994). Apoptotic SMCs were visualized using a Bio-Rad 1024UV confocal system (Bio-Rad, Hercules, CA) attached to a Zeiss Axiovert 100 microscope and a 63x oil immersion Plan-Apochromatic objective (1.4 numerical aperture). Images were acquired through a pinhole aperture 2.0 µm in diameter, displayed on a personal computer, and printed on a Codonics NP1600 printer (Codonics, Inc., Middleburg Heights, OH).

Electron Microscopy. SMC culture, seeded on to Matrigel membrane matrix (BD Biosciences), and tissues sections of GPISMLL were fixed in modified Karnovsky medium (2.5% glutaraldehyde and 2.0% formaldehyde in 0.1 M sodium cacodylate, pH 7.3, for 1 h. The SMCs were postfixed for 1 h in 1% osmium tetra-oxide in 0.1 M sodium cacodylate buffer, then treated with propylene oxide to remove the Matrigel. Cells were dehydrated in ethanol and embedded in araldite resin. Thin ultrasections were cut on a Leica Ultratome (Leica, Wetzlar, Germany), placed on carbon-coated Formvar grids, and stained with uranyl acetate for 15 min and lead citrate for 2 min at room temperature 27°C (Overgaard, 1976). Samples were examined in a JEOL-1200 EXII scanning electron microscope (JEOL, Tokyo, Japan).

Isometric Contraction. Strips from GPISMLL unexposed or exposed to -radiation were equilibrated for 1 h under 1g tension in a tissue bath containing 5 ml of Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 0.4 mM NaH₂Po₄H₂O, 1.36 mM CaCl₂, 11.9 mM NaHC0₃) at 37°C. The strips were stimulated with 80 mM KCl, washed three times with Tyrode's, then stimulated with acetylcholine (Ach; 1 µM), and the changes in tension were recorded (Kanashiro and Khalil, 1998b). The same protocol was repeated in tissues pretreated with PDBu (1 µM) for 1 h or with the PKC inhibitor Gö (1 µM) or -PKC V1–2 (250 nM) in culture medium for 72 h (Martiny-Baron et al., 1993; Gray et al., 1997).

Tissue Fractions. GPISMLL slices unexposed or exposed to -radiation were stimulated with 1 µM PMA or Ach, then transferred to ice-cold equilibrating buffer A. The tissues were homogenized in buffer B, centrifuged at 100,000 rpm for 20 min, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in homogenization buffer containing 1% Triton X-100, centrifuged at 100,000 rpm for 20 min, and the supernatant was used as the particulate fraction (Murthy and Makhlouf, 1995; Kanashiro and Khalil, 1998b). Protein concentrations were determined using a protein assay kit (Bio-Rad). Buffer A contained 5 mM EGTA, 25 mM Tris-HCl, pH 7.5, 20 µl of leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (Sigma). Buffer B had the same composition as buffer A plus 250 mM sucrose.

PKC Activity Assay. PKC activation is associated with its translocation from the cytosolic to the particulate fraction. The cytosolic and particulate fractions were applied to DEAE-cellulose columns (Bio-Rad), and the protein was eluted with 0.1 M NaCl. PKC activity in the aliquots was determined by measuring ³²P incorporation from [³²P]ATP (MP Biomedicals, Irvine, CA) into histone III-S or myelin basic protein as described previously (Kanashiro and Khalil, 1998b). The assay mixture contained 25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 200 µg/ml histone IIIS, 80 µg/ml PS, 30 µg/ml diolein, [³²P]ATP (1 to 3 x 10⁵ cpm/nmol), and 0.5 to 3 µg of protein. After a 5-min incubation at 30°C, the reaction was stopped by spotting 25 µl of the assay mixture onto phosphocellulose discs. The discs were washed 3 x 5 min with 5% trichloroacetic acid, placed in 4 ml Ecolite (MP Biomedicals), and radioactivity was measured in a scintillation counter. Specific PKC activity was defined as PKC activity in the presence minus that in the absence of PS.

Immunoblotting. GPISMLL slices were homogenized in a buffer containing protease inhibitors. Protein-matched samples of the cytosolic and particulate fractions were subjected to electrophoresis on 8% SDS-polyacrylamide gels, then transferred to nitrocellulose membranes. The membranes were incubated in 5% nonfat dry milk in PBS-Tween for 1 h, then in the anti-PKC antibody solution at 4°C overnight. Monoclonal antibody to -PKC (1:100; Seikagaku America, Brockville, ON, Canada) and polyclonal antibodies to -, -, -, - -, and -PKC (1:500; Invitrogen) were used. To maintain the labeling conditions constant, we used the same concentration of anti-PKC antibodies and the same amount of protein (10 µg) in all samples. Actin was used as internal control. The membranes were washed 5 x 15 min, then incubated in horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG for 1.5 h. The blots were washed 5 x 15 min and visualized with the enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The reactive bands corresponding to PKC isoforms were analyzed using an optical densitometer (GS-700; Bio-Rad) (Kanashiro and Khalil, 1998b).

Statistical Analysis. Data were expressed as means ± S.D. and analyzed using unpaired Student's t test. Differences were considered statistically significant when p < 0.05.

	Results

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Effect of -Radiation on SMC Apoptosis. We first tested whether a single absorption dose of -irradiation causes SMC apoptosis. Irradiated SMCs were labeled with annexin V-fluorescein. The fluorescence intensity in the irradiated cells was not uniform but rather confined in sites on the membrane surface, particularly on membrane blebs and on nuclei giving the appearance of apoptotic bodies (Fig. 1A). The increased annexin V-fluorescence at the outer membrane leaflet suggested that -radiation was associated with PS externalization (Fig. 1A).

View larger version (66K): [in this window] [in a new window]   Fig. 1. Effect of -radiation in cultured SMCs and sections from GPISMLL. A, SMCs on coverslips were exposed to 50 Gy, fixed in 2% formaldehyde, and labeled with 1:100 annexin/V-fluorescein and 1:2000 4,6-diamidino-2-phenylindole. Images were acquired using confocal microscopy. SMCs irradiated with 50 Gy for 24 h show PS externalization in the outer leaflet of the surface membrane and apoptotic bodies (arrow). Control cells show pycnotic condensed masses (arrow) but no increase in fluorescence. B, TUNEL assay in paraffin tissue sections from GPISMLL 72 h postradiation show labeling of the nucleus with brown diaminobenzidine staining on DNA fragments localized in apoptotic bodies that contrast with methyl green. Representative sections from tissues exposed to 30 Gy show crescent masses in the nucleus (magnification, 100x), tissues exposed to 50 Gy show scattered apoptotic bodies (magnification, 40x), tissues exposed to 50 Gy without the enzyme TdT as negative control show apoptotic bodies (magnification, 40x), and control (0 Gy) presents normal SMCs (magnification, 40x). C, GPISMLL tissue slices stained with toluidine blue for 5 min show typical features of apoptosis in 50 Gy irradiated tissues and examined 72 h postradiation (magnification, 100x).

Time Course of Apoptotic Bodies. Due to the difficulty in quantifying apoptotic bodies by the TUNEL method, DNA content was quantified by flow cytometry assay. In SMCs irradiated with 50 Gy, the percent DNA content of apoptotic cells in the sub-G₀/G₁ phase (M₂ region) significantly increased (***, p < 0.001) when the postradiation period was extended from 24 to 48 and 72 h (71.3 ± 1.8, 72.1 ± 0.4, and 78.9 ± 3.6%, respectively; Fig. 2). The population of apoptotic cells in the control samples was 34.6 ± 5.9%. No significant change in DNA content was observed in SMCs irradiated with 10 and 30 Gy, but periods longer than 72 h showed significant decrease in M₁ region for all absorption doses.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Time course of cell apoptosis after 24-, 48-, and 72-h exposure to -radiation. DNA content was detected using flow cytometry of suspended cultured SMCs from GPISMLL, stained with propidium iodide, and analyzed using FACS Calibur software and FL3 channel. Apoptotic cells (percentage) correspond to the sub-G0/G1 phase. Data are expressed as means ± S.D. ***, p < 0.001.

Effects of -Radiation on SMC Ultrastructure. We examined the cell ultrastructure in control and -irradiated cultured SMCs of GPISMLL using scanning electron microscopy. In control (0 Gy) SMCs, all the intracellular structures and organelles were preserved. Ultrastructural alterations were observed in irradiated SMCs. SMCs irradiated at 20 and 30 Gy exhibited lysosome-like electron-dense elements throughout the cytosol, preserved nucleus and mitochondria, the myofilaments compacted near intact plasma membrane, and apoptotic body formation (as indicated by an arrowhead in Fig. 3). Similar findings were observed in SMCs irradiated with 50 Gy, but the myofilaments appeared distant from the plasma membrane, which was still preserved.

View larger version (105K): [in this window] [in a new window]   Fig. 3. Effect of -radiation on ultrastructure of SMCs of GPISMLL. Cultured SMCs seeded on Matrigel were fixed 72 h postradiation and stained in preparation for examination by transmission electron microscope. Control SMCs (0 Gy) present preserved intracellular structures and organelles. Cells irradiated with 20 or 30 Gy show normal nucleus (N) and the myofilaments (M) compacted near plasma membrane, and 20 Gy shows apoptotic bodies (arrowhead). Cells with 50 Gy show compacted myofilaments (M) but are distant from the plasma membrane. For all absorption doses of -radiation, the plasma membrane was preserved.

Effects of -Radiation on Smooth Muscle Contraction. GPISMLL strips showed significant contraction to both KCl and Ach presented as phasic followed by tonic responses (Fig. 4). The Ach-induced tonic contraction was slightly increased in tissues exposed to 10 Gy absorption dose (0.34 ± 0.04 g/mg, n = 10) versus control (0.29 ± 0.09 g/mg, n = 10) but was decreased with exposure to absorption doses of 30 Gy (0.26 ± 0.09 g/mg, n = 10) and 50 Gy (0.17 ± 0.08 g/mg, n = 10) (Fig. 4). Pretreating the tissues with the PKC activator PDBu did not cause any significant changes in the contraction of irradiated tissues, suggesting that PDBu is already activated under these conditions. In GPISMLL strips exposed to 50 Gy -radiation, the Ach contractile response was recovered to levels similar to or even greater than control levels (0 Gy) in the presence of the PKC inhibitors Gö (0.48 ± 0.11 g/mg, n = 3) or -PKC V1–2 (0.38 ± 0.03 g/mg, n = 6). The -PKC peptide V1–2 alone did not significantly affect Ach contraction (0.29 ± 0.09 g/mg, n = 10, p = 1.00).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of -radiation on GPISMLL contraction. GPISMLL strips were exposed to different absorption doses of -radiation for 3 h, then transferred to tissue baths filled with Tyrode's solution at 37°C. The tissues were stimulated with KCl (80 mM) and Ach (10–6 M), and the changes in isometric contraction were measured. The figure shows representative traces from experiments on nonirradiated strips (0 Gy) (A and C), on tissues exposed to 50 Gy (B and D), and nontreated or treated with PDBu (10–6 M). Cumulative data show decreased tonic response to Ach in tissues exposed to -radiation in the presence (+) or absence (–) of PDBu or the PKC inhibitor -PKC V1–2 (250 nM). E, data are expressed as means ± S.D. ***, p < 0.004.

View larger version (12K): [in this window] [in a new window]   Fig. 5. PKC activity in the cytosolic and particulate fractions of GPISMLL exposed to -radiation. A, PKC activity showed an increase in the particulate fraction and a decrease in the cytosolic fraction in tissues exposed to increasing absorption doses of -radiation and processed 72 h postradiation. B, effect of -radiation on the particulate/cytosolic PKC activity ratio was measured in tissues nontreated or treated with the PKC activator PMA (1 µM) or pretreated with the PKC inhibitor staurosporine (1 µM) for 1 h. Data are expressed as means ± S.D. *, measurements in irradiated tissues and treated with staurosporine are significantly different from irradiated tissues. *, p < 0.05.

Effect of -Radiation on the Distribution of PKC Isoforms. Immunoblots were performed to verify the expression of PKC isoforms in GPISMLL and to test their possible involvement in -radiation-induced apoptosis. Significant immunoreactive band was detected with specific antiserum to -PKC at 80 kDa and with antibody to -PKC at 90 kDa. The specificity of the reactive bands was confirmed by the loss of signal when the PKC antibody was not included in the immunoblot protocol. In control tissues, - and -PKC were mainly cytosolic. Exposure of GPISMLL to -radiation (10–50 Gy) was associated with an increase in - and -PKC immunoreactivity in the particulate fraction and a decrease in the cytosolic fraction (Fig. 6). In tissues treated with the PKC activator PMA (1 µM), - and -PKC were mainly in the particulate fraction, and -radiation did not cause a significant change in PKC distribution (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Western blot analysis of -PKC (A) and -PKC (B) in the cytosolic (Cyt) and particulate (Part) fractions of GPISMLL irradiated with -radiation and examined 72 h postradiation. GPISMLL was either nontreated (circles) or treated for 1 h with 1 µM PMA (triangles). The cytosolic and particulate fractions were separated and electrophoresed on 8% SDS-polyacrylamide gel electrophoresis followed by Western blotting using antibodies to specific PKC isoforms. Immunoreactive bands for -PKC (80 KDa) and -PKC (90 KDa) were analyzed by densitometry. Data represent means ± S.D. *, measurements in cytosolic and particulate fractions of tissues exposed to 10, 30, or 50 Gy and nontreated with PMA are significantly different from those in control tissues (0 Gy). *, p < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of PKC activator and inhibitors on cell cycle in SMCs exposed to -radiation. Cultured SMCs from GPISMLL were irradiated with 0, 10, 30, and 50 Gy, stained with propidium iodide, and the DNA content was analyzed using FACS Calibur software and FL3 channel of flow cytometry. A, cultured SMCs were treated with (+) or without (–) the PKC activator, PDBu (1 µM), for 3 h. B, representative histograms of single assays of control (0 Gy) and 50 Gy with or without -PKC V1–2 inhibitor peptide (250 nM). C, cultured SMCs were irradiated in the presence (+) or absence (–) of PKC inhibitors for 72 h: 1 µM Gö, 1 µM GF-109203X (GF), 1 µM benzophenanthridine alkaloid chelerythrine (Chel), and 250 nM -PKC V1–2 peptide (V1–2). Data are expressed as means ± S.D. *, measurement at 10, 30, or 50 Gy is significantly greater than that at 0 Gy. ***, p < 0.001. #, measurement at 30 or 50 Gy is significantly greater than that at 10 Gy. **, p < 0.01. , measurement at 10, 30, or 50 Gy in presence of PDBu is significantly different from that at 0 Gy and from the corresponding measurement in the absence of PDBu. ***, p < 0.002. , measurement in the presence of the PKC inhibitor is significantly less than control. *, p < 0.05.

	Discussion

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The present experiments with annexin V-fluorescein demonstrated that -radiation of SMCs is associated with PS externalization (Fig. 1A). In viable cells, PS is a normal constituent of the inner leaflet of plasma membrane. PS translocation to outer plasma membrane is an early event in apoptosis (Zhou et al., 1997). It has also been suggested that changes in intracellular Ca²⁺ concentration are involved in PS externalization and cell apoptosis (Zhao et al., 1998; Frasch et al., 2000; Leist and Jäättelä, 2001; Cui et al., 2004). The present study demonstrates an increase in PKC activity in SMCs exposed to -radiation. PS is a known activator of PKC (Nishizuka, 1992). The observations that -radiation of SMCs was associated with both PS externalization to the outer plasma membrane leaflet and increased PKC activity raise the possibility that the two events may be interrelated.

Other observed morphological and biological changes indicative of radiation-induced SMC apoptosis included DNA fragmentation and the appearance of brown-color apoptotic bodies as shown in the TUNEL assay (Fig. 1B), the condensed chromatin around the nuclear periphery (Fig. 1C), the increased apoptotic bodies as shown by electron microscopy (Fig. 3), as well as the increased cell population in sub-G₀/G₁ phase as indicated by propidium iodide staining and FACS analysis (Fig. 2). The -radiation-induced increase in SMC apoptosis appears to involve PKC because: 1) the PKC activator PDBu potentiated the effects of low 10 Gy radiation on increasing the number of apoptotic cells; 2) PDBu did not cause further increase in apoptotic cells with higher 30 and 50 Gy radiation, suggesting that PKC is already activated under these conditions (Fig. 7A); and 3) PKC inhibitors decreased the number of apoptotic cells induced by -radiation (Fig. 7C).

Differentiated intestinal SMCs have the ability to contract. In GPISMLL, activation of muscarinic receptors by acetylcholine causes contraction by stimulating Ca²⁺ influx (Murthy, 2006). In addition to Ca²⁺, PKC plays a significant role in SMC contraction (Murthy et al., 2000; Salamanca and Khalil, 2005). Studies in longitudinal smooth muscle strips of the rat proximal and distal colon suggest that activation of muscarinic receptors by Ach produces significant contractile response partly due to Ca²⁺ sensitization via activation of PKC (Takeuchi et al., 2004). The present experiments demonstrated that exposure of GPISMLL to 10 Gy -radiation enhanced Ach contraction. These observations can be partly explained by the possibility that 10 Gy -radiation may cause Ca²⁺ sensitization of the contractile myofilament to Ach-stimulated Ca²⁺ influx. This is supported by studies in vascular smooth muscle that demonstrated that 6 Gy ionizing radiation alters myofilament Ca²⁺ sensitivity and contraction through a potential role of PKC (Soloviev et al., 2005). The Ca²⁺ sensitization mechanism was not evident in GPISMLL strips exposed to 30 and 50 Gy; instead, a significant decrease in contraction was observed (Fig. 4). In addition, the observed apoptotic signs in GPISMLL strips exposed to 30 and 50 Gy -radiation suggest that the SMC apoptosis and the decreased contraction may share a common signaling pathway. The inhibitory effects of 30 and 50 Gy -radiation on GPISMLL contraction appear to involve PKC because: 1) the PKC activators phorbol esters did not cause further reduction in contraction in 30 and 50 Gy irradiated GPISMLL, suggesting that PDBu decreases myofilament calcium sensitivity through a PKC-dependent pathway; 2) the inhibitory effects of 30 and 50 Gy -radiation on GPISMLL contraction were prevented in tissues treated with PKC inhibitors (1 µMGö, not shown, and 250 nM V1–2); and 3) -radiation of GPISMLL was associated with an increase in PKC activity (Fig. 5).

PKC is a family of several isoforms. Previous studies suggest that the PKC isoenzymes and are important regulators of SMC growth and proliferation (Carlin et al., 1999; Salamanca and Khalil, 2005; Skaletz-Rorowski et al., 2005). The present experiments provided evidence that activation of - and -PKC isoforms may play a role in the -radiation-induced SMC apoptosis. This is supported by the observation that the number of apoptotic cells was reduced in irradiated SMCs treated with the isoform-specific PKC inhibitors Gö and -PKC V1–2. In addition, Gö and -PKC V1–2 restored the reduced GPISMLL contraction in irradiated tissues to the normal control levels. Furthermore, -radiation was associated with activation and translocation of - and -PKC from the cytosolic to the particulate fraction (Fig. 6).

In many cell types, radiation-induced apoptosis is regulated by a balance between pro- and antiapoptotic signals (Belka et al., 2004). Nevertheless, we should caution that cell death by apoptosis may not be linked to PKC activation (Knauf et al., 1999). Some studies suggest that activation of PKC causes cell death by apoptosis (Spitaler et al., 1999; Gonzalez-Guerrico et al., 2005; Griner and Kazanietz, 2007). Other studies suggest that PKC isoenzymes may exert different effects on apoptotic and survival pathways and participate in both pro- and antiapoptotic signaling cascades (Nakajima, 2006). For example, -PKC appears to be involved in both anti- and proapoptotic signaling (Spitaler et al., 1999; Lee et al., 2003). The dichotomy in the effects of -PKC on the apoptotic process (Nakajima, 2006) may partly explain some of the present observations. For example, PBDu did not cause significant apoptosis in control SMCs but enhanced the apoptosis induced by 10 Gy -radiation. This can be explained by the possibility that PDBu activation of -PKC would activate both pro- and antiapoptotic effects, with a net lack of effect on SMC apoptosis. In contrast, exposure to 10 Gy may prime/potentiate the -PKC-mediated proapoptotic pathway. This is supported by the observation that about 15% of apoptosis inhibition was related to -PKC. Thus, although the present study identified the potential role of - and -PKC in -radiation-induced SMC apoptosis, the quantitative aspect of their role particularly with regard to potential antiapoptotic effects may require further investigation.

Members of the PKC family respond differently to various combinations of lipids, particularly PS and diacylglycerol, and Ca²⁺ (Nishizuka, 1992; Kanashiro and Khalil, 1998a). Furthermore, Ras and Raf-1 can phosphorylate PKC isoforms, which in turn activate MAP kinases through a protein kinase cascade (Salamanca and Khalil, 2005). PKC activity may also be determined by the agonist used and intensity of stimulus such as the absorption dose of ionizing radiation and LET. The PKC inhibitors tested in this study acted as potent radioprotectors in vitro, as evidenced by their ability to reduce the blockade of the G₁ phase and the restored cell proliferation even at 50 Gy. However, although the M₂ region that represents the sub-G₀/G₁ phase was significantly suppressed in the presence of PKC inhibitors, it did not reach the levels observed in control nonirradiated SMCs, suggesting that other pathways in addition to PKC are involved in -radiation-induced apoptosis. Our data are in agreement with studies in adult rat ventricular myocytes, which showed that apoptosis was attenuated by inhibition of not only -PKC but also extracellular signal-regulated kinase activity (Shizukuda and Buttrick, 2001).

Our findings suggest that sublethal damages in normal SMCs occur during exposure to single high-absorption doses of -radiation. The data suggest that radiotherapy of pelvic cancer may cause injury of normal surrounding SMCs in addition to the well documented injuries to epithelial and submucosal layers. In view of the observed -radiation-induced PKC activation, the use of PKC inhibitors to amend radiation-induced SMC apoptosis and visceral complications may warrant further examination. In addition, further understanding of the cellular mechanisms involved in radiation-induced SMC apoptosis and the factors determining their radiosensitivity relative to other cell types and biological systems may be useful in determining the total absorption dose of radiation (single or fractionated), the type of radiation (low or high LET), and the postradiation period.

In conclusion, in guinea pig ileum smooth muscle longitudinal layer, -radiation induces SMC apoptosis, growth arrest, and contractile dysfunction. The increased activity of - and -PKC isoforms suggests a role for PKC-mediated pathway in -radiation-induced visceral smooth muscle apoptosis.

	Acknowledgements

 
We thank Renato Mortara, Esper G. Kallas, Edna Freymuller, and Paulo Sergio Cerri for assistance with confocal microscopy, flow cytometry, and electronic microscopy analysis, respectively.

	Footnotes

 
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.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125930.

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;-PKC V1–2, -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.

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

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