DISRUPTION OF SKELETAL MYOCYTES INITIATES SUPEROXIDE RELEASE: CONTRIBUTION OF NAD(P)H OXIDASE

Generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has frequently been predicted. However, although it is known that severe inflammatory reactions occurring after major muscle trauma originate mainly from early local incidents within the injured tissue, no detailed studies exist on the local generation of ROS in response to myocyte destruction thus far. Therefore, in this study, ROS formation after lethal mechanical damage was examined using a model of scraping injury to cultured C2C12 skeletal myocytes and superoxide detection by lucigenin chemiluminescence, nitrotetrazolium blue chloride reduction, or electron spin resonance spectroscopy. Mechanical rupture of myocytes resulted in an immediate release of superoxide from the damaged cells that could be substantially blocked by the superoxide scavengers superoxide dismutase (51%), tiron (95%), and MAMA/NO (93%) and by hypoxia (83% inhibition). Superoxide generation was primarily confined to the myocytes' membrane fraction and 7- to 8-fold enhanced by the addition of NADH or NADPH. The NADPH-enhanced superoxide generation could largely be diminished by the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin in cell lysates (97% and 35% inhibition, respectively) and in isolated membrane fractions (61% and 63% inhibition). We thus conclude that immediately after myocyte damage, large amounts of superoxide are formed that predominantly originate from membrane-bound electron-transferring enzymes, especially NAD(P)H oxidase. This suggests a decisive role of ROS in the pathogenesis of tissue trauma, with superoxide being an initiator of the signaling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.ABBREVIATIONS-DPI, diphenyleneiodonium chloride; ESR, electron spin resonance; NBT, nitrotetrazolium blue chloride; NOX, NADPH oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase

[1]  M. Jackson,et al.  Microdialysis studies of extracellular reactive oxygen species in skeletal muscle: factors influencing the reduction of cytochrome c and hydroxylation of salicylate. , 2005, Free radical biology & medicine.

[2]  H. de Groot,et al.  ATP-INDUCED CALCIUM INCREASE AS A POTENTIAL FIRST SIGNAL IN MECHANICAL TISSUE TRAUMA. A LASER SCANNING MICROSCOPIC STUDY ON CULTURED MOUSE SKELETAL MYOCYTES , 2005, Shock.

[3]  I. Kang,et al.  Nox 2 stimulates muscle differentiation via NF-κB/iNOS pathway , 2005 .

[4]  S. Black,et al.  Cyclic stretch increases VEGF expression in pulmonary arterial smooth muscle cells via TGF-1 and reactive oxygen species: a requirement for NAD(PH) oxidase , 2005, The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society.

[5]  D. Gozal,et al.  Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury , 2005, Spinal Cord.

[6]  N. Patel,et al.  INHIBITORS OF NADPH OXIDASE REDUCE THE ORGAN INJURY IN HEMORRHAGIC SHOCK , 2005, Shock.

[7]  J. Lambeth.  NOX enzymes and the biology of reactive oxygen , 2004, Nature Reviews Immunology.

[8]  D. Giddens,et al.  Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion. , 2003, The Journal of biological chemistry.

[9]  M. Saran.  To what end does nature produce superoxide? NADPH oxidase as an autocrine modifier of membrane phospholipids generating paracrine lipid messengers. , 2003, Free radical research.

[10]  Z. Ungvari,et al.  High Pressure Induces Superoxide Production in Isolated Arteries Via Protein Kinase C–Dependent Activation of NAD(P)H Oxidase , 2003, Circulation.

[11]  R. Clempus,et al.  Vascular NAD(P)H oxidases: specific features, expression, and regulation. , 2003, American journal of physiology. Regulatory, integrative and comparative physiology.

[12]  P. Kaminski,et al.  Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase–Mediated Activation of the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Cascade , 2003, Circulation research.

[13]  D. Harrison,et al.  Detection of Superoxide in Vascular Tissue , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[14]  J. Tidball.  Interactions between muscle and the immune system during modified musculoskeletal loading. , 2002, Clinical orthopaedics and related research.

[15]  A. Nagano,et al.  Spinal Tuberculosis: Report of an Atypical Presentation , 2002, Clinical orthopaedics and related research.

[16]  S. Hussain,et al.  Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles. , 2002, American journal of respiratory and critical care medicine.

[17]  J. Lambeth,et al.  The neutrophil NADPH oxidase. , 2002, Archives of biochemistry and biophysics.

[18]  J. Wilcox,et al.  Upregulation of Nox‐Based NAD(P)H Oxidases in Restenosis After Carotid Injury , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[19]  L. Mazzolai,et al.  Flow Pulsatility Is a Critical Determinant of Oxidative Stress in Endothelial Cells , 2001, Hypertension.

[20]  G. Shao,et al.  Prediction of amorphous phase stability in the metal–silicon systems , 2001 .

[21]  Erwin G. Van Meir,et al.  Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. , 2001, Gene.

[22]  A. Zalewski,et al.  Increased NAD(P)H Oxidase and Reactive Oxygen Species in Coronary Arteries After Balloon Injury , 2001, Arteriosclerosis, thrombosis, and vascular biology.

[23]  D. Harrison,et al.  Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells. , 2001, Free radical biology & medicine.

[24]  M. Reid,et al.  highlighted topics Plasticity in Skeletal, Cardiac, and Smooth Muscle Invited Review: Redox modulation of skeletal muscle contraction: what we know and what we don’t , 2001 .

[25]  J. Lambeth,et al.  Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase. , 2000, Biochemical and biophysical research communications.

[26]  J. Krieger,et al.  Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity. , 2000, Free radical biology & medicine.

[27]  D. Sorescu,et al.  NAD(P)H oxidase: role in cardiovascular biology and disease. , 2000, Circulation research.

[28]  C. Dahlgren,et al.  Particles binding beta(2)-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis. , 1999, Biochimica et biophysica acta.

[29]  L. Ji,et al.  Aging and acute exercise enhance free radical generation in rat skeletal muscle. , 1999, Journal of applied physiology.

[30]  J. Lederer,et al.  The effects of injury on the adaptive immune response. , 1999, Shock.

[31]  I. Marzi,et al.  Mediators in polytrauma – pathophysiological significance and clinical relevance , 1998, Langenbeck's Archives of Surgery.

[32]  J. Zweier,et al.  Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems* , 1998, The Journal of Biological Chemistry.

[33]  T. Lüscher,et al.  Pulsatile stretch stimulates superoxide production in human aortic endothelial cells. , 1997, Circulation.

[34]  T. Lüscher,et al.  Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-κB in Human Coronary Smooth Muscle , 1997 .

[35]  R. van Wijk,et al.  NADPH-oxidase-dependent superoxide production by myocyte-derived H9c2 cells: influence of ischemia, heat shock, cycloheximide and cytochalasin D. , 1997, Journal of molecular and cellular cardiology.

[36]  W. Pryor,et al.  Oxidative stress following traumatic brain injury in rats. , 1997, Surgical neurology.

[37]  A. Fisher,et al.  Membrane depolarization and NADPH oxidase activation in aortic endothelium during ischemia reflect altered mechanotransduction. , 2005, American journal of physiology. Heart and circulatory physiology.

[38]  P. Tsao,et al.  Cyclic strain induces reactive oxygen species production via an endothelial NAD(P)H oxidase , 2001, Journal of cellular biochemistry. Supplement.

[39]  RJ Goris.  Pathophysiology of shock in trauma. , 2000, The European journal of surgery = Acta chirurgica.

[40]  H. Bergmeyer.  Methoden der enzymatischen Analyse , 1962 .