The effects of extracellular nitric oxide and reactive oxygen intermediates on intracellular oxidant levels of skeletal muscle

In order to investigate the potential for paracrine exchange of nitric oxide derivatives (NOx) and reactive oxygen intermediates (ROI) between capillary endothelial cells and skeletal muscle cells we exposed rut diaphragm to donors of NOx and ROI and measured the intracellular oxidant levels with 2',7'-dichlorohydrofluoroscein diacetate (DCFH-DA). Diaphragm fibre bundles were incubated in oxygenated Krebs-Ringer solution containing 50 uM DCFH-DA, which concentrates intracellularly and emits at 520 nm when oxidized; emissions were quantified using a fluorescence microscope. Experimental muscles were exposed to a NOx donor (400 uM NOC-7) or ROI donors (100 uM hydrogen peroxide, H₂O₂; 100 uM tert-butyl hydroperoxide, tBOOH; 1mM hypoxanthine combined with 0.01 U/ml xanthine oxidase, HX-XO) for 40 mins while the controls bundles remained in Krebs-Ringer solution. Control muscle emissions increased linearly over 40 mins. Emissions were significantly increased from control by NOC-7 (303.1±31.82%), H₂O₂ (319.8±30.7%), tBOOH (1396.2±282.9%), and HX-XO (409.2±89.6%). These observations demonstrate that 1) resting diaphragm muscle produces oxidants at a steady rate and 2) extracellular NOx and ROI can cross skeletal muscle membranes and significantly alter the intracellular redox state. These observations suggest that cells adjacent to skeletal muscle that release NOx and ROI can effect the oxidant status of the muscle cells.