Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres

To test whether exogenous oxidants alter intracellular oxidant levels in sk eletal muscle fibres, we exposed rat diaphragm to donors of nitric oxide (N Ox), reactive oxygen species (ROS) or hyperoxia, and monitored intracellula r oxidant levels using a fluorescent probe. Fibre bundles were dissected fr om the diaphragm and loaded with 2',7'-dichlorodihydrofluorescein (DCFH); e missions were monitored using a fluorescence microscope. DCFH-loaded muscle s were exposed to either a NOx donor (1 mM S-nitroso-N-acetyl penicillamine . SNAP: 1 mM sodium nitroprusside, SNP; 400 mu M 1-hydroxy-2-oxo-3-(N-3-met hyl-aminopropyl)-3-methyl-1-triazen, NOC-7), an ROS donor (100 mu M hydroge n peroxide, H2O2; 100 mu M tert-butyl hydroperoxide; 1 mM hypoxanthine plus 0.01 U mL(-1) xanthine oxidase, HXXO) or a range of PO(2)s (25, 60 Or 95% O-2 oxygenating Krebs-Ringer solution) for 40 min; time-matched control bun dles remained in Krebs-Ringer solution. Control muscles oxidized DCFH at a rate of 0.32 +/- 0.1 greyscale units min(-1). SNAP (766%), SNP (1244%). NOC -7 (851%). H2O2 (543%), and HXXO (541%) increased DCFH oxidation from contr ol levels. The increase in emissions caused by NOC-7 and SNP were blunted b y the NO, scavenger haemoglobin (1 mu M). DCFH oxidation by HXXO was unaffe cted by 1000 U mL(-1) superoxide dismutase but was significantly decreased by 1000 U mL(-1) catalase and 1 mM salicylate. Po-2 had no effect on intrac ellular oxidant levels. Therefore, extracellular NOx and ROS can alter intr acellular oxidant status in skeletal muscle fibres. These observations sugg est that intrafibre oxidant levels could be the result of both intracellula r and extracellular oxidant production.