Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle.

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3-dimethoxy-1-naphthoquinone (DMNQ; 1, 10, 100 microm; a mitochondrial O(2)(.-)/H2O2 generator) or exogenous H2O2 (5, 50, 500 microm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr-PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 microm H2O2, no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 microm and reduced PTP at 1 microm. Similar reductions in Ser/Thr-PPase activity were seen in response to 50 and 500 microm H2O2 and PTP at 500 microm H2O2. ROS treatments resulted a dose-dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed.