Abstract-Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC).Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca 2ϩ channel ␣1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca 2ϩ -ATPase, ␣-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase ␣1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart. (Circ Res. 2007;101:1155-1163.) Key Words: preconditioning Ⅲ S-nitrosylation Ⅲ cardioprotection I schemic preconditioning (IPC) is a cellular adaptive phenomenon whereby brief episodes of myocardial ischemia and reperfusion (I/R) render the heart resistant to subsequent prolonged ischemic injury. 1 Through activation of a complex cascade of signaling events, IPC has been shown to reduce arrhythmias, infarct size, and postischemic contractile dysfunction. [2][3][4][5] Nitric oxide (NO) has been shown to be an important signal in cardioprotection. 6 -8 In acute IPC, NO has been shown to mediate protection at least in part by activation of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) and the activation of protein kinase G, which in turn leads to the opening of the mitochondrial K ATP channel. 6 Recently, it has also been shown that protein kinase G results in activation of an endogenous mitochondrial protein kinase C that is involved in activation of the mitochondrial K ATP channel. 9,10 The opening of the mitochondrial K ATP channel is reported to reduce mitochondrial Ca 2ϩ loading 11,12 and also to lead to generation of reactive oxygen species, which activate signaling cascades in a feed-forward manner to elicit cardioprotection. 13,14 In addition to activating cGMP/protein kinase G-dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which ha...
The differences in drug therapy found in this study may indicate that patients with TBI and PTSD respond differently to treatment than patients with PTSD alone.
Using proteomic technologies, we identified 62 proteins that are oxidized to carbonyl derivatives during growth of Escherichia coli under nitrogen starvation (NS), carbon starvation (CS), and phosphate starvation (PS) conditions. The carbonylated proteins were converted to 2,4-dinitrophenylhydrazone derivatives and these were identified using Western blotting and mass spectrometry by searching E. coli proteins in the Swiss-Prot and/or NCBI databases. Fourteen of the oxidized proteins were formed under both NS and CS conditions, and only three proteins were specifically oxidized under PS conditions. Interestingly, the carbonyl content of proteins in crude extracts of cells harvested after 48 h of stationary growth under NS and CS was significantly lower than that observed at mid-log and end-log phases of growth. In contrast, the carbonyl content of proteins in extracts of cells grown under PS conditions was fairly constant during comparable periods of growth. proteomics A ging, oxidative stress, nutritional factors, and some diseases are associated with the accumulation of oxidized proteins [formation of protein carbonyl (RCϭO) derivatives] (1-10). In studies to determine how protein turnover is regulated, it was demonstrated that oxidation of proteins makes them susceptible to proteolytic degradation (11-21). Taking advantage of proteomic technologies, we have now identified proteins that are oxidized in Escherichia coli during the exponential and stationary phases of growth under nitrogen-, carbon-, or phosphatelimiting conditions and determined the effects of starvation conditions on the ability to hydrolyze a pure preparation of oxidized glutamine synthetase. Results Protein Carbonyl (RC؍O) and Protease Activities of Cell-Free Extracts.The protein carbonyl content and abilities of crude extracts to degrade oxidized proteins of wild-type (CSH7) E. coli grown on normal medium (M9) and harvested at mid-log phase of growth and those harvested at mid-log, end-log, and after 24 h or 48 h of stationary growth in nitrogen starvation (NS), carbon starvation (CS), or phosphate starvation (PS) media are summarized in Fig. 1 Upper. It is apparent from these data that the level of RCϭO during mid-log and end-log phases of growth on NS media was appreciably higher than those observed for the corresponding phases under CS or PS conditions. However, the level of RCϭO in both NS and CS declined during 48 h of stationary growth, whereas the level in PS cells was affected little by prolonged incubation. In view of the fact that oxidized proteins are more susceptible to proteolytic degradation (13-21), we determined the effect of starvation conditions on the ability of cells to degrade oxidized 14 C-labeled glutamine synthetase. As shown in Fig. 1 Lower, the protease activity of CS cells declined significantly during the 24-48 h of stationary growth, but decreased slightly in the NS and PS cells exposed to these conditions. Identification of Proteins Oxidized During Starvation Conditions.Protein spots from 2,4-dinitrophenylhyd...
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