Adverse Outcomes Associated with Cigarette Smoke Radicals Related to Damage to Protein-disulfide Isomerase.

Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset.

Cigarette smoking affects oxidative protein folding in endoplasmic reticulum by modifying protein disulfide isomerase.

The endoplasmic reticulum (ER) stress response (ERSR) and associated protein aggregation, is under investigation for its role in human diseases, including chronic obstructive pulmonary disease (COPD) where cigarette smoking (CS) is a risk factor for disease development. Our hypothesis states that CS-associated oxidative stress interferes with oxidative protein folding in the ER and elicits ERSR. We investigated ERSR induction following acute CS exposure and delineated mechanisms of CS-induced ERSR. Lung lysates from mice exposed or not to one cigarette were tested for activation of the ERSR. Up to 4-fold increase in phosphorylation of eIF2α and nuclear form of ATF6 was detected in CS-exposed animals. CS affected the formation of disulfide bonds through excessive posttranslational oxidation of protein disulfide isomerase (PDI). Increased amounts of complexes between PDI and its client proteins persisted in CS-exposed samples. BiP was not a constituent of these complexes, demonstrating the specificity of the early effects of CS exposure on ER. Disturbances in protein folding were accompanied by changes in the organization of ER network and ER exit sites. Our results provide evidence that ERSR is induced early in response to CS exposure and identifies the first known ER-resident target of CS PDI, demonstrating that CS affects oxidative protein folding.

Mitigation of chlorine gas lung injury in rats by postexposure administration of sodium nitrite.

Nitrite (NO(2)(-)) has been shown to limit injury to the heart, liver, and kidneys in various models of ischemia-reperfusion injury. Potential protective effects of systemic NO(2)(-) in limiting lung injury or enhancing repair have not been documented. We assessed the efficacy and mechanisms by which postexposure intraperitoneal injections of NO(2)(-) mitigate chlorine (Cl(2))-induced lung injury in rats. Rats were exposed to Cl(2) (400 ppm) for 30 min and returned to room air. NO(2)(-) (1 mg/kg) or saline was administered intraperitoneally at 10 min and 2, 4, and 6 h after exposure. Rats were killed at 6 or 24 h. Injury to airway and alveolar epithelia was assessed by quantitative morphology, protein concentrations, number of cells in bronchoalveolar lavage (BAL), and wet-to-dry lung weight ratio. Lipid peroxidation was assessed by measurement of lung F(2)-isoprostanes. Rats developed severe, but transient, hypoxemia. A significant increase of protein concentration, neutrophil numbers, airway epithelia in the BAL, and lung wet-to-dry weight ratio was evident at 6 h after Cl(2) exposure. Quantitative morphology revealed extensive lung injury in the upper airways. Airway epithelial cells stained positive for terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL), but not caspase-3. Administration of NO(2)(-) resulted in lower BAL protein levels, significant reduction in the intensity of the TUNEL-positive cells, and normal lung wet-to-dry weight ratios. F(2)-isoprostane levels increased at 6 and 24 h after Cl(2) exposure in NO(2)(-)- and saline-injected rats. This is the first demonstration that systemic NO(2)(-) administration mitigates airway and epithelial injury.

Oxidative stress-mediated macromolecular damage and dwindle in antioxidant status in aged rat brain regions: role of L-carnitine and DL-alpha-lipoic acid.

BACKGROUND: The free radical theory of aging has significant relevance in a number of age-related neurological disorders. Too many free radicals create cellular pollution that shuts down energy levels. They have also been implicated in the loss of physiological functioning associated with the aging of post mitotic cells such as the brain. The activities of enzymatic antioxidative defenses decrease in rat brain may be possible causes of age-associated increase in oxidative damage to macromolecules. METHODS: We determined whether DL-alpha-lipoic acid (100 mg/kg body weight/day), and L-carnitine (300 mg/kg body weight/day) together when administered for 30 days declines the rate of oxidative stress-mediated macromolecular damages such as lipid peroxidation (LPO), protein carbonyl (PCO) and DNA protein cross-links in different anatomic regions (cortex, striatum and hippocampus). The activities of antioxidant enzymes in programmed aging were evaluated in the cortex, striatum and hippocampus of young and aged rat brain regions. RESULTS: Aged rats elicited a significant decline in the antioxidant status and increase in LPO, PCO and DNA protein cross-links as compared to young rats in all the 3 brain regions. The increase in LPO, PCO and DNA protein cross-links were (35.8%, 35.6%, 43.5%) in cortex, (32.5%, 40.3%, 29.8%) in striatum and (62.7%, 42.4%, 34.9%) in hippocampus, respectively, in aged rats as compared to young rats. Co-supplementation of carnitine and lipoic acid was found to be effective in reducing brain regional LPO, PCO and DNA protein cross-links and in increasing the activities of enzymatic antioxidants in aged rats to near normalcy. CONCLUSION: The combination of l-carnitine and lipoic acid overcame the oxidative stress induced rate of lipid peroxidation, protein carbonyl formation, accumulation of DNA protein cross-links and deficits in antioxidant enzyme activities in various brain regions of aged rats.