Reactive oxygen species (ROS)‐induced cellular injury contributes to the pathogenesis of neuronal disorders. Uncoupling proteins (UCPs) facilitate electron flows on the mitochondrial membrane and are expected to decrease ROS production. We previously showed that silencing of UCP4, a central nervous system‐specific UCP, led to apoptosis in PC12 cells. The purpose of the current study was to define the effects of UCP4 on ROS homeostasis and on mitochondrial antioxidants by contrasting the effects of UCP4‐silencing and overexpression. ROS levels were significantly increased in UCP4‐silencing (S2) cells (251.4±1.4% of. controls) but decreased in UCP4‐overexpressing (hUCP4) cells (73.3±1.4% of. controls). MnSOD was upregulated in S2 cells but downregulated in hUCP4 cells. Peroxiredoxin III was significantly downregulated in S2 cells but maintained in hUCP4 cells. Furthermore, mitochondrial levels of GSH were significantly decreased in S2 cells (51.9±8.4% of controls), which was associated with reduced expression of dicarboxylate carrier, a mitochondrial GSH transporter, but increased in hUCP4 cells (155.6±23.1% of. controls). These findings suggest that UCP4 may regulate ROS levels by modifying mitochondrial antioxidants, especially those that are important in reducing H2O2 and preventing the formation of hydroxyl radicals. UCP4 may thus be a therapeutic target for ROS‐induced neuronal disorders.

Vascular inflammation is a hallmark of Type‐1 and to a lesser extent, Type‐2 diabetes mellitus. We investigated the status of arterial cyclooxygenase‐2 (COX‐2), prostaglandin D and E synthases (PGDS and mPGES) and P450 2J2 proteins in healthy and streptozotocin (STZ) diabetic rats. Age‐matched male Sprague Dawley control and STZ‐diabetic rats were used. After 6 or 12 weeks STZ or vehicle, mesenteric arteries were harvested from rats, cleaned and prepared for Western blot analyses of target proteins. Serum levels of 6‐keto‐PGF1a, nitrite/nitrate (NOx), PGE2 and 8‐epi‐isoprostanes were determined with enzyme immunoassay kits. STZ‐diabetic were persistently hyperglycemia; COX‐2, mPGES proteins, 8‐epi‐isoprostanes, PGE2, 6‐keto‐PGF1a and NOx were significantly elevated versus control rats. CYP 2J and PGDS proteins were down‐regulated in diabetic versus control arteries. The significant increases in the expression of COX‐2 and mPGES proteins as well as enhanced serum PGE2 and NOx levels and reduced expressions of CYP 2J and PGDS are compatible with development of arterial inflammation. Support: University of Louisville Intramural Research Incentive Grant.

Excessive accumulation of amyloid beta (Abeta) has been proposed as a pivotal event in the pathogenesis of Alzheimer's disease. Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excess production of reactive oxidative species (ROS) and apoptosis. Neuroglobin (Ngb), a newly discovered globin in vertebrates that exhibits neuroprotective functions, may have a potential role in scavenging ROS. To examine the potential protective role of Ngb in Abeta-induced cytotoxicity, PC12 cells were treated with Abeta (1-42 fragment) for 24h. Abeta treatments increased ROS production in PC12 cells. Overexpression of Ngb but not Ngb mutant in the PC12 cells significantly attenuated Abeta-induced ROS production and lipids peroxidation. Furthermore, overexpression of Ngb also attenuated Abeta-induced mitochondrial dysfunction and apoptosis, and promoted cell survival in PC12 cells. Therefore, Ngb may act as an intracellular ROS scavenger, and such antioxidant properties may play a protective role against Abeta-induced cell injury.

WHPC extends the survival of mice exposed to lethal environmental hypoxia by preserving structural and functional integrity of key organs. We now examined whether WHPC would also prolong survival in response to hemorrhagic shock. Conscious male rats were subjected to bleeding of 60% of their estimated blood volume (~37.2 ml/kg) over 30 min. We found that naïve rats died 76±9 min after the completion of bleeding (n=15) and this process was associated with progressively increasing metabolic acidosis and cell/organ injury. In contrast, more than 80% of rats treated with WHPC (6 cycles of 8% O2x10-min/21% O2x10-min) 2 h before bleeding survived >6 h without fluid resuscitation (n=14, p<0.001) and displayed significantly reduced acidosis and cell/organ injury. Although it decreased drastically following bleeding in all rats, O2 consumption in those treated with WHPC was significantly higher, suggesting better tissue perfusion in these animals. Furthermore, surviving animals appeared to be recovering from the initial impact of massive blood loss, with accelerated compensation and sustained maintenance of key variables above critical levels (mean arterial pressure >60 mmHg, blood base excess >−6 mmol/L, and plasma lactate <5 mmol/L). WHPC treatment 8 h before bleeding was associated with similar beneficial effects. We conclude that WHPC provides a robust protective mechanism against acute hemorrhagic shock.

Objective To test the hypothesis that the enhanced vascular responsiveness to norepinephrine that occurs during deoxycorticosterone acetate (DOCA)-salt induced hypertension is causally related to increased expression of cyclo-oxygenase (COX)-2 and oxidative stress, which diminishes the vasomodulatory influence of endothelium-derived nitric oxide.Methods Four groups of age-matched, male Sprague-Dawley rats were studied: Sham (normotensive); DOCA-salt (hypertensive); DOCA-salt treated with manganese(ill) tetra(4-benzoic acid) porphyrin chloride [MnTBAP, an antioxidant; 15 mg/kg intraperitoneally (i.p.) for 21 days]; DOCA-salt treated with [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methane sulfonamide) (NS-398, a COX-2 selective blocker; 5 mg/kg i.p. for 7 days). Contraction and relaxation were measured with FT03 force transducers coupled to a Grass polygraph in aortic rings bathed with physiologic salt solution (37 degrees C) and bubbled with a 5%CO2/95%O-2 gas mixture. Aortic sensitivities (pD(2) values) to norepinephrine and serum isoprostanes (8-isoprostaglandin F-2 alpha, a marker of oxidative stress) were measured for each experimental paradigm.Results NS-398 significantly reduced maximal contractions in response to norepinephrine in aortic rings from Sham (44 +/- 3%) and DOCA-salt (96 +/- 2%) group rats. Expression of COX-2 protein increased significantly in vessels from DOCA-salt rats compared with those from Sham group rats. Treatment of DOCA-salt rats with either MnTBAP or NS-398 alleviated hypertension, normalized aortic pD(2) values for norepinephrine and restored serum 8-isoprostane concentrations towards those observed in Sham group rats.Conclusions COX-2 expression increases during DOCA-salt hypertension, and mediates production of factors that enhance rat aortic contractility in response to norepinephrine. Our data also suggest a role for increased oxidative stress, which is at least in part dependent on enhanced COX-2 expression, in the mechanism(s) of enhanced aortic contractility in response to norepinephrine during DOCA-salt hypertension. (c) 2005 Lippincott Williams & Wilkins.

NS-398 (N-(2-cyclohexyloxy-4-nitrophenyl)-methane sulfonamide) is a selective inhibitor of the cyclooxygenase-2 isozyme in vitro and in vivo. This study reports on acute inhibition of receptor-mediated contractions of isolated rat aorta by NS-398 and its modulation by endothelium-derived nitric oxide. NS-398 (1-10 microM) blocked norepinephrine, and 5-hydroxytryptamine (5-HT) evoked contractions and suppressed E(max) responses for both agonists. E(max) changes occurred in endothelium-intact vessel rings and in the absence, as well as in the presence of cycloheximide or dexamethasone in the physiological salt solution (PSS) bathing the tissues. NS-398 altered contractions to these receptor agonists in denuded rings only at 10 microM, and did not significantly alter contractions to KCl and sodium fluoride in all situations. NS-398 (3 and 10 microM) reduced aortic contractions initiated by cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca(2+)-ATPase blocker, in endothelium intact rings bathed with PSS with/without nitro-D-arginine methyl ester (D-NAME; 100 microM), but did not alter contractions to the compound in endothelium-denuded aortic rings and in vessel rings bathed with PSS+L-NAME (100 microM). Western blot analyses reveal significantly denser cyclooxygenase-2 protein expressions in freshly isolated endothelium-intact, compared to, denuded vessel segments. We conclude that: (1) cyclooxygenase-2 is constitutively expressed in rat aortic endothelial and smooth muscle cells, and (2) NS-398 modulates aortic contractions principally through an action on endothelial cyclooxygenase-2. Our data strongly suggest that cyclooxygenase-2 and/or its product(s), in concert with endothelium-derived nitric oxide, regulates the sarcoplasmic reticulum Ca(2+) pump activity in rat aorta.