 The Role of HSP90 and ATM in the Radiation- Induced eNOS Activation Masaki Nagane 1 , Hironobu Yasui 1 , Tohru Yamamori 1 , Yuri Sakai 1 , Koichi Niwa 2 , and Osamu Inanami 1 1 Hokkaido University, Japan, 2 Tokyo University of Agriculture, Japan Nitric oxide (NO) serves multiple functions such as vasodilation, neurotransmission, and immune defense. Endothelial NO synthase (eNOS) is the major source of NO production in endothelial cells. Recently, several studies have demonstrated that ionizing radiation (IR) increases eNOS activity. However, the mechanism of radiation-induced eNOS activation remains unclear. to decipher it, bovine aortic endothelial cells (BAEC) were used as a model of vascular endothelial cells. When BAEC were irradiated, NOS activity was significantly upregulated at 12- 24 h after IR. in addition, eNOS-Ser1179 phosphorylation was increased at 6-24 h after IR. Treatment with an HSP90 inhibitor geldanamycin abrogated the eNOS activation and ATM-Ser1981 phosphorylation after IR. Furthermore, an ATM inhibitor Ku-60019 attenuated eNOS phosphorylation and activation after IR. These results suggested that ATM and HSP90 were involved in the radiation-induced eNOS activation.  Nitrite regulation of Hydrogen Sulfide Metabolism in the Endothelial Cells Sibile Pardue 1 , Xinggui Shen 1 , and Christopher G Kevil 1 1 LSU Health Sciences Center- Shreveport, United States Hypoxia critically alters numerous endothelial cell functions including NO bioavailability and redox status. We have previously shown that during hypoxia endothelial cells can mediate a one- electron reduction of nitrite to NO. However, the effect of nitrite dependent NO formation on endothelial cell hydrogen sulfide metabolism remains unknown. the aim of the current study was to determine the effect of nitrite on hydrogen sulfide bioavailability in human umbilical vein endothelial cell (HUVECS) under normoxia (21%O2) or hypoxia (0.1% O2). Free sulfide, acid-labile sulfide and bound sulfane sulfur in HUVECs was measured after treatment with 0.01-ȝ0 of nitrite using the monobromobimane derivatization and RP-HPLC method. We found that free sulfide and acid-labile sulfide levels were decreased but that bound sulfane sulfur levels were increased after four hours of hypoxia. Importantly, treatment with exogenous nitrite significantly enhanced free sulfide, acid-labile sulfide and bound sulfane sulfur during hypoxia. We further examined the regulation of sulfide bioavailability through NOS enzyme, xanthine oxidase, or GCL pathways. Inhibition of these pathways did not affect sulfide levels due to nitrite treatment during hypoxia, but did decrease free sulfide bioavailability under normoxia. Inhibition of these pathways also results in the decrease of bound sulfane sulfur in HUVECS treated with nitrite under either normoxia or hypoxia. Together, our findings highlight a novel role of nitrite in regulating hydrogen sulfide bioavailability in endothelial cells indicating a new dimension in gaseous signal molecule regulation of endothelial cell dysfunction during hypoxia.  Conditioning Media to Target Dissolved Oxygen Levels Victoria Eon 1 and Brian Parent 1 1 The Baker Company, United States The fundamentals of a good research model include the environment of the cells and how the media affects the outcome. Exposing cells native to low-O2 (2-8%) environments to normal atmosphere (21% O2) causes abnormal cell interactions and reduces cell viability. Moreover, a controlled environment for achieving and maintaining atmosphere and media conditions within nontoxic pH parameters (7.0-7.4) is crucial for cellular- based work. Therefore, gas controlled incubators and controlled atmosphere workstations are often used to simulate normal conditions in the ERG\ｶV RUJDQ V\VWHPV and provide a better understanding of WRGD\ｶV HSLGHPLRORJLFDO SUREOHPV Gas controlled incubators and workstations help control the atmosphere surrounding cell and tissue cultures, yet they remain prey to oxidative stress. the problem is contrasting concentrations of dissolved O2 between the cells and growth media. New media contains approximately 10-12% dissolved O2 content by mass, but certain tissue cells are typically plated best at 1-3% O2, depending on the research application. to combat this, most researchers use their own methods to condition media to a desired concentration of O2 for their research application. Little documented research is available that examines these methods, including the precision and accuracy of the actual dissolved O2 content within the media. The objective of this trial was 1) to determine if media conditioned through a commercially-available media conditioning system +\SR[\&22/ LQIOXHQFHV the cell culture on a molecular level in any way (gene expression); 2) to assess the feasibility and ease of use of the conditioning system and testing protocol in practice; 3) to confirm deoxygenation behavior of various media and degree of O2 depletion; 4) to gather data on shelf life and long-term behavior of the conditioned media with respect to pH and O2 concentration.  nNOS is Necessary for Adaptive Hypertrophy and Resistance to Contraction-Induced Injury in the MDX Mouse Model of Duchenne Muscular Dystrophy Sarah Reed 1 , Stanley Froehner 1 , and Justin Percival 1,2 1 University of Washington, United States, 2 University of Miami Miller School of Medicine, United States Loss of neuronal nitric oxide synthase mu (nNOSP) activity is a feature of many neuromuscular diseases including Duchenne Muscular Dystrophy (DMD) which is caused by loss of dystrophin. This makes nNOS an attractive clinical target for the treatment of muscle disease especially DMD. However, the role of nNOS in dystrophin-deficient muscle remains unclear and is more complex than previously appreciated because the nNOSE splice variant is also expressed in muscle and may compensate for loss of nNOSP. to address nNOS splice variant function in dystrophin- deficient muscle, we examined the impact of the loss of dystrophin on nNOSE and the impact of nNOSP/nNOSE deficiency on dystrophin-deficient muscle pathology in mdx mice. Like nNOSP, nNOSE localization was disrupted in mdx muscle 6 6)5%0  doi: 10.1016/j.freeradbiomed.2013.10.645 doi: 10.1016/j.freeradbiomed.2013.10.646 doi: 10.1016/j.freeradbiomed.2013.10.647