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Show SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS MEASURING NITRIC OXIDE PRODUCTION BY ENDOTHELIAL CELLS USING ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY Jenn Nhan (John David Symons) Division of Endocrinology, Diabetes & Metabolism University of Utah The overall goal of our laboratory is to elucidate mechanisms whereby endothelial nitric oxide (NO) synthase (eNOS) enzyme function and N O bioavailability are impaired in individuals with diet-induced obesity (DIO), type 2 diabetes (T2DM), and insulin resistance. This is a clinically relevant issue because all of these conditions are associated with poor vascular function that might be precipitated by disrupted eNOS enzyme function and N O bioavailability. To study this issue w e use model systems including cell culture, isolated arteries, and rodents. It is important to have an accurate, reproducible, and sensitive method to detect N O in each of these systems. The purpose of m y work was to optimize the technique of electron paramagnetic resonance spectroscopy (EPR) to assess N O in bovine aortic endothelial cells (BAECs). N O contains an unpaired electron. This structural feature facilitates the measurement of N O via EPR. N O is detected with a spin trap of FeS04 and diethyldithiocarbamate trihydrate (DETC). In the presence of NO, the spin trap forms a NO-Fe(DETC) complex which is detected by EPR. BAECs were grown to 70-80% confluency in six-well plates, and treated with: (i) vehicle (buffer only), (ii) 600 u M of insulin (an N O agonist), (iii) 10 u M of NG-monomethyl-L-arginine acetate salt [LNMMA, an N O synthase (NOS) inhibitor], and (iv) three doses (1,10,100 uM) of diethylammonium (DEA) NONOate (an N O donor) to serve as a positive control. All treatments contained L-arginine [the substrate for endothelial NOS (eNOS)] and FeS04 (the spin trap). After a 60-min incubation with treatments (i) - (iv), cells were dislodged from each plate, collected, frozen in liquid nitrogen, and N O was detected using EPR. Relative to vehicle treatment, 1 uM, 10uM, and 10OuM DEA NONOate increased N O production by 50±8%, 150±2%, and 700±26% (p<0.05) respectively. Insulin increased (p<0.05) N O production by 73±4% vs. vehicle, and this response was prevented in cells treated with insulin + LNMMA. These results indicate EPR is a suitable method to detect cellular N O production. M y next project is to optimize this technique for use in isolated arteries from experimental animal models. NO Concentration via EPR 10 i NO Concentration via EPR n=9 P 7 at 5 Veh 1 10 100 uM NONOate Ins LNMMA John David Symons 127 |
