Jennifer Nhan, University of Utah
College of Health, Division of Endocrinology, Metabolism, and Diabetes
The overall goal of our laboratory is to elucidate mechanisms whereby endothelial nitric oxide (NO) synthase (eNOS) enzyme function and NO 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 NO bioavailability. To study this issue we use model systems including cell culture, isolated arteries, and rodents. It is important to have an accurate, reproducible, and sensitive method to detect NO in each of these systems. The purpose of my work was to optimize the technique of electron paramagnetic resonance spectroscopy (EPR) to assess NO in bovine aortic endothelial cells (BAECs). NO contains an unpaired electron. This structural feature facilitates the measurement of NO via EPR. NO is detected with a spin trap of FeSO4 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 uM of insulin (an NO agonist), (iii) 10 uM of NG-monomethyl-L-arginine acetate salt [LNMMA, an NO synthase (NOS) inhibitor], and (iv) three doses (1, 10, 100 uM) of diethylammonium (DEA) NONOate (an NO donor) to serve as a positive control. All treatments contained L-arginine [the substrate for endothelial NOS (eNOS)] and FeSO4 (the spin trap). After a 60-min incubation with treatments (i) (iv), cells were dislodged from each plate, collected, frozen in liquid nitrogen, and NO was detected using EPR. Relative to vehicle treatment, 1uM, 10uM, and 100uM DEA NONOate increased NO production by 50±8%, 150±2%, and 700±26% (p<0.05) respectively. Insulin increased (p<0.05) NO 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 NO production. My next project is to optimize this technique for use in isolated arteries from experimental animal models.