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Show 135 school of medicine and health sciences Individuals with type 2 diabetes and hypertension are more susceptible to cardiovascular complica-tions such as impaired blood vessel function. Due to the elevation of free fatty acids (FFAs) in obese individuals, ceramide, a lipid metabolite, accumulates and might contribute to the inability of a blood vessel to constrict or relax appropriately. Vessel dysfunction is caused, at least in part, by the inability of the endothelium, the innermost protective lining of blood vessels, to synthesize and release nitric oxide (NO). Our data indicate that ceramide impairs endothelial NO synthase (eNOS), the enzyme that synthesizes NO. In order to study mechanisms by which ceramide might impair eNOS, it is important to measure cellular ceramide production in response to physiological, pathophysiological, pharmaco-logical, and genetic manipulations. Previously, we used P-32 radioactive assays to measure ceramide accumulation. However, the use of radioactivity is expensive, potentially hazardous to the experimen-talist, and waste disposal is an environmental concern. Therefore, I sought to import an accurate, cost effective method that poses minimal threat to the individual or the environment. Immunofluorescence (IF) allows ceramide to be tagged with a primary antibody which can be detected by a secondary antibody conjugated with a fluorescent dye. Bovine aortic endothelial cells were grown to 50% conflu-ency in two-chambered glass slides. Next, the cells were treated with vehicle or the physiologically relevant saturated FFA palmitate. I have observed that 250, 500, and 750 uM palmitate incubation for 3 h increases (p<0.05) endothelial cell ceramide accrual in a dose-dependent manner. Further, a FFA-independent method to alter ceramide accrual i.e., 3 h incubation of cells with N-oleoylethanolamine, also elevates (p<0.05) ceramide production. Importantly, I have shown that 500 uM palmitate-induced ceramide accrual can be prevented (p<0.05) by two structurally dissimilar inhibitors (10 uM myrio-cin, 1mM L-cycloserine) of the rate-limiting enzyme responsible for ceramide biosynthesis i.e., serine palmitoyl transferase (SPT). None of these inhibitors impairs cell viability. The next step is to determine the effectiveness of gene silencing techniques to inhibit palmitate-induced ceramide accumulation. Once we confirm our ability to consistently manipulate and accurately measure ceramide accumula-tion, we can assess the molecular mechanisms whereby ceramide causes vascular dysfunction. MEASURING CELLULAR CERAMIDE ACCRUAL USING IMMUNOFLUORESCENCE Anindita Ravindran (John David Symons) Division of Endocrinology, Metabolism, and Diabetes, School of Medicine University of Utah UNDERGRADUATE RESEARCH ABSTRACTS Anindita Ravindran John David Symons |