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Show SCHOOL OF MEDICINE AND HEALTH SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS Ting Ruan J. David Symons CERAMIDE INITIATES PP2A COLOCALIZATION WITH ENOS BY DISSOCIATING PP2A FROM I2PP2A Ting Ruan, (J. David Symons) College of Health and Division of Endocrinology, Metabolism and Diabetes University of Utah In whole cell lysates w e showed that palmitate disrupts the interaction between inhibitor 2 (12) of protein phosphatase 2A (PP2A) and PP2A and that this event initiates PP2A colocalization with eNOS. Both responses were negated when ceramide synthesis was inhibited. W e sought to confirm this palmitate evoked, ceramide-mediated effect using immunofluorescent approaches. Bovine aortic endothelial cells (BAECs) were treated for 3 h with BSA (V), V + 500 p M palmitate (P), V + ceramide inhibition (10 u M myrio-cin; M), or P + M (PM). P-induced increases (p<0.05) in ceramide were prevented (p<0.05) in cells exposed to P + M (n=8). P increased PP2A association with eNOS (1.7 ± 0.1 -fold vs.V, p<0.05, n=6) and decreased p-eNOS(S)1177 to eNOS (0.7± 0.06 -fold vs. V) in isolated membrane fractions (p<0.05, n=8), and these responses were prevented in cells exposed to P+M. W e then permeabilized cells that were treated as above, and incubated them with 1 ° antibodies for eNOS, PP2A, I2PP2A, or ceramide, followed by incubation with fluorescent 2° antibodies to assess subcellular localization in intact cells using confocal microscopy. Relative to V-treatment, P: (i) increased ceramide association with I2PP2A (1.7 ± 0.06 -fold) and decreased the association between PP2A and I2PP2A (1.0 ± 0.05 -fold) in the cytosol; and (ii) increased PP2A association with eNOS (1.8 ± 0.09 -fold; all p<0.05) in the membrane. All responses were negated in P + M -treated cells (n=6 per combination). Endogenous ceramide disrupts the cytosolic interaction between I2PP2A and PP2A and causes translocation of PP2A to the membrane where it associates with eNOS. |
