| Description |
In preliminary studies we showed that basal and insulin-mediated endothelial nitric oxide synthase phosphorylation (p-eNOS) and nitric oxide (NO) production are impaired when Bovine Aortic Endothelial Cells (BAECs) are treated with the saturated fatty acid palmitate (pal, 500 uM) vs. vehicle (veh) for 3 hours (h). Co-incubation of BAECs with the ceramide synthesis inhibitor myriocin (myr, 10 uM) negated these effects. Insulin-evoked 1.73 ± 0.09-fold increases in p-eNOS SI 177 that were abolished by pal, but were restored by concurrent treatment with myr (western immunoblotting, n=T6-32, p<0.05). Thus, pal incubation impairs insulin-stimulated p-eNOS SI 177. 100 nM insulin evoked 1.65 ± 0.10-fold increases in NO production by BAECs. These increases were negated by pal, and were restored by concurrent treatment of pal with myr (amperometric probes, n=9-31, p<0.05). Thus, we determined that palmitate incubation impairs insulin-stimulated NO production. ROS can lower NO bioavailability. The purpose of this study was to determine whether pal increases ROS production in a ceramide-dependent manner and if so, to determine the source of ROS production. DCFDA fluorescence was used to assess ROS after BAECs were incubated for 3h with veh ± pal, myr, 10 uM potassium cyanide (KCN, a mitochondrial ROS inhibitor), or 1 uM N-vanillylnonanamide (NVN, an NADPH oxidase inhibitor). Relative to veh, palevoked increases (2.8±0.1-fold, p<0.05, n=48) in ROS production were blunted (15±5%; p<0.05) but were not completely abolished by myr, suggesting the existence of ceramidedependent and -independent pathways. KCN, NVN, and KCN + NVN lowered (p<0.05) pal-evoked ROS production by 37±4%, 18±7%, and 39±12% (n=8-32), respectively, suggesting that in addition to mitochondrial and NADPH oxidase-dependent sources of ROS, additional sources of pal-induced ROS exist. Pal-induced ROS production decreased further when BAECs were co-incubated with myr + NVN (46±4%, p<0.05, n=16) vs. NVN alone, but not when myr was co-incubated with KCN (n=12) or KCN + NVN (n=16). Moreover, pal-evoked increases (p<0.05, n=12) in NADPH oxidase activity were similar in the absence (1.6±0.2-fold) and presence (2.1±0.2-fold) of myr. Thus, pal-evoked and ceramide-mediated ROS production in BAECs is not derived from NADPH oxidase but may arise from mitochondria. |
