Location
Philadelphia, PA
Start Date
9-5-2018 1:00 PM
Description
Protein kinase C epsilon (PKCε) is known to increase endothelial nitric oxide synthase (eNOS) activity and subsequent nitric oxide (NO) release via phosphorylation at serine 1177 of eNOS in previous in vitro and in vivo animal studies under normoxic conditions. However, the role of PKCε regulating eNOS activity and NO release is not well known in human endothelial cells. Moreover, understanding the role of PKCε in regulating eNOS activity would be essential in the clinical setting of myocardial infarction. Re-establishing blood flow after thrombus removal results in endothelial dysfunction and is characterized by decreased NO bioavailability and excess reactive oxygen species (ROS) generated by uncoupled eNOS during reperfusion. Therefore, determining NO release using selective cell permeable PKCε activator (Myr-PKCε+) and inhibitor peptides (Myr-PKCε- ) in cultured human umbilical vein endothelial cells (HUVECs) under normoxic conditions would provide a foundation to test these peptides under hypoxic-reoxygenation conditions when eNOS would be uncoupled and produce ROS instead of NO release. Single-donor HUVECs at passages 3-4 were grown to confluence in 6-well (106 cells/well) plates. NO release was measured in real time using a calibrated NO electrode following the administration of 10 μM Myr-PKCε+ or 10 μM Myr-PKCε- treatments in the absence or presence of 10 μM acetylcholine (Ach) stimulation of NO release. Basal NO release (83±12 pM) was determined by measuring the difference between wells with and without HUVECs (n=9, P<0.05 compared to wells with no cells). In the absence of Ach stimulation, Myr-PKCε+ significantly enhanced NO release to 136±13 pM (n=6, p<0.05) and Myr-PKCε- attenuated total NO release to 16±27 pM (n=7, p<0.05) compared to basal levels. Similar to Myr-PKCε+, 10 μM Ach significantly enhanced NO release to 153±11 pM above basal levels (p<0.05, n=20). Moreover, in the presence of 10 μM Ach, Myr-PKCε+ still significantly increased NO release (129±17 pM, p<0.05, n=8) and Myr-PKCε- decreased NO release (34±12 pM, p<0.05, n=7) compared to basal levels. In summary, these results suggest that in cultured HUVECs, Myr-PKCε+ presumably increase NO release via activation of eNOS; whereas Myr-PKCε- attenuates eNOS activity by inhibiting its phosphorylation. Therefore, the effects of Myr-PKCε+ and Myr-PKCε- are translational across species in regulating NO release.
Embargo Period
5-30-2018
Modulation of Nitric Oxide Release in Cultured Human Umbilical Vein Endothelial Cells by Myristoylated-PKC Epsilon Activator/Inhibitor Peptides
Philadelphia, PA
Protein kinase C epsilon (PKCε) is known to increase endothelial nitric oxide synthase (eNOS) activity and subsequent nitric oxide (NO) release via phosphorylation at serine 1177 of eNOS in previous in vitro and in vivo animal studies under normoxic conditions. However, the role of PKCε regulating eNOS activity and NO release is not well known in human endothelial cells. Moreover, understanding the role of PKCε in regulating eNOS activity would be essential in the clinical setting of myocardial infarction. Re-establishing blood flow after thrombus removal results in endothelial dysfunction and is characterized by decreased NO bioavailability and excess reactive oxygen species (ROS) generated by uncoupled eNOS during reperfusion. Therefore, determining NO release using selective cell permeable PKCε activator (Myr-PKCε+) and inhibitor peptides (Myr-PKCε- ) in cultured human umbilical vein endothelial cells (HUVECs) under normoxic conditions would provide a foundation to test these peptides under hypoxic-reoxygenation conditions when eNOS would be uncoupled and produce ROS instead of NO release. Single-donor HUVECs at passages 3-4 were grown to confluence in 6-well (106 cells/well) plates. NO release was measured in real time using a calibrated NO electrode following the administration of 10 μM Myr-PKCε+ or 10 μM Myr-PKCε- treatments in the absence or presence of 10 μM acetylcholine (Ach) stimulation of NO release. Basal NO release (83±12 pM) was determined by measuring the difference between wells with and without HUVECs (n=9, P<0.05 compared to wells with no cells). In the absence of Ach stimulation, Myr-PKCε+ significantly enhanced NO release to 136±13 pM (n=6, p<0.05) and Myr-PKCε- attenuated total NO release to 16±27 pM (n=7, p<0.05) compared to basal levels. Similar to Myr-PKCε+, 10 μM Ach significantly enhanced NO release to 153±11 pM above basal levels (p<0.05, n=20). Moreover, in the presence of 10 μM Ach, Myr-PKCε+ still significantly increased NO release (129±17 pM, p<0.05, n=8) and Myr-PKCε- decreased NO release (34±12 pM, p<0.05, n=7) compared to basal levels. In summary, these results suggest that in cultured HUVECs, Myr-PKCε+ presumably increase NO release via activation of eNOS; whereas Myr-PKCε- attenuates eNOS activity by inhibiting its phosphorylation. Therefore, the effects of Myr-PKCε+ and Myr-PKCε- are translational across species in regulating NO release.
Comments
Presented by Tameka Dean.