Modulation of Nitric Oxide Release in Human Umbilical Vein Endothelial Cells by Myristolated‐PKC Epsilon Activator/Inhibitor Peptides

Abstract

Ischemia/reperfusion (I/R) injury plays a critical role in endothelial dysfunction as a consequence of limited nitric oxide (NO) bioavailability and excess generation of reactive oxygen species (ROS) generated by endothelial nitric oxide synthase (eNOS) during reperfusion. Activated protein kinase C epsilon (PKCɛ) binds to a specific receptor for activated C kinase (RACK‐1), facilitating its translocation from the cytosol to the cell membrane for phosphorylation of targets, such as eNOS, at serine‐1177 to augment activity. Previous in vitro and in vivo animal studies have shown that PKCɛ activation stimulates eNOS activity, increasing NO release; whereas PKCɛ inhibition using a peptide inhibitor that interferes with its interaction with RACK‐1 results in decreased NO release. However, the modulation of PKCɛ‐mediated eNOS activity has yet to be demonstrated in human cells. In this study, we aim to demonstrate enhancement and attenuation of NO release in human umbilical vein endothelial cells (HUVECs) using cell‐permeable, myristic‐acid conjugated PKCɛ activator (Myr‐PKCɛ+), Myr‐HDAPIGYD (MW=1097 g/mol), and inhibitor (Myr‐PKCɛ−), Myr‐EAVSLKPT (MW=1054 g/mol), peptides respectively. Single‐donor HUVECs at passages 3–4 were grown to confluence in 6‐well (35 mm) plates. NO release was measured in real time using a calibrated NO electrode following the administration of 10 μM Myr‐PKCɛ+ or 10 μM Myr‐PKCɛ− treatments in the absence or presence of 10 μM acetylcholine (Ach) stimulation of NO release. Basal NO release (375±175 pM) was determined by measuring the difference between wells with and without cells (n=4). Myr‐PKCɛ+ increased NO levels by 36±13 pM (p<0.01, n=5) and Myr‐PKCɛ− decreased NO levels by 41±55 pM, p<0.01, n=5) significantly compared to baseline in the absence of Ach stimulation. In the presence of 10 μM Ach only, NO release significantly increased by 61±15 pM above baseline (p<0.01, n=5). In Ach‐stimulated cells, there was an additional significant increase in NO release by 70±23 pM (p<0.01, n=5) when treated with Myr‐PKCɛ+ compared to baseline, although there was no significance compared to the initial Ach‐stimulated NO release. Following initial 10 μM Ach administration, Myr‐PKCɛ− significantly reduced NO release by 48±16 pM, (p<0.01, n=5) compared to baseline. These results suggest Myr‐PKCɛ+ presumably increases NO release via activation of eNOS, whereas Myr‐PKCɛ− attenuates eNOS activity by inhibiting its phosphorylation in HUVECs; thus, eNOS modulation by these peptides demonstrate comparable effects across species. Elucidating the role of eNOS modulation is important for developing therapeutic interventions in the context of human organ I/R injury, in which uncoupled eNOS produces ROS instead of NO. Future studies will evaluate the effects of Myr‐PKCɛ+ and Myr‐PKCɛ− on NO release in HUVECs during reperfusion following prolonged ischemia compared to non‐ischemic controls.Support or Funding InformationThis study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Biomedical Sciences at Philadelphia College of Osteopathic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.