The Effects of Novel Myristoylated PKC Epsilon Peptide Modulators on Real‐Time Nitric Oxide Release in Human Umbilical Vein Endothelial Cells
Authors: Castro Jeremy , Dean Tameka , Suarez Michael , Lloyd Michael , Verwoert Alexis , Madison Ian , Martorana Rose , Andre Claudine , Thompson Chantel , Heller Gordon , Major Ahsal , Chen Qian , Barsotti Robert , Jenney Francis , Young Lindon 
Published: April 2020

Endothelial dysfunction is a major consequence of ischemia‐reperfusion (I/R) injury, such as during the re‐establishment of blood flow after endovascular thrombus removal. It is characterized by limited nitric oxide (NO) bioavailability and production of superoxide (SO) instead of NO, in part from uncoupled endothelial NO synthase (eNOS) activity when the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is elevated during reperfusion following prolonged ischemia. The protein kinase C epsilon (PKCɛ) isoform is known to participate in pre‐conditioning (PKCɛ activation treatment prior to ischemia) and post‐conditioning (PKCɛ inhibition treatment during reperfusion) to attenuate rat myocardial I/R injury. Normally following diacylglycerol‐dependent activation, PKCɛ binds to isoform‐specific receptors for activated C‐kinase (RACK) and translocates from the cytosol to the cell membrane to phosphorylate its targets, such as eNOS at Ser‐1177 to augment NO release when the BH2 to BH4 ratio is reduced to promote coupled eNOS activity that is normally seen prior to prolonged ischemia. Cell permeable myristic acid conjugated PKCɛ peptide activator (myr‐HDAPIGYD; myr‐PKCɛ+) and inhibitor (N‐myr‐EAVSLKPT; myr‐PKCɛ‐) has been shown to increase and decrease NO release, respectively, in rat aortic tissue by regulating PKCɛ translocation to eNOS. However, modulation of PKCɛ‐mediated NO release with these peptides in human cells remains undetermined. We hypothesize that myr‐PKCɛ+ will augment and myr‐PKCɛ‐ will attenuate NO release in cultured human umbilical vein endothelial cells (HUVECs). Real‐time HUVEC NO release (pmol) was measured using a calibrated NO electrode following administration of myr‐PKCɛ+ or myr‐PKCɛ‐ ± acetylcholine (Ach; positive control). Data were analyzed by ANOVA using Fishers post‐hoc analysis. Basal NO levels (66±6; n=35) were determined as the pmol difference between cell‐populated (106 cells/well) and reagent only wells. Control Ach (10 μM) enhanced NO release (102±7; n=37), myr‐PKCɛ+ (10 μM) enhanced NO release with Ach (107±16; n=8) and without Ach (107±13; n=7), and myr‐PKCɛ‐ (10 μM) attenuated NO release with Ach (37±9; n=13) and without Ach (7±29; n=9) compared to basal levels (all p<0.05). Results suggest that myr‐PKCɛ +/− effects on NO release is translational across mammalian species, presumably through activation and inhibition of PKCɛ‐mediated phosphorylation of eNOS. Thus, therapeutic intervention that targets inhibition of PKCɛ to reduce uncoupled eNOS activity during reperfusion may yield protective effects via attenuating the extent of I/R injury seen in clinical myocardial infarction, coronary bypass, coronary angioplasty, and organ transplantation.

Support or Funding Information

This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC.