Myristoylated PKC β II Peptide Inhibitor Exerts Dose-Dependent Inhibition of N-Formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP) Induced Leukocyte Superoxide Release

Abstract

Phosphorylation of polymorphonuclear leukocyte (PMN) NADPH oxidase by protein kinase C (PKC) is essential to generate superoxide (SO) release. Inhibition of PMN SO release attenuates inflammation mediated vascular tissue injury during myocardial ischemia/ reperfusion (MI/R) injury. PMNs express five isoforms of PKC (alpha (α), beta I (βI), beta II (βII), delta (δ) and zeta (ζ)) and their role regulating this response have not been fully elucidated. PKC α, βII and ζ are thought to positively regulate PMN SO release, whereas PKC δ negatively regulates PMN SO release. [1,2] PKC βI, in contrast to the other four isoforms, translocates to the nucleus after second messenger stimulation [1]. PKC βII, a classical isoform, is activated by calcium and diacylglycerol (DAG) following PMN chemotactic receptor stimulation with fMLP peptide (Figure 1) [1]. Activated PKC βII will phosphorylate PMN NADPH oxidase to produce SO. Selective PKC βII peptide inhibitor has been developed based on its binding sites to receptor for activated C kinase (RACK) domain (Figure 2) [3]. RACK shuttles cytosolic PKC βII to interact with cell membrane substrates (e.g., NADPH oxidase). Myristoylation of peptides is known to be an effective strategy to enable simple diffusion through cell membranes to affect PKC function [4,5].The cell permeable myristoylated (myr) PKC βII peptide inhibitor is known to inhibit PMN SO release at doses that correlated with restoration of postreperfused cardiac function following global MI(20 min)/R(45 min) in leukocyte mediated cardiac MI/R dysfunction and more recently in prolonged MI(30 min)/R(90 min) in isolated rat hearts [1,6,7]. However, a full dose-response curve with Myr-PKC βII peptide inhibitor has not been indicated previously. Characterizing the full dose-response effects is essential in identifying putative mechanisms responsible for attenuating vascular and tissue injury following I/R.