Abstract 447: Myristoylation of Protein Kinase C Beta II Peptide Inhibitor Facilitates Rapid Attenuation of Phorbol 12-myristate 13-acetate in Activated Superoxide Release in Isolated Rat Polymorphonuclear Leukocytes

Abstract

Protein kinase C beta II (PKCβII) activates polymorphonuclear leukocyte (PMN) superoxide (SO) production via NADPH oxidase (NOX-2) phosphorylation to exacerbate myocardial ischemia/reperfusion (I/R) injury. In prior studies, myristoylation (myr) of PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-), which disrupts PKCβII translocation/phosphorylation of NOX-2, was shown to dose-dependently attenuate PMN SO release induced by phorbol 12-myristate 13-acetate (PMA), a broad-spectrum PKC agonist. However, the role of myr on the inhibitory effects of myr-PKCβII- has yet to be elucidated. We hypothesized that myr-PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) would augment, myr-PKCβII- would attenuate, and scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram), a control for myr, would not affect PMA-induced PMN SO release compared to unconjugated peptides and nontreated controls. Rat PMNs (5х10 6 ) were incubated for 15 min at 37 o C in the presence/absence of SO dismutase (SOD; 10 μg/mL), unconjugated PKCβII+/-, myr-PKCβII+/-, or myr-PKCβII-scram (all 20 μM). SO release was measured by the change in absorbance at 550 nm via ferricytochrome c reduction after PMA (100 nM) stimulation for 390 sec. Data were analyzed by ANOVA using Student-Newman-Keuls post hoc analysis. Myr-PKCβII- significantly attenuated SO release (0.30±0.02; n=27; p<0.05) compared to nontreated controls (0.46±0.01; n=73), myr-PKCβII+ (0.46±0.03; n=25), unconjugated PKCβII+ (0.43±0.04; n=15), PKCβII- (0.43±0.02; n=22) and myr-PKCβII-scram (0.65±0.04; n=22). SOD (n=8), which rapidly converts SO to H 2 O 2 , significantly reduced absorbance by 94±7%, indicating that absorbance increased mainly due to PMA stimulation. Cell viability (trypan blue exclusion) was similar in all groups (94±2%). Unexpectedly, myr-PKCβII-scram significantly stimulated the highest increase in absorbance compared to all groups (p<0.01). Future studies will determine whether myr-PKCβII-scram augments absorbance by a different mechanism. Results suggest that myr improves myr-PKCβII- delivery compared to unconjugated PKCβII- but does not affect inhibition of PMA-induced PMN SO release. Myr-PKCβII- may thus effectively limit inflammation-induced I/R injury.