Abstract
Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previously, myristic acid conjugated (or myristoylated) PKCβII inhibitor (myr-PKCβII-) significantly attenuated PMN SO release when dual conjugated to myristic acid and Trans-activator of transcription (myr-Tat-PKCβII-; N-myr-Tat-CC-SLNPEWNET) compared to myr-conjugation alone. However, the optimal concentration of myr-Tat-PKCβII- has yet to be determined. We hypothesized that myr-Tat conjugation would enhance the intracellular delivery of PKCβII- cargo and attenuate SO release in a concentration-dependent manner while retaining greater cell viability at lower concentrations.
This study tested the concentration-dependent effects of myr-Tat-PKCβII- on SO release and cell viability compared to myr-Tat-PKCβII scrambled (myr-Tat-PKCβII- scram; N-myr-Tat-CC-WNPESLNTE), unconjugated PKCβII-, and untreated control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII- (20μM), myr-Tat-PKCβII- (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr-Tat-PKCβII-scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after phorbol-12-myristate-13 acetate (PMA) stimulation (100nM). Intracellular delivery was evaluated by the magnitude of PMA-induced PMN SO release attenuation with the PKCβII- cargo. Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis.
Myr-Tat toxicity occurs at concentrations higher than 2 μM. Myr-Tat PKCβII- significantly decreased SO release compared to control while retaining similar cell viability at 5 μM (n=15, 0.384±0.03) and 7.5μM (n=11, 0.391±0.05) concentrations. Myr-Tat PKCβII-scram 5μM (n=7, 0.44±0.07) and 7.5μM (n=5, 0.409±0.11) were not different from control.
Results suggest that Myr-Tat-PKCβII- 5μM significantly attenuates PMA-induced PMA SO release while retaining cell viability at > 80%. Future studies will assess the effects of myr- or myr-Tat-PKCβII- peptides on PKCβII- translocation activity.
Original language | American English |
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State | Published - May 11 2022 |
Keywords
- Superoxide
- ischemia-reperfusion
Disciplines
- Medicine and Health Sciences