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Protein Kinase C Beta II Peptide Modulation of Superoxide Release in Rat Polymorphonuclear Leukocytes
Author(s) -
Ebo Chinyere,
Lipscombe Christina,
Metellus Daphne,
Humayun Annam,
Nair Arjun,
Patel Harsh,
Michaels Megan,
Dang Jennifer,
Finnegan Matthew,
Checchio Lucy,
MuftauLediju Faosat,
Martorana Rose M,
Chen Qian,
Barsotti Robert,
Young Lindon
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.836.5
Subject(s) - protein kinase c , superoxide , activator (genetics) , nadph oxidase , chemistry , respiratory burst , phorbol , peptide , n formylmethionine leucyl phenylalanine , agonist , staurosporine , chemotaxis , superoxide dismutase , bisindolylmaleimide , microbiology and biotechnology , biochemistry , phosphorylation , reactive oxygen species , receptor , biology , antioxidant , enzyme
Previous studies have shown that protein kinase C (PKC) alpha, beta II (βII) and zeta inhibition attenuates polymorphonuclear leukocyte (PMN) superoxide (SO) release, whereas PKC delta inhibition augments this response via inhibiting phosphorylation of PMN NADPH oxidase (NOX‐2). However, to date, there is no information regarding the role of PKCβII peptide activator on regulating PMN SO release. PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation after stimulation by N‐formyl‐L‐methionyl‐Lleucyl‐L‐phenylalanine (fMLP; a PMN chemotactic receptor agonist) or phorbol 12‐myristate 13‐acetate (PMA; a diacylglycerol mimetic). It is known that myristic acid (myr‐) conjugation is an effective means to facilitate intracellular delivery of the cargo sequence to regulate PKCβII activity. We hypothesize that myr‐PKCβII peptide‐activator ( N ‐myr‐SVEIWD; myr‐PKCβ+) would increase PMA/fMLP‐induced rat PMN SO release, whereas, myr‐PKCβII peptideinhibitor ( N‐ myr‐SLNPEWNET; myr‐PKCβ−) would attenuate this response compared to non‐drug treated controls or unconjugated native PKCβ+/− peptide sequences. PMNs were isolated via peritoneal lavage from male Sprague‐Dawley rats (~500g) and were incubated (5x10 6 ) for 15min at 37°C in the presence/absence of native/myr‐PKCβ+/− (20 mM; n=8[fMLP]) or SO dismutase (SOD;10mg/mL; n=8) as positive control. fMLP or PMA induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 90 sec (fMLP;1mM; n=19) or 360 sec (PMA;100nM; n=17). fMLP‐induced PMN SO release increased absorbance to 0.155±0.018 in non‐drug controls, 0.162±0.037 in myr‐PKCβ+, 0.136±0.025 in native PKCβ+, 0.070±0.012 in myr‐PKCβ− and 0.146±0.018 in native PKCβ− treated PMNs at 30 sec. Myr‐PKCβ− significantly decreased fMLP‐induced PMN SO release compared to nondrug controls, myr‐PKCβ+ and native PKC β+/− peptides (p<0.05). PMA‐induced PMN SO release increased absorbance to 0.41±0.039 in non‐drug controls, 0.517±0.047(n=7) in myr‐PKCβ+, 0.345±0.071(n=5) in native PKCβ+, 0.241±0.055(n=6) in myr‐PKCβ−, and 0.308±0.052(n=6) in native PKC β− at 360 sec. Myr‐PKCβ‐significantly attenuated PMA‐SO release compared to non‐drug controls and myr‐PKCβ+ (p<0.05). SOD‐treated samples showed >90% reduction of fMLP or PMA‐induced SO release and native PKCβ+/− was not different from non‐drug controls. Cell viability ranged between 94±2% and 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr‐PKCβ‐significantly attenuates fMLP/PMA‐induced SO release, whereas myr‐PKCβ+ only shows a trend to augment fMLP/PMA‐induced SO release. Additional studies will be conducted to determine if myr‐PKCβ+ will significantly augment PMN SO release. Support or Funding Information This research was supported by the Department of Bio‐Medical Sciences and the Division of Research at Philadelphia College of Osteopathic Medicine (PCOM) and by Young Therapeutics, LLC. 4170 City Avenue, Philadelphia, PA 19131 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .