TY - CONF
T1 - The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes
AU - Lipscombe, Christina
AU - Ebo, Chinyere
AU - Metellus, Daphne
AU - Dang, Jennifer
AU - Humayun, Annam
AU - Nair, Arjun
AU - Patel, Harsh
AU - Michaels, Megan
AU - Finnegan, Matthew
AU - Checchio, Lucy
AU - Muftau-Lediju, Faosat
AU - Martorana, Faosat
AU - Chen, Qian
AU - Barsotti, Robert J.
AU - Young, Lindon H.
PY - 2019/5/8
Y1 - 2019/5/8
N2 - Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p<0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p<0.05). SOD-treated samples showed >90% reduction of PMA-induced SO release and was significantly different from all groups (p<0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC.
AB - Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p<0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p<0.05). SOD-treated samples showed >90% reduction of PMA-induced SO release and was significantly different from all groups (p<0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC.
KW - Phorbol 12-myristate 13-acetate (PMA)
KW - polymorphonuclear leukocyte (PMN)
KW - superoxide (SO) release
KW - protein kinase C (PKC) beta II (βII) (PKCβII)
UR - https://digitalcommons.pcom.edu/research_day/research_day_PA_2019/researchPA2019/29
M3 - Presentation
ER -