2 research outputs found
The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes
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 (Ī²II) mediating this response is not known. Itās known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCĪ²II activator and inhibitor peptides work by augmenting or attenuating PKCĪ²II translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCĪ²II peptide-activator (N-myr-SVEIWD; myr-PKCĪ²+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCĪ²II peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCĪ²-) 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-PKCĪ²+/- (20 Ī¼M) or SO dismutase (SOD;10Ī¼g/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 0.39Ā±0.04 in non-drug treated controls (n=28), and 0.49Ā±0.05 in myr-PKCĪ²+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCĪ²- (0.26Ā±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCĪ²+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94Ā± to 98Ā±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCĪ²- significantly attenuates PMA-induced SO release, whereas myr-PKCĪ²+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCĪ²+/- 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
Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury
Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCĪ²II) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCĪ²II-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCĪ²II during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCĪ²II- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCĪ²II peptide activator (N-myr-SVEIWD; myr-PKCĪ²II+) will show no improvement compared to control. Myristoylation of PKCĪ²II peptides facilitate their entry into the cell in order to affect PKCĪ²II activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCĪ²II+ (20Ī¼M; n=9), myr-PKCĪ²II- (20ĀµM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCĪ²II- significantly improved LV end-diastolic pressure 37Ā±7 mmHg compared to control (58Ā±5; p\u3c0.01) and myr-PKCĪ²II+ (58Ā±4; p\u3c0.01). Myr-PKCĪ²II- significantly reduced infarct size to 14Ā±3% compared to control (26Ā±5%; p\u3c0.01), while myr-PKCĪ²II+ (25Ā±3%) showed no difference. The data indicate that myr-PKCĪ²II- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis