Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH ) is oxidized to dihydrobiopterin (BH2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor (myr-PKCϵ ; N-myr-EAVSLKPT) and activator (myr-PKCϵ+; N-myr-HDAPIGYD). We hypothesize that myr-PKCϵ with uncoupled eNOS (i.e., bound to BH ) should reduce leukocyteendothelial interactions in postcapillary venules and H2O2 levels after SWL. Myr-PKCϵ+ should promote similar effects when eNOS is coupled (i.e., bound to BH4). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH2 and test solutions (Krebs' buffer, 10 μM myr-PKCϵ+, or 10 μM myr-PKCϵ ) with or without 100 μM BH 4. Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail (myr-PKCϵ+ [0.9 mg/kg]/myr-PKCϵ- [0.8 mg/kg] with BH2 [2mg/kg] or BH4 [0.8 mg/kg]) was given i.v. NO or H2 O2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student- Newman-Keuls test. BH -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr- PKCϵ+ (n = 6) and attenuated with myr-PKCϵ (n = 5) compared to control (n=5) at 120 min (p