The Role of Protein Kinase C Epsilon in the Regulation of Endothelial Nitric Oxide Synthase (eNOS) during Oxidative Stress caused by Extracorporeal Shock Wave Lithotripsy (ESWL)

BACKGROUND: Clinical ESWL treatment to ablate kidney stones can cause acute to chronic damage in renal microvasculature leading to decreased renal blood flow and hypertension. Shockwaves can stimulate endothelial cells to release superoxide resulting in decreased nitric oxide (NO) bioavailability and increased oxidative stress, causing vascular endothelial dysfunction in the kidney. When the dihydrobiopterin:tetrahydrobiopterin ratio is increased during oxidative stress such as ESWL, eNOS becomes uncoupled and produces superoxide instead of NO. Superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase. Protein kinase C epsilon (PKC-ε) is known to positively regulate endothelial NO synthase (eNOS) activity. In order to establish controls for the effects of PKC-ε activator and inhibitor, the effect of ESWL was tested by the comparison of ESWL-treated rats to those with no ESWL exposure, both with a saline infusion. We hypothesized that the PKC-ε peptide inhibitor (Myr-EAVSLKPT, MW = 1054.6) would decrease ESWL-induced H2O2 release and decreased the attenuation of NO release compared to ESWL-saline control rats. PKC-ε activator (Myr-NDAPIGYD, MW = 1098.5) was expected to show no effect on H2O2 or NO release, displaying a similar trend to ESWL-saline control rats

The Role of Endothelial Nitric Oxide Synthase (eNOS) Uncoupling on Leukocyte-Endothelial Interactions in Rat Mesenteric Postcapillary Venules

BACKGROUND: Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte-endothelial interactions associated with vascular injury. Endothelial NO synthase (eNOS) is coupled to L-arginine in the presence of tetrahydrobiopetrin (BH4) to produce NO. However, when BH4 is oxidized to dihydrobiopetrin (BH2) under conditions of oxidative stress, the ratio of BH2 to BH4 is increased causing the uncoupling of eNOS to use molecular oxygen as a substrate, instead of L-arginine, to produce superoxide

The Effects of Dihydrobiopterin and Tetrahydrobiopterin on Hydrogen Peroxide and Nitric Oxide Release During Extracorporeal Shockwave Lithotripsy

Extracorporeal shockwave lithrotripsy (ESWL) is an effective, non-invasive clinical therapy utilized to break up stones in the kidney and urinary tract. A lithotripter generates high-energy acoustic pulses and propagates those shock waves through a lens on a region that focuses on the location of the stone, in turn breaking up the stone. The successive pulses generate shearing forces and cavitation bubbles. Cavitation bubbles are the formation and implosion of liquid free zones. The cavitation bubbles implode rapidly to create their own shockwaves that also put pressure on the stone. After treatment, fragmentation of the stone allows the debris to be cleared by the flow of the urinary tract. The problem is that to break up the kidney stone, it requires many repetitive shock waves that not only hit the kidney stone but also the surrounding tissue. Although lithotripsy provides a safer alternative to invasive treatments for removing harmful stones, ESWL may cause prolonged vasoconstriction after ESWL treatment, reducing renal blood flow, and subsequent endothelial dysfunction, which may cause kidney damage leading to acute to chronic hypertension clinically. ESWL-induced vascular oxidative stress and further endothelial dysfunction may be mediated by reduced levels of endothelial-derived nitric oxide (NO) and/or increased reactive oxygen species. Previous studies have shown that ESWL can induce oxidative stress, which can cause an increase in blood hydrogen peroxide (H2O2) and a decrease in endothelial-derived NO bioavailability release. Under normal conditions, tetrahydrobiopterin (BH4) is the cofactor to promote eNOS coupling, and endothelial-derived NO is produced. When the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is increased during oxidative stress, such as ESWL, BH2 promotes eNOS uncoupling and produces superoxide (SO) instead of NO. (1,2) (Figure 1) SO is then later converted to H2O2 by superoxide dismutase. BH4 and BH2 bind to eNOS with equal affinity, therefore the ratio will determine whether eNOS principally produces NO or SO

Effects of NADPH oxidase inhibitor apocynin on real-time blood hydrogen peroxide release in femoral artery/vein ischemia and reperfusion

Background: Vascular endothelial dysfunction can initiate oxidative stress during ischemiaJreperfusion (IIR). Endothelial dysfunction is characterized by an increase in blood hydrogen peroxide (H20 ]) and a decrease in endothelial-derived nitric oxide (NO) bioavailability. Previous studies using Go 6983, a broad-spectrum protein kinase C inhibitor that can inhibit NADPH oxidase activity, has attenuated blood H20 ] levels during femoralliR in vivo. This study examines the effects of apocynin, a direct NADPH oxidase inhibitor, on real-time blood H20 ] levels in femoral I1R in vivo. H20 ] microsensors (100 Mm) were inserted into both femoral veins in anesthetized rats

The Effects of Modulating eNOS Activity and Coupling on Leukocyte-endothelial Interactions in Rat Mesenteric Postcapillary Venules

Background: Leukocyte-endothelial interactions associated with vascular injury are attenuated by endothelial-derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of tetrahydrobiopterin (BH4) produces NO from L-arginine and is termed eNOS coupling. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide instead of NO. Protein kinase C epsilon (PKC ε) positively regulates eNOS activity. This study examined modulating eNOS activity and coupling by superfusing BH2 (100 μM) by itself, combined with PKC ε activator (10μM) or PKC ε inhibitor, or combined with BH4 (100μM) and PKC ε activator in rat mesenteric venules

Effects of protein kinase C broad spectrum inhibitor Gö 6983 on real-time blood nitric oxide and hydrogen peroxide release in femoral artery/vein ischemia and reperfusion

BACKGROUND: Vascular endothelial dysfunction is one of the earliest recognizable events under hyperglycemic conditions. It is characterized by decreased endothelium-derived nitric oxide (NO) and increased oxidative stress, such as superoxide and hydrogen peroxide (H2O2). However, the real-time changes in blood NO and H2O2 levels under acute hyperglycemia have not been evaluated

Gö 6983: A Fast Acting Protein Kinase C Inhibitor That Attenuates Myocardial Ischemia/Reperfusion Injury

Reperfusion injury is characterized by a decrease in endothelial release of nitric oxide within 5 min after reperfusion, increased leukocyte-endothelium interaction, and transmigration of leukocytes into the myocardium, producing cardiac contractile dysfunction. Gö 6983 is a fast acting, lipid soluble, broad spectrum protein kinase C inhibitor. When administered at the beginning of reperfusion, it can restore cardiac function within 5 min and attenuate the deleterious effects associated with acute ischemia/reperfusion. Gö 6983 may offer greater cardioprotection than other broad-spectrum PKC inhibitors in postischemic reperfusion injury because it inhibits PKC(zeta) as well as four other isoforms. The cardioprotection is associated with decreased leukocyte superoxide release and increased endothelial derived nitric oxide from vascular tissue. In vitro studies of human tissue showed that Gö 6983 significantly inhibited antigen-induced superoxide release from leukocytes of patients previously sensitized to tree pollen. In human vascular tissue, Gö 6983 inhibited intracellular Ca(2+) accumulation, suggesting a mechanism for its vasodilator properties. These studies suggest that Gö 6983 would be an effective compound to use in a clinical ischemia/reperfusion setting of organ transplantation and/or cerebral ischemia where inhibiting superoxide release and vasoconstriction in postischemic tissues would allow for better restoration of organ function during reperfusion. However, given the broad-spectrum action of Gö 6983, careful titration of the dose regimen would be recommended to ensure a successful outcome in the setting of organ transplantation and/or cerebral ischemia