Nitric Oxide Synthase Function Research Articles

Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

A Hypothesis for Examining Skeletal Muscle Biopsy-Derived Sarcolemmal nNOSμ as Surrogate for Enteric nNOSα Function.

The pathophysiology of gastrointestinal motility disorders is controversial and largely unresolved. This provokes empiric approaches to patient management of these so-called functional gastrointestinal disorders. Preliminary evidence demonstrates that defects in neuronal nitric oxide synthase (nNOS) expression and function, the enzyme that synthesizes nitric oxide (NO), the key inhibitory neurotransmitter mediating mechano-electrical smooth muscle relaxation, is the major pathophysiological basis for sluggishness of oro-aboral transit of luminal contents. This opinion is an ansatz of the potential of skeletal muscle biopsy and examining sarcolemmal nNOSμ to provide complementary insights regarding nNOSα expression, localization, and function within enteric nerve terminals, the site of stimulated de novo NO synthesis. The main basis of this thesis is twofold: (a) the molecular similarity of the structures of nNOS α and μ, similar mechanisms of localizations to “active zones” of nitrergic synthesis, and same mechanisms of electron transfers during NO synthesis and (b) pragmatic difficulty to routinely obtain full-thickness biopsies of gastrointestinal tract, even in patients presenting with the most recalcitrant manifestations of stasis and delayed transit of luminal contents. This opinion attempts to provoke dialog whether this approach is feasible as a surrogate to predict catalytic potential of nNOSα and defects in nitrergic neurotransmission. This discussion makes an assumption that similar molecular mechanisms of nNOS defects shall be operant in both the enteric nerve terminals and the skeletal muscles. These overlaps of skeletal and gastrointestinal dysfunction are largely unknown, thus meriting that the thesis be validated in future by proof-of-principle experiments.

Secondhand Smoking Is Associated With Vascular Inflammation

The relative risk for cardiovascular diseases in passive smokers is similar to that of active smokers despite almost a 100-fold lower dose of inhaled cigarette smoke. However, the mechanisms underlying the surprising susceptibility of the vascular tissue to the toxins in secondhand smoke (SHS) have not been directly investigated. The aim of this study was to investigate directly vascular endothelial cell function in passive smokers. Using a minimally invasive method of endothelial biopsy, we investigated directly the vascular endothelium in 23 healthy passive smokers, 25 healthy active smokers, and 23 healthy control subjects who had never smoked and had no regular exposure to SHS. Endothelial nitric oxide synthase (eNOS) function (expression of basal eNOS and activated eNOS [phosphorylated eNOS at serine1177 (P-eNOS)]) and expression of markers of inflammation (nuclear factor-κB [NF-κB]) and oxidative stress (nitrotyrosine) were assessed in freshly harvested venous endothelial cells by quantitative immunofluorescence. Expression of eNOS and P-eNOS was similarly reduced and expression of NF-κB was similarly increased in passive and active smokers compared with control subjects. Expression of nitrotyrosine was greater in active smokers than control subjects and similar in passive and active smokers. Brachial artery flow-mediated dilation was similarly reduced in passive and active smokers compared with control subjects, consistent with reduced endothelial NO bioavailability. Secondhand smoking increases vascular endothelial inflammation and reduces active eNOS to a similar extent as active cigarette smoking, indicating direct toxic effects of SHS on the vasculature.

A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from L-arginine or nitrite?

The NO/sGC/cGMP/PKG system is one of the most powerful mechanisms responsible for platelet inhibition. In numerous publications, expression of functional NO synthase (NOS) in human and mouse platelets has been reported. Constitutive and inducible NOS isoforms convert L-arginine to NO and L-citrulline. The importance of this pathway in platelets and in endothelial cells for the regulation of platelet function is discussed since decades. However, there are serious doubts in the literature concerning both expression and functionality of NOS in platelets. In this review, we aim to present and critically evaluate recent data concerning NOS expression and function in platelets, and to especially emphasise potential pitfalls of detection of NOS proteins and measurement of NOS activity. Prevailing analytical problems are probably the main sources of contradictory data on occurrence, activity and function of NOS in platelets. In this review we also address issues of how these problems can be resolved. NO donors including organic nitrites (RONO) and organic nitrate (RONO2) are inhibitors of platelet activation. Endogenous inorganic nitrite (NO2 (-)), the product of NO autoxidation, and exogenous inorganic nitrite are increasingly investigated as NO donors in the circulation. The role of platelets in the generation of NO from nitrite is also discussed.

Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina: Implications for Nitric Oxide Synthase Function in Retinopathy

Retinopathy of prematurity is a sight-threatening complication of premature birth caused by nitro-oxidative insult to the developing retinal vasculature during therapeutic hyperoxia exposure and later ischemia-induced neovascularization on supplemental oxygen withdrawal. In the vasodegenerative phase, during hyperoxia, defective endothelial nitric oxide synthase (NOS) produces reactive oxygen and nitrogen free radicals rather than vasoprotective nitric oxide for unclear reasons. Crucially, normal NOS function depends on availability of the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4). Because BH4 synthesis is controlled enzymatically by GTP cyclohydrolase (GTPCH), we used GTPCH-depleted mice [hyperphenylalaninemia strain (hph1)] to investigate the impact of hyperoxia on BH4 bioavailability and retinal vascular pathology in the neonate. Hyperoxia decreased BH4 in retinas, lungs, and aortas in all experimental groups, resulting in a dose-dependent decrease in NOS activity and, in the wild-type group, elevated NOS-derived superoxide. Retinal dopamine levels were similarly diminished, consistent with the dependence of tyrosine hydroxylase on BH4. Despite greater depletion of BH4, the hph(+/-) and hph1(-/-) groups did not show exacerbated hyperoxia-induced vessel closure, but exhibited greater vascular protection and reduced progression to neovascular disease. This vasoprotective effect was independent of enhanced circulating vascular endothelial growth factor (VEGF), which was reduced by hyperoxia, but to local retinal ganglion cell layer-derived VEGF. In conclusion, a constitutively higher level of VEGF expression associated with retinal development protects GTPCH-deficient neonates from oxygen-induced vascular damage.

Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤20Gy) and time-dependent (6–72h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-l-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.

Plasma arginine metabolites reflect airway dysfunction in a murine model of allergic airway inflammation.

L-arginine metabolism is important in the maintenance of airway tone. Shift of metabolism from the nitric oxide synthase to arginase pathways contributes to the increased airway responsiveness in asthma. We tested the hypothesis that systemic levels of L-arginine metabolites are biomarkers reflective of airway dysfunction. We used a mouse model of acute allergic airway inflammation to OVA that manifests with significant airway hyperresponsiveness to methacholine. To determine tissue arginase activity in vivo, the isotopic enrichment of an infused L-arginine stable isotope and its product amino acid L-ornithine were measured in lung and airway homogenates using liquid chromatography-tandem mass spectrometry. Tissue and plasma concentrations of other L-arginine metabolites, including L-citrulline and symmetric and asymmetric dimethylarginine, were measured and correlated with lung arginase activity and methacholine responsiveness of the airways. The effectiveness of intratracheal instillation of an arginase inhibitor (boronoethylcysteine) on pulmonary arginase activity and circulating concentrations of L-arginine metabolites was also studied. We demonstrate that 1) plasma indexes of L-arginine bioavailability and impairment of nitric oxide synthase function correlate with airway responsiveness to methacholine; 2) plasma levels of L-ornithine predict in vivo pulmonary arginase activity and airway function; and 3) acute arginase inhibition reduces in vivo pulmonary arginase activity to control levels and normalizes plasma L-ornithine, but not L-arginine, bioavailability in this model. We conclude that plasma L-ornithine may be useful as a systemic biomarker to predict responses to therapeutic interventions targeting airway arginase in asthma.

Vasorelaxant effect of 17α-ethynylestradiol on human saphenous vein.

A protective effect for estrogens against cardiovascular problems has long been known. The aim of this study was to investigate the vasorelaxant effect of 17α-Ethynylestradiol (17α-EE) on human saphenous vein. The veins were suspended horizontally between two triangular stainless steel hooks for the measurement of isometric tension in individual organ baths containing 10ml Krebs solution, at 37°C and gassed with carbogen under 3gr optimum tension. The effect of different concentrations of 17α-EE (2-40 μM) on vascular tone was investigated in veins precontracted with PGF2α. Relaxation was measured after 40min and expressed as the percent decrease of initial contraction. To determine the involvement of potassium channels, endothelium, nitric oxide synthase, guanylylcyclase and prostaglandins in the vasorelaxant effect of estrogen, the veins were incubated with tetraethyl ammonium, N-nitro-L-arginine methyl ester, methylene blue or indomethacin, respectively for 20min prior to experimentation. Responses to 17α-EE were directly compared to those obtained in the same tissues in the absence of the inhibitors. The mean relaxations induced by 17α-EE with concentrations of 2, 5, 10, 20 and 40μM in tissues precontracted with PGF2α were 19.8 ±5.5%, 26.1±10.8%, 32.2±7.4%, 48.6±10.8%and56±7.6%, respectively. The results of the inhibition of potassium channels, nitric oxide synthase, guanylylcyclase, cyclooxygenase and removing endothelium in relaxation induced by 17α-EE on precontracted veins with PGF2α proved no significant differences. This study showed that 17α-EE has significant vasorelaxant effect on human saphenous vein in a concentration-dependent manner. This effect is probably independent of potassium channels, nitric oxide synthase, guanylylcyclase, prostaglandin synthesis and endothelium functions.

Kenneth D. Bloch, MD (1956–2014)

On a sunny, beautiful day in September 2014, we gathered in a funeral home in the town of Brookline, Massachusetts, to say a sad good-bye to our beloved friend and colleague, Dr. Kenneth D. Bloch. Ken was born in New York City on May 17, 1956, and raised in Brookline. After receiving his undergraduate and medical education at Brown University, he began his career as a medical resident at Massachusetts General Hospital (MGH), followed by a cardiology fellowship and then a position on the division staff in 1990. Two years later, he became an investigator in the Cardiovascular Research Center of MGH. Ken was a brilliant researcher who made a number of seminal contributions. He characterized the mechanisms of atrial natriuretic peptide and endothelial nitric oxide synthase function as well as the downstream mechanisms of nitric oxide and cyclic guanosine monophosphate (cGMP) signaling. More recently, Ken and colleagues in his laboratory discovered novel functions and mechanisms of action of the bone morphogenetic protein family in relation to cardiovascular disease and anemia of chronic inflammation, identifying this pathway as an important target in human disease. Ken was appointed the William T. G. Morton Professor of Anesthesia at Harvard Medical School in 2007 and Professor of Medicine in 2008. Ken was much more than a gifted molecular biologist and cardiologist. He had many personal gifts that were truly exceptional and that made him a rare mentor, colleague, and friend. Here are just a few stories. In our lab meetings, he would listen as we analyzed a problem of applied physiology or biochemistry or molecular biology and struggled with the tougher ones. Almost always, as the discussion ensued Ken would come up with a key insight unlocking the path to the next step in the research. If we got off track, Ken was always the one to gently point the way back to the core problem. Ken never failed to remember a past study by us or others. Even in these last years, as he struggled courageously with cancer, never once complaining, his memory was extraordinary and unfailing. Ken was a Pied Piper of scientists. He loved to work with young scientists, always had a flock of young students and physicians/scientists, and spent many hours each day guiding them and counseling them. He did this with the patience of a saint. He also spent untold hours correcting the drafts of papers, and it was not just his fellows’ work. We would send Ken what we considered to be a just-about-perfect draft of a new paper, and in about a week we would get it back with 100 or 200 red editorial balloon comments. Ken was unflappable. He possessed equanimity in the face of any stress. He could logically and sequentially dissect and deal with any issue (and not only the scientific ones) thoughtfully and without losing his calm demeanor. We cannot remember Ken raising his voice, even with the most exasperating people. He dealt sensitively with the toughest problems arising in our lab administration, and we knew he would find a just and proper solution with the fewest ruffled feathers. Ken managed every challenge. Ken spent his happiest hours in the lab. Each day, including Saturday and Sunday, he would arrive early, before anyone else. He loved to get his hands wet with an experiment or clean out the ice from a freezer. When he was happy all of us knew it—because Ken whistled. Not a tune we recognized, but his happiness was evident to all around him. He glowed when an experiment went well or a fellow or student gave a great talk. We sought his compliments, and he often gave them to us after a great presentation or scientific advance. An “attaboy” from Ken was a proud gold star to any recipient. Ken leaves behind him a hundred intellectual children from nations around the globe. He brightened our lives with his wisdom, his amazing grasp of science, his carefully and gently articulated insights and opinions, and his remarkable way of helping us express ourselves. Ken was a bright, steady beacon who lit the way for many of us. So strong and true, his beacon will continue to guide us and give us strength as we carry on our lives, scientific and personal. We can hear him admonishing us to do that and asking us to stop talking about his very private life. Let us go forward, do our science, live our lives and make him proud. To quote Dr. Seuss, that famous American philosopher we often quoted to our children, “Don't cry because it's over, laugh because it happened!”

Endothelial nitric oxide synthase uncoupling: A novel pathway in OSA induced vascular endothelial dysfunction

The mechanism of vascular endothelial dysfunction (VED) and cardiovascular disease in obstructive sleep apnea (OSA) is unknown. We performed a comprehensive evaluation of endothelial nitric oxide synthase (eNOS) function directly in the microcirculatory endothelial tissue of OSA patients who have very low cardiovascular risk status. Nineteen OSA patients underwent gluteal biopsies before, and after effective treatment of OSA. We measured superoxide (O2•−) and nitric oxide (NO) in the microcirculatory endothelium using confocal microscopy. We evaluated the effect of the NOS inhibitor l-Nitroarginine-Methyl-Ester (l-NAME) and the NOS cofactor tetrahydrobiopterin (BH4) on endothelial O2•− and NO in patient endothelial tissue before and after treatment. We found that eNOS is dysfunctional in OSA patients pre-treatment, and is a source of endothelial O2•− overproduction. eNOS dysfunction was reversible with the addition of BH4. These findings provide a new mechanism of endothelial dysfunction in OSA patients and a potentially targetable pathway for treatment of cardiovascular risk in OSA.

Abstract 16763: Mechanical Circulatory Support Promotes Myocardial Nitric Oxide Signaling in Heart Failure Patients

Introduction: The integrity of the cardiovascular system is dependent on the continuous generation of nitric oxide (NO). Reduction in NO bioavailability is central to the development of cardiovascular disorders and patients with heart failure (HF) exhibit blunted endothelium-dependent flow-mediated dilation in indicating diminished NO release. We have previously reported that patients with heart failure have marked reductions in circulating NO metabolites and that NO-based therapies significantly reverse left ventricular (LV) dysfunction in preclinical animal models of HF. Methods: Plasma and left ventricular (LV) tissue were collected in HF patients (n=10) at the time of left ventricular assist device (LVAD) implantation. Plasma and LV tissue were again collected in the same 10 patients at the time of cardiac transplantation. Plasma and cardiac nitrite concentrations were determined by ion chromatography methods and nitrosylated protein (RXNO) levels were measured by chemiluminescence detection. Cyclic guanosine 5’-monophosphate (cGMP) was analyzed using radioimmunoassay technique. Results: Results: Following LVAD implantation, RXNO (NO-bound protein) levels were significantly elevated in both circulation and in the LV (10.2 nM vs. 33.3 nM, p < 0.05). Downstream NO signaling activates soluble guanylyl cyclase to form cGMP. Interestingly, LV cGMP levels were significantly augmented post-LVAD implantation (p<0.01). In contrast, plasma and cardiac nitrite (NO2) levels remained unchanged. Conclusions: These data strongly suggest that cardiovascular remodeling accompanying LVAD implantation promotes endothelial nitric oxide synthase (eNOS) function and increases NO bioavailability and signaling.

Endothelial dysfunction abrogates the efficacy of normobaric hyperoxia in stroke.

Hyperoxia has been uniformly efficacious in experimental focal cerebral ischemia. However, pilot clinical trials have showed mixed results slowing its translation in patient care. To explain the discordance between experimental and clinical outcomes, we tested the impact of endothelial dysfunction, exceedingly common in stroke patients but under-represented in experimental studies, on the neuroprotective efficacy of normobaric hyperoxia. We used hyperlipidemic apolipoprotein E knock-out and endothelial nitric oxide synthase knock-out mice as models of endothelial dysfunction, and examined the effects of normobaric hyperoxia on tissue perfusion and oxygenation using high-resolution combined laser speckle and multispectral reflectance imaging during distal middle cerebral artery occlusion. In normal wild-type mice, normobaric hyperoxia rapidly and significantly improved tissue perfusion and oxygenation, suppressed peri-infarct depolarizations, reduced infarct volumes, and improved neurological function. In contrast, normobaric hyperoxia worsened perfusion in ischemic brain and failed to reduce infarct volumes or improve neurological function in mice with endothelial dysfunction. These data suggest that the beneficial effects of hyperoxia on ischemic tissue oxygenation, perfusion, and outcome are critically dependent on endothelial nitric oxide synthase function. Therefore, vascular risk factors associated with endothelial dysfunction may predict normobaric hyperoxia nonresponders in ischemic stroke. These data may have implications for myocardial and systemic circulation as well.

IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals.

Human blood platelets lack nitric oxide synthase activity

Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography–mass spectrometry (GC–MS) method to detect NOS activity in human platelets. The method measures simultaneously [15N]nitrite and [15N]nitrate formed from oxidized 15N-labeled nitric oxide (15NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-15N2]-arginine. Using this GC–MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.

Polymorphisms in endothelial nitric oxide synthase gene in early and late severe preeclampsia

Preeclampsia (PE) is characterized by hypertension and proteinuria, occurring after the 20th week of pregnancy in women who have had no previous symptoms. The disease progresses with generalized vasoconstriction and endothelial dysfunction. Clinically, it is important to diagnose the severe form of the disease (sPE), in which blood pressure and proteinuria are much higher. Recently, the gestational age (GA) of the onset of PE has led to the classification of this disease as early (GA <34 weeks) and late (GA ≥34 weeks). Several genetic polymorphisms affecting endothelial nitric oxide synthase (eNOS) levels or function were described, including G894T (Glu298Asp), VNTR b/a (variable-number 27-bp tandem repeat) and T-786C (promoter) polymorphisms. Thus, the aim of this study was to compare the distribution of G894T, VNTR b/a and T-786C polymorphisms and their haplotypes in Brazilian early and late sPE, as well as in normotensive pregnant. A total of 201 women were evaluated, 53 with early sPE, 45 with late sPE and 103 as normotensive pregnant women. The frequency of 894T allele was higher in late sPE vs normotensive pregnant, and 894TT genotype was higher in late sPE vs early sPE and normotensive pregnant. For VNTR b/a polymorphism, higher frequencies of aa genotype and a allele were observed in early sPE vs late sPE and normotensive pregnant. Besides, the frequency of haplotype T-b-C was higher in late sPE vs early sPE and normotensive pregnant. Considering the results found for eNOS polymorphisms, it is possible to suggest that the functional alterations induced by these two polymorphisms may influence the time of severe PE onset, although both alterations are putatively associated with low NO bioavailability. However, other studies are necessary to validate these findings and clarify this issue.

209 Beta-catenin Functions as an Endogenous Negative Regulator of Endothelial Nitric Oxide Synthase Function

IntroductionNitric oxide (NO) derived from endothelial NO synthase (eNOS) has multiple anti-atherogenic actions in the vasculature. eNOS activity is controlled by association with a number of regulatory proteins. We here...

Abstract 167: A Cell-Autonomous Role for Endothelial GTP Cyclohydrolase 1 and Tetrahydrobiopterin in Blood Pressure Regulation

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and nitric oxide (NO) generation. Augmentation of BH4 levels can prevent eNOS uncoupling and improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de-novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele ( GCH1 fl/fl ) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1 fl/fl Tie2cre mice demonstrated virtually absent NO bioactivity and significantly greater O 2 • - production. GCH1 fl/fl Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1 fl/fl Tie2cre aortas were in part mediated by NOS-derived hydrogen peroxide (H 2 O 2 ), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H 2 O 2 production in GCH1 fl/fl Tie2cre aortas. GCH1 fl/fl Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalised by NOS inhibitor, L-NAME. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure, and identify endothelial cell BH4 as a pivotal regulator of NO vs. H 2 O 2 as alternative eNOS-derived endothelial derived relaxing factors.

P83: Hydrogen sulfide cytoprotective signaling is nitric oxide dependent

Introduction Consensus has formed that hydrogen sulfide (H2S) has robust cytoprotective actions in multiple organ systems. The pro-survival actions and biological profiles of H2S are remarkably similar to those of nitric oxide (NO). Although there is recent evidence of cross-talk between H2S and NO, these molecules are thought to modulate independent signaling pathways. Methods/Results Mice devoid of the key H2S producing enzyme, cystathionine gamma-lyase (CSE KO), exhibited impaired endothelial nitric oxide synthase (eNOS) function and diminished circulating nitrite (0.41 uM vs 0.73 μM, p Conclusion These findings reveal that H2S modulates eNOS activity and nitric oxide signaling. Moreover, the inability of H2S therapy to protect eNOS dysfunctional mice against myocardial ischemia/reperfusion injury indicates that the cardioprotective actions of H2S are dependent on eNOS function and NO signaling.

Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation.

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (•-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.

A study of the mechanisms for antiaggregant activity of pyrrolidone derivatives in rats with chronic hyperglycemia.

In rats with chronic alloxan-induced hyperglycemia, pramiracetam and phenylpiracetam (but not piracetam) had a strong antiaggregant effect that was mediated by various mechanisms of platelet aggregation modulation. The effect of pramiracetam is mainly realized via the inhibition of thromboxane A2 metabolism, while activity of phenylpiracetam is primarily associated with a normalizing effect on function of constitutive NO synthase in platelets and vascular endothelium.