Nitric Oxide Synthase Inhibitor L-NMMA Research Articles

Suppressive effect of nitric oxide synthase (NOS) inhibitor L-NMMA acetate on choroidal fibrosis in experimental myopic guinea pigs through the nitric oxide signaling pathway

Myopia is one of the most prevalent eye diseases that seriously threaten the eyesight of children and adolescents worldwide. However, the pathogenesis is still unclear, and effective drugs are still scarce. In the present study, the guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a NOS inhibitor (L-NMMA) injection group, and a NOS inhibitor solvent phosphate-buffered saline (PBS) group and the animals received relevant treatments. After 2- and 4-week different treatments, we noted that the refraction and choroidal thickness in the LIM group decreased compared with the NC group, whereas the ocular axial length increased significantly, and the choroid showed a fibrotic trend. The expression of NOS1, NOS3, TGF-β1, COLI, and α-SMA at gene and protein levels was increased significantly in the choroid (all P < 0.05). After intravitreal injection of NOS inhibitor L-NMMA, we found that compared with the LIM group, the refraction and the choroidal thickness significantly increased, whereas the axial length reduced significantly, accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis. The expression levels of choroidal NOS1, NOS3, TGF-β, COLI, and α-SMA were significantly reduced (all P < 0.05). In conclusion, the trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase in axial length. The NOS inhibitor L-NMMA can alleviate the process of choroidal fibrosis in myopic guinea pigs by inhibiting NO signaling pathway.

Arginase 1 in erythrocytes is a critical modulator of cardioprotective signalling by nitric oxide and soluble guanylyl cyclase

Abstract Background Arginase is involved in the development of ischemia-reperfusion injury by regulating nitric oxide (NO) bioactivity via competition with NO synthase (NOS) for their common substrate L-arginine. Erythrocytes are known to contain high levels of arginase that may reduce export of cardioprotective NO bioactivity. Aim To determine the role of arginase 1 in erythrocytes for cardiac protection against myocardial ischemia-reperfusion injury. Methods A tie2 cre-flox mouse model, in which arginase 1 was deleted (Arg 1-KO) in hematopoietic and endothelial cells, was developed. In vivo ischemia-reperfusion was performed in anaesthetized mice by left anterior descending coronary artery ligation (30 min ischemia and 120 min reperfusion). To determine the specific role of erythrocytes, an isolated perfused mouse heart model was applied. Erythrocytes from Arg 1-KO and wild type (WT) mice were given to isolated hearts from WT mice at the onset of global ischemia. After 40 min ischemia, the recovery of left ventricular developed pressure (LVDP) during 60 min reperfusion was recorded as an indicator of cardiac functional recovery. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996) Results Following in vivo ischemia-reperfusion, infarct size was smaller in Arg 1-KO mice than in WT mice (Fig. A). A similar degree of infarct size reduction was obtained by i.v. administration of an arginase inhibitor in WT mice. The cardioprotective effect observed in Arg 1-KO mice was abolished by the NOS inhibitor L-NMMA (Fig. A). In isolated buffer-perfused hearts, there was no difference in the post-ischemic recovery of LVDP between Arg 1-KO and WT hearts. When erythrocytes from Arg 1-KO mice were administrated to isolated WT mouse hearts, the post-ischemic recovery of LVDP was significantly improved compared to hearts given erythrocytes from WT mice. Also, the cardioprotective effect of erythrocytes from Arg 1-KO mice was abolished by pre-incubation of the erythrocytes with the NOS inhibitor L-NAME (Fig. B) or the inhibitor of soluble guanylyl cyclase ODQ (Fig. C). Conclusion Arginase 1 in erythrocyte plays an important role in regulating cardioprotection mediated via the NO-soluble guanylyl cyclase pathway. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation (20190266),The Swedish Research Council (2020-01372)

Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis.

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells.

Trichophyton rubrum (T. rubrum) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum, investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells.

L-NAME releases nitric oxide and potentiates subsequent nitroglycerin-mediated vasodilation

L-NG-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature.

PL - 026 Mismatch between skeletal muscle glucose delivery, interstitial concentration and membrane permeability may limit insulin sensitivity after exercise

Objective The relationship between skeletal muscle perfusion, interstitial glucose concentration and sarcolemmal permeability to glucose in exercise-induced increases in muscle insulin sensitivity is not well established. A single bout of exercise increases skeletal muscle insulin sensitivity through coordinated increases in insulin-stimulated microvascular perfusion and insulin signalling Reducing leg and muscle microvascular blood flow with local nitric oxide synthase (NOS) inhibition during a hyperinsulinaemic euglycaemic clamp reduces leg glucose uptake in a previously exercised, but not in a contralateral non-exercised leg, without affecting insulin signalling in either leg (Sjoberg et al. 2017). Therefore, it is possible that the reduction in muscle perfusion decreases muscle interstitial glucose concentration to a point that limits skeletal muscle insulin-stimulated glucose uptake following exercise. We examined this using microdialysis of vastus lateralis muscle.&#x0D; Methods Ten healthy males (Age: 27±1 yr., Weight: 77.7±2.3 kg, BMI 23.9±0.5, VO2 peak: 50.7±1.5 ml·kg-1·min-1) performed 60 min of 1-legged knee extensor exercise at 80% of 1-legged peak work load with three 5 min intervals at 100% 1-legged peak work load. Participants then rested for 4 hours and catheters were inserted into the femoral artery and vein of both legs for subsequent measurement of leg glucose uptake and for femoral artery infusion of the NOS inhibitor NG-monomethyl L-arginine acetate (L-NMMA) and the vasodilator ATP. Catheters were also placed in antecubital veins for infusion of insulin and glucose. Three microdialysis catheters, with a semi-permeable membrane the length of 30 mm and a molecular cut-off at 20,000 dalton, were inserted into the vastus lateral muscle of both legs. Glucose and D-[6-3H(N)]glucose were added to the perfusate. Four hours after discontinuing the exercise a 225 minute euglycaemic hyperinsulinaemic clamp was initiated (insulin infusion 1.4 mU-1kg-1min). Ninety min into the clamp L-NMMA was infused at a constant rate (0.4 mg·kg-1 leg mass·min-1) into both femoral arteries for 45 min. The insulin infusion was maintained for another 90 min and during the last 45 min ATP (0.3 μmol∙ml-1) was infused locally into both femoral arteries at a rate of 200-350 μl∙min-1 to obtain a leg blood flow that was double the blood flow during insulin only infusion. A second control protocol was undertaken that was identical in regards to exercise and recovery but no insulin, L-NMMA or ATP was infused.&#x0D; Results During the clamp leg glucose uptake and leg blood flow were higher (P&lt;0.05) in the previously exercised than the control leg whereas the interstitial glucose concentration decreased to lower (P&lt;0.05) values in the exercised (~3.1mM) than the control (~4.8mM) leg. Estimated sarcolemmal glucose permeability was twice as high (P&lt;0.05) in the exercised compared with the rested leg. The NOS inhibitor L-NMMA decreased LBF in both legs and interstitial glucose concentration dropped to ~2.3 mM in the exercised but only to ~3.7 mM in non-exercised muscle. This abrogated the augmented effect of insulin on LGU in the exercised leg while apparent sarcolemmal permeability to glucose remained unchanged with L-NMMA in both legs. Doubling leg blood flow by local infusion of ATP increased leg glucose uptake in both legs without any major change in interstitial glucose concentration or sarcolemmal permeability to glucose.&#x0D; Conclusions These findings suggest that during flow restriction due to L-NMMA, the interstitial glucose concentration becomes limiting for leg glucose uptake in exercised but not in non-exercised muscle. Therefore, the vasodilatory effect of insulin is an important component of the increased insulin sensitivity to stimulate glucose uptake following exercise by limiting the drop in the interstitial glucose concentration that occurs due to the increased sarcolemmal permeability to glucose.&#x0D; Reference&#x0D; Sjoberg, K. A., C. Frosig, R. Kjobsted, L. Sylow, M. Kleinert, A. C. Betik, C. S. Shaw, B. Kiens, J. F. P. Wojtaszewski, S. Rattigan, E. A. Richter, and G. K. McConell. Exercise Increases Human Skeletal Muscle Insulin Sensitivity via Coordinated Increases in Microvascular Perfusion and Molecular Signaling. Diabetes 66: 1501-10, 2017.

OP.8C.02] NORADRENERGIC RECEPTOR FUNCTION IN HEALTHY AND OBESE PERIVASCULAR ADIPOSE TISSUE

Objective: Healthy perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating blood pressure, and evidence suggests that sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of beta3-adrenoceptors. In obesity there is sympathetic over-activity, which may result in a loss of PVAT function and subsequent hypertension. Therefore we examined beta3-adrenoceptor function in obesity. Design and method: Electrical field stimulation (EFS) profiles of healthy and obese mouse mesenteric arteries (<200um, +/−PVAT) were characterised using wire myography (0.1–30 Hz, 20 V, 0.2ms pulse duration, 4 s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. Beta3-adrenoceptor function was investigated using the agonist CL-316,243 (10uM) and antagonist SR59203A (100 nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100uM), and NOS activator histamine (100uM). Results: During EFS healthy PVAT elicited an anti-contractile effect; which was lost in obesity. Solution transfer from stimulated healthy exogenous PVAT to a –PVAT vessel significantly reduced contraction, confirming that stimulated PVAT releases a transferable anti-contractile factor. Solution transfer from obese PVAT had no effect on contraction. Inhibition of beta3-adrenoceptors in healthy PVAT using SR59230A significantly reduced the anti-contractile effect, whereas activation of beta3-adrenoceptors in obese PVAT using CL-316,243 did not restore function. Using immunohistochemistry, expression of beta3-adrenoceptors is reduced in obesity. In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect. In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function. Conclusions: These results demonstrate that in health PVAT releases an anti-contractile factor via activation of beta3-adrenoreceptors, which downstream triggers the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by beta3-activation, but is restored by activation of NOS. This reveals that in obesity beta3-adrenoreceptors are downregulated, leading to a loss of anti-contractile function, which may contribute to the development of hypertension. Targeting the NOS pathway may restore anti-contractile function, and treat the vascular complications of obesity.

Silencing of argininosuccinate lyase inhibits colorectal cancer formation.

Arginine and nitric oxide (NO) are important mediators of tumorigenesis in various types of cancer. Dysregulation of NO content by argininosuccinate lyase (ASL) has been previously demonstrated to inhibit the proliferation of liver and breast cancer cells. However, the function of ASL in colon cancer is not well defined. The present study aimed to determine the effect of ASL on colon cancer. Western blot analysis indicated that ASL expression was induced by endoplasmic reticulum stress in HCT116 and SW480 colon cancer cells. Additionally, the expression of ASL in colon cancer tissues was enhanced compared with that in the adjacent normal tissues, and the patients with colon cancer with higher ASL expression exhibited poorer survival rates. Transfection of ASL-targeting short hairpin RNA (shRNA) into HCT116 cells inhibited cell proliferation and decreased anchorage-independent growth in a soft agar assay. In addition, when injected subcutaneously into NOD/SCID mice, stable transfectant ASL-downregulated HCT116 cells exhibited decreased invivo tumorigenic ability. Flow cytometric analysis of cell cycle progression indicated that ASL-targeting shRNA induced G2/Marrest, and western blot analysis showed that the inhibition of ASL was accompanied by cyclinA2 degradation. Furthermore, ASL-targeting shRNA resulted in increased autophagosomes and decreased NO levels. Inhibition of NO by the NO synthase inhibitor L-NMMA significantly reduced cell proliferation and colony formation. In summary, the results of the present study indicated that ASL-targeting shRNA-induced growth inhibition is associated with decreased cyclinA2 expression and NO content in colon cancer.

YIA 03-09 NORADRENERGIC RECEPTOR FUNCTION IN HEALTHY AND OBESE PERIVASCULAR ADIPOSE TISSUE

Objective: Perivascular adipose tissue (PVAT) exerts an anti-contractile effect vital to regulating blood pressure. Evidence suggests that the sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of β3-adrenoceptors. Therefore it was decided to examine β3-adrenoceptor function in obesity. Design and Method: Electrical field stimulation (EFS) of healthy and obese mouse mesenteric arteries (<200 μm, +/-PVAT) were characterised using wire myography (0.1–30 Hz, 20V, 0.2ms pulse duration, 4s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. β3-adrenoceptor function was investigated using the agonist CL-316,243 (10 μM) and antagonist SR59203A (100nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100 μM), and NOS activator histamine (100 μM). Results: Healthy PVAT elicits an anti-contractile effect which was lost in obesity. Inhibition of β3-adrenoceptors significantly reduced the anti-contractile effect, whereas activation of β3-adrenoceptors in obese PVAT using CL-316,243 did not restore function. Solution transfer from stimulated healthy exogenous PVAT to a –PVAT vessel significantly reduced contraction, confirming the release of a transferable factor. The release of this factor could be inhibited using SR59230A. Solution transfer from obese PVAT had no effect on contraction, and again could not be restored using CL-316,243. In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect. In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function. Conclusions: These results demonstrate that healthy PVAT releases an anti-contractile factor via activation of β3-adrenoreceptors, which downstream triggers the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by β3-activation, but is restored by activation of NOS. This suggests that in obesity β3-adrenoreceptors must be downregulated or desensitised, leading to a loss of anti-contractile function.

Nitric oxide regulation of migrating motor complex: randomized trial of N(G)-monomethyl-L-arginine effects in relation to muscarinic and serotonergic receptor blockade.

The migrating motor complex (MMC) propels contents through the gastrointestinal tract during fasting. Nitric oxide (NO) is an inhibitory neurotransmitter in the gastrointestinal tract. Little is known about how NO regulates the MMC. In this study, the aim was to examine nitrergic inhibition of the MMC in man using N(G)-monomethyl-L-arginine (L-NMMA) in combination with muscarinic receptor antagonist atropine and 5-HT3 receptor antagonist ondansetron. Twenty-six healthy volunteers underwent antroduodenojejunal manometry for 8 h with saline or NO synthase (NOS) inhibitor L-NMMA randomly injected I.V. at 4 h with or without atropine or ondansetron. Plasma ghrelin, motilin and somatostatin were measured by ELISA. Intestinal muscle strip contractions were investigated for NO-dependent mechanisms using L-NMMA and tetrodotoxin. NOS expression was localized by immunohistochemistry. L-NMMA elicited premature duodenojejunal phase III in all subjects but one, irrespective of atropine or ondansetron. L-NMMA shortened MMC cycle length, suppressed phase I and shifted motility towards phase II. Pre-treatment with atropine extended phase II, while ondansetron had no effect. L-NMMA did not change circulating ghrelin, motilin or somatostatin. Intestinal contractions were stimulated by L-NMMA, insensitive to tetrodotoxin. NOS immunoreactivity was detected in the myenteric plexus but not in smooth muscle cells. Nitric oxide suppresses phase III of MMC independent of muscarinic and 5-HT3 receptors as shown by nitrergic blockade, and acts through a neurocrine disinhibition step resulting in stimulated phase III of MMC independent of cholinergic or 5-HT3 -ergic mechanisms. Furthermore, phase II of MMC is governed by inhibitory nitrergic and excitatory cholinergic, but not 5-HT3 -ergic mechanisms.

184 Temporospatial Effects of Endothelium Specific Inhibition of Shc Homology 2 -containing Inositol 5´ Phosphatase 2 on Nitric Oxide Bioavailability and Whole Body Insulin Sensitivity

IntroductionAgeing is an important risk factor for diabetes and cardiovascular disease. Integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Insulin signalling in endothelial cells modulates the generation...

EP News: Basic and Translational

Cheng et al (JMCC 61:102,2013) investigated the mutations in CAV3-encoding caveolin-3 (Cav3) implicated in LQT9 and SIDS. This study addressed the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A.HEK-293 cells expressing SCN5A and Cav3-F97C resulted in a 2-fold increase in late INa versus Cav3-WT, which was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin, nNOS, and Cav3.

Angiotensin II Type 2 Receptor–Mediated and Nitric Oxide–Dependent Renal Vasodilator Response to Compound 21 Unmasked by Angiotensin-Converting Enzyme Inhibition in Spontaneously Hypertensive Rats In Vivo

Angiotensin II type 2 receptor (AT2R)-mediated vasodilation has been demonstrated in different vascular beds in vitro and in perfused organs. In vivo studies, however, consistently failed to disclose renal vasodilator responses to compound 21, a selective AT2R agonist, even after angiotensin II type 1 receptor blockade. Here, we investigated in vivo whether angiotensin-converting enzyme inhibition, reducing endogenous angiotensin II levels, could unmask the effects of selective AT2R stimulation on blood pressure and renal hemodynamics in normotensive and hypertensive rats. After pretreatment with the angiotensin-converting enzyme inhibitor captopril, intravenous administration of compound 21 did not affect blood pressure and induced dose-dependent renal vasodilator responses in spontaneously hypertensive but not in normotensive rats. The D1 receptor agonist fenoldopam, used as positive control, reduced blood pressure and renal vascular resistance in both strains. The AT2R antagonist PD123319 and the nitric oxide synthase inhibitor L-NMMA (N(G)-monomethyl-L-arginine acetate) abolished the renal vasodilator response to compound 21 without affecting responses to fenoldopam. The cyclooxygenase inhibitor indomethacin partially inhibited the renal vascular response to compound 21, whereas the bradykinin B2 receptor antagonist icatibant was without effect. Angiotensin-converting enzyme inhibition unmasked a renal vasodilator response to selective AT2R stimulation in vivo, mediated by nitric oxide and partially by prostaglandins. AT2R may have a pathophysiological role to modulate renal hemodynamic effects of angiotensin II in the hypertensive state.

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: A role for reactive oxygen species

ObjectiveSystemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species. MethodsWe cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE−/− mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis. ResultsApoE−/−/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE−/− mice. Atherosclerosis was significantly increased in ApoE−/−/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 103 μm2, P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE−/−/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE−/−/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (−12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (−8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (−23 ± 0.04%, P = 0.006). ConclusionsInsulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.

17β-Estradiol Reduces Nitric Oxide Production in the Guinea Pig Cochlea

Intense noise exposure and the application of ototoxic substances result in increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO). In order to reduce the free NO concentration in the inner ear under pathological conditions, the use of natural cytoprotective substances such as 17β-estradiol is a promising therapeutic concept. In male guinea pigs the organ of Corti and the lateral wall were isolated from the cochlea and afterwards incubated for 6 h in cell-culture medium. 17β-Estradiol was adjusted in 2 concentrations to organ cultures of the right ears (12 animals per concentration). The left ears were used as controls. The NO production was quantified in the supernatant by chemiluminescence after incubation. Depending on the concentration, 17β-estradiol reduced NO in the organ of Corti by 43% (p=0.015) and 46% (p=0.026), respectively. In the lateral wall, the NO concentration was reduced by 24%, but without statistical significance (p=0.86). However, when analyzing the association between the 2 cochlear regions for each animal separately, the NO concentrations were lower in nearly all 17β-estradiol-treated ears compared to controls. In order to demonstrate the flexibility of the organ culture system, the NO donor DETA NONOate and the nitric oxide synthase inhibitors L-NAME and L-NMMA were applied. The electron microscopic analysis revealed a well-preserved cochlear cell morphology after incubation. The ability of 17β-estradiol to influence the NO production preferentially in the organ of Corti might offer new therapeutic perspectives for inner ear protection.

Arginase inhibition reduces infarct size via nitric oxide, protein kinase C epsilon and mitochondrial ATP-dependent K+ channels

Reduced bioavailability of nitric oxide (NO) contributes to the development of myocardial ischemia-reperfusion (I/R) injury. Increased activity of arginase is a potential factor that reduces NO bioavailability by competing for the substrate l-arginine. The aim of the study was to test the hypothesis that inhibition of arginase after coronary artery occlusion protects from I/R injury and to explore possible mechanisms behind this effect.Male Sprague-Dawley rats subjected to 30min of coronary artery ligation and 2h reperfusion were given i.v. before the reperfusion: 1) saline; 2) the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA); 3) nor-NOHA with the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA); 4) nor-NOHA with the mitochondrial ATP-dependent K+ (mitoKATP) channel blocker 5-hydroxydecanoic acid (5-HD); 5) nor-NOHA with the protein kinase C epsilon (PKCε) inhibitor ε-V1-2 or 6) ε-V1-2 alone.Infarct size in the control groups was 61±3% and it was reduced to 47±3% (P<0.01) by nor-NOHA. The cardioprotective effect was blocked by the NOS inhibitor l-NMMA. PKCε expression was reduced by I/R and this reduction was attenuated by nor-NOHA. Furthermore, the PKCε inhibitor ε-V1-2 abolished the protective effect of nor-NOHA (infarct size 69±6%). In addition, the cardioprotective effect of nor-NOHA was also abolished following blockade of the mitoKATP channel (infarct size 62±1%).Inhibition of arginase before reperfusion protects the heart from I/R injury via a NOS-dependent pathway, increased expression of PKCε and activation of mitoKATP channels.

241 ENDOTHELIAL SHIP2 CONFERS AGE-DEPENDENT CONTRASTING AFFECTS ON WHOLE BODY GLUCOSE HOMEOSTASIS AND VASCULAR FUNCTION

<h3>Introduction</h3> Aging is an important risk factor for diabetes and cardiovascular disease and integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Although the vascular implications of endothelial insulin resistance are well understood, the effect of <i>enhanced</i> endothelial insulin signaling on whole body glucose regulation and vascular function remains poorly characterized. We therefore generated mice with endothelial-specific downregulation of the lipid phosphatase SHIP2 (a negative regulator of insulin signaling) to investigate whether enhanced insulin signaling in the endothelium modulates vascular function and whole body glucose regulation. <h3>Methods</h3> Exons 18–19 of the ship 2 gene were deleted using Cre-Lox technology under control of the Tie2 promoter generating a catalytically inactivate protein. Male heterozygotes for the inactive protein (EC-SHIP2^+/−) were compared to sex-matched littermate controls. <h3>Results</h3> EC-SHIP^+/− mice were morphologically indistinguishable from controls, exhibiting normal development. At 8 weeks of age EC-SHIP2^+/− mice displayed increased glucose tolerance after glucose challenge (P=0.03) and improved insulin sensitivity (P=0.02) after insulin challenge compared to controls. Surprisingly however, by 40 weeks of age this phenotype was reversed; EC-SHIP2^+/− mice revealed significant insulin resistance 60 min after insulin challenge (P=&lt;0.05). This phenotype was confirmed by euglycemic hyperinsulinemic clamping showing whole body insulin resistance in EC-SHIP^+/− mice (decreased glucose infusion rate of 26% P&lt;0.05). In young mice <i>ex vivo</i> aortic vasomotor studies in both controls and EC-SHIP^+/− revealed similar contractile responses to phenylephrine and displayed decreased contraction after insulin incubation (Emax 0.88±0.05g vs 0.62±0.05g P=0.002 and 0.83±0.05g vs 0.69±0.05g P=0.025 respectively). Both groups displayed increased contraction after NO synthase inhibitor LNMMA incubation (Emax 0.88±0.05g vs 1.31±0.11g P=0.01 and 0.83±0.05g vs 1.28±0.02g P=&lt;0.0001 respectively). However, at 40 weeks old in EC-SHIP2^+/− mice the vasodilatory aortic ring response to insulin was abolished (Emax controls 0.59±0.04g vs 0.47±0.03g P=0.04, EC-SHIP2^+/− 0.64±0.04g vs 0.63±0.06g P=0.9) and EC-SHIP2^+/− displayed no increase in contraction to LNMMA incubation (Emax controls 0.59±0.04g vs 0.82±0.08g P=0.02, EC-SHIP2^+/− 0.64±0.04g vs 0.69±0.07g P=0.5) indicating insulin resistance and lower basal NO production, suggesting the change in glucose homeostasis may be mediated by changes in NO bioavailability. <h3>Conclusion</h3> Endothelial functional downregulation of SHIP2 augments whole body glucose disposal in young mice but attenuates whole body glucose disposal in older mice. Although further studies are required to elucidate the molecular mechanisms our data suggest a previously unrecognised age dependent role for the vascular endothelium in whole body glucose regulation.

Haemoglobin

Haemoglobin

Caveolin-3 suppresses late sodium current by inhibiting nNOS-dependent S-nitrosylation of SCN5A

Aims: Mutations in CAV3-encoding caveolin-3 (Cav3) have been implicated in type 9 long QT syndrome (LQT9) and sudden infant death syndrome (SIDS). When co-expressed with SCN5A-encoded cardiac sodium channels these mutations increased late sodium current (INa) but the mechanism was unclear. The present study was designed to address the mechanism by which the LQT9-causing mutant Cav3-F97C affects the function of caveolar SCN5A. Methods and results: HEK-293 cells expressing SCN5A and LQT9 mutation Cav3-F97C resulted in a 2-fold increase in late INa compared to Cav3-WT. This increase was reversed by the neural nitric oxide synthase (nNOS) inhibitor L-NMMA. Based on these findings, we hypothesized that an nNOS complex mediated the effect of Cav3 on SCN5A. A SCN5A macromolecular complex was established in HEK-293 cells by transiently expressing SCN5A, α1-syntrophin (SNTA1), nNOS, and Cav3. Compared with Cav3-WT, Cav3-F97C produced significantly larger peak INa amplitudes, and showed 3.3-fold increase in the late INa associated with increased S-nitrosylation of SCN5A. L-NMMA reversed both the Cav3-F97C induced increase in late and peak INa and decreased S-nitrosylation of SCN5A. Overexpression of Cav3-F97C in adult rat cardiomyocytes caused a significant increase in late INa compared to Cav3-WT, and prolonged the action potential duration (APD90) in a nNOS-dependent manner. Conclusions: Cav3 is identified as an important negative regulator for cardiac late INa via nNOS dependent direct S-nitrosylation of SCN5A. This provides a molecular mechanism for how Cav3 mutations increase late INa to cause LQT9. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Nitric Oxide/Peroxynitrite Redox Imbalance in Endothelial Cells Measured with Amperometric Nanosensors

The cytoprotective messenger nitric oxide (NO) and cytotoxic peroxynitrite (ONOO-) are the main components of oxidative stress and can be generated by endothelial cells. A tandem of electrochemical nanosensors (diameter 200-300 nm) were used to measure, in situ, the balance between NO and ONOO-produced by human umbilical vein endothelial cells (HUVEC’s). The amperometric nanosensors were placed 5 ± 2 μm from the surface of the endothelial cells and the concentration of NO and ONOO- was measured at 630 mV and -300 mV (vs Ag/AgCl) respectively. Normal, functional, endothelial cells produced maximal 450 ± 25 nmol.L-1 of NO and 180 ± 15 nmol.L-1 of ONOO- in about 3 s, after stimulation with calcium ionophore. The in situ measurements of NO and ONOO- were validated using nitric oxide synthase inhibitor L-NMMA, ONOO- scavenger Mn(III) porphyrin, and superoxide dismutase (PEG-SOD). The ratio of NO concentration to ONOO- concentration ([NO]/[ONOO-]) was introduced for quantification of both, the redox balance and the level of the nitroxidative stress in the endothelium. [NO]/[ONOO-] was 2.7 ± 0.1 in a functional endothelium. The model of the dysfunctional endothelium was made by the treatment of HUVEC’s with angiotensin II for 20 min. Dysfunctional HUVEC’s produced only 115 ± 15 nmol.L-1 of NO, but generated a significantly higher concentration of ONOO- of 490 ± 30 nmol.L-1. The [NO]/[ONOO-] ratio decreased to 0.23 ± 0.14 in the dysfunctional endothelium. Electrochemical nanosensors can be effectively used for in situ monitoring of changing levels of nitroxidative/ oxidative stress, and may be useful in early medical diagnosis of the cardiovascular system.