Cholinergic regulation of vascular endothelial function by human ChAT+ T cells.

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human T cells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ T cells in blood. These findings on ChAT+ human T cells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

NO-ferroheme is a signaling entity in the vasculature.

Despite wide appreciation of the biological role of nitric oxide (NO) synthase (NOS) signaling, questions remain about the chemical nature of NOS-derived bioactivity. Here we show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species, which can transfer between proteins, partition into a hydrophobic phase and directly activate the sGC-cGMP-PKG pathway without intermediacy of free NO. The NO-ferroheme species (with or without a protein carrier) efficiently relax isolated blood vessels and induce hypotension in rodents, which is greatly potentiated after the blockade of NOS activity. While free NO-induced relaxations are abolished by an NO scavenger and in the presence of red blood cells or blood plasma, a model compound, NO-ferroheme-myoglobin preserves its vasoactivity suggesting the physiological relevance of NO-ferroheme species. We conclude that NO-ferroheme behaves as a signaling entity in the vasculature.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice.

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

Cardiovascular characterization of the novel organic mononitrate NDIBP in rats.

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 µM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 µM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.

AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis.

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate-nitrite-NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (l-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate-nitrite-NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Head-to-head comparison of inorganic nitrate and metformin in a mouse model of cardiometabolic disease.

BACKGROUND/PURPOSE: Unhealthy dietary habits contribute to the increasing incidence of metabolic syndrome and type 2 diabetes (T2D), which is accompanied by oxidative stress, compromised nitric oxide (NO) bioavailability and increased cardiovascular risk. Apart from lifestyle changes, biguanides such as metformin are the first-line pharmacological treatment for T2D. Favourable cardiometabolic effects have been demonstrated following dietary nitrate supplementation to boost the nitrate-nitrite-NO pathway. Here we aim to compare the therapeutic value of inorganic nitrate and metformin alone and their combination in a model of cardiometabolic disease. EXPERIMENTAL APPROACH: Mice were fed control or high fat diet (HFD) for 7 weeks in combination with the NO synthase (NOS) inhibitor l-NAME to induce metabolic syndrome. Simultaneously, the mice were treated with vehicle, inorganic nitrate, metformin or a combination of nitrate and metformin in (drinking water). Cardiometabolic functions were assessed in vivo and tissues were collected/processed for analyses. KEY RESULTS: HFD + L-NAME was associated with cardiometabolic dysfunction, compared with controls, as evident from elevated blood pressure, endothelial dysfunction, impaired insulin sensitivity and compromised glucose clearance as well as liver steatosis. Both nitrate and metformin improved insulin/glucose homeostasis, whereas only nitrate had favourable effects on cardiovascular function and steatosis. Mechanistically, metformin and nitrate improved AMPK signalling, whereas only nitrate attenuated oxidative stress. Combination of nitrate and metformin reduced HbA1c and trended to further increase AMPK activation. CONCLUSION/IMPLICATIONS: Nitrate and metformin had equipotent metabolic effects, while nitrate was superior regarding protection against cardiovascular dysfunction and liver steatosis. If reproduced in future clinical trials, these findings may have implications for novel nutrition-based strategies against metabolic syndrome, T2D and associated complications.

Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice.

BACKGROUND/AIMS: Canonical Wnt signaling is involved in oxidative stress, vasculopathy and diabetes mellitus but its role in diabetic renal microvascular dysfunction is unclear. We tested the hypothesis that enhanced canonical Wnt signaling in renal afferent arterioles from diabetic mice increases reactive oxygen species (ROS) and contractions to endothelin-1 (ET-1). METHODS: Streptozotocin-induced diabetes or control C57Bl/6 mice received vehicle or sulindac (40 mg·kg-1·day-1) to block Wnt signaling for 4 weeks. ET-1 contractions were measured by changes of afferent arteriolar diameter. Arteriolar H2O2, O2 -, protein expression and enzymatic activity were assessed using sensitive fluorescence probes, immunoblotting and colorimetric assay separately. RESULTS: Compared to control, diabetic mouse afferent arteriole had increased O2- (+ 84%) and H2O2 (+ 91%) and enhanced responses to ET-1 at 10-8 mol·l-1 (-72±4% of versus -43±4%, P< 0.05) accompanied by reduced protein expressions and activities for catalase and superoxide dismutase 2 (SOD2). Arteriolar O2 - was increased further by ET-1 and contractions to ET-1 reduced by PEG-SOD in both groups whereas H2O2 unchanged by ET-1 and contractions were reduced by PEG-catalase selectively in diabetic mice. The Wnt signaling protein ß-catenin was upregulated (3.3-fold decrease in p-ß-catenin/ß-catenin) while the glycogen synthase kinase-3ß (GSK-3ß) was downregulated (2.6-fold increase in p-GSK-3ß/ GSK-3ß) in preglomerular vessels of diabetic mice. Sulindac normalized the Wnt signaling proteins, arteriolar O2 -, H2O2 and ET-1 contractions while doubling microvascular catalase and SOD2 expression in diabetic mice. CONCLUSION: Increased ROS, notably H2O2 contributes to enhanced afferent arteriolar responses to ET-1 in diabetes, which is closely associated with Wnt signaling. Antioxidant pharmacological strategies targeting Wnt signaling may improve vascular function in diabetic nephropathy.

Red blood cells from endothelial nitric oxide synthase-deficient mice induce vascular dysfunction involving oxidative stress and endothelial arginase I.

BACKGROUND & AIMS: Nitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. METHODS & RESULTS: RBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. CONCLUSIONS: RBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia.

Inhibitory effects of hydroxysafflor yellow A on the formation of advanced glycation end products in vitro.

To investigate the inhibitory effects of hydroxysafflor yellow A (HSYA) on the protein glycation in vitro. Using bovine serum albumin (BSA)-glucose assay, BSA-methylglyoxal (MGO) assay, and N-acetylglycyl-lysine methyl ester (G.K.) peptide-ribose assay, inhibitory effects of HSYA were investigated. Advanced glycation end products (AGEs) production was assessed by AGEs-specific fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In BSA-glucose assay, HSYA concentration dependently decreased AGEs formation, with maximum inhibitory effects at 1 mM by 95%. Further more, HSYA also showed significant inhibitory effects on MGO-medicated protein modification and subsequent cross-linking of proteins. Finally, when co-incubated with G.K. peptide and ribose, HSYA exhibited its antiglycation effects, and the maximum inhibitory effects of HSYA at 1 mM were 84%. Overall, our present study provides the first evidence of the antiglycation effects of HSYA on AGEs formation in vitro.

Inorganic nitrate and nitrite ameliorate kidney fibrosis by restoring lipid metabolism via dual regulation of AMP-activated protein kinase and the AKT-PGC1α pathway.

BACKGROUND: Renal fibrosis, associated with oxidative stress and nitric oxide (NO) deficiency, contributes to the development of chronic kidney disease and renal failure. As major energy source in maintaining renal physiological functions, tubular epithelial cells with decreased fatty acid oxidation play a key role in renal fibrosis development. Inorganic nitrate, found in high levels in certain vegetables, can increase the formation and signaling by bioactive nitrogen species, including NO, and dampen oxidative stress. In this study, we evaluated the therapeutic value of inorganic nitrate treatment on development of kidney fibrosis and investigated underlying mechanisms including regulation of lipid metabolism in tubular epithelial cells. METHODS: Inorganic nitrate was supplemented in a mouse model of complete unilateral ureteral obstruction (UUO)-induced fibrosis. Inorganic nitrite was applied in transforming growth factor ß-induced pro-fibrotic cells in vitro. Metformin was administrated as a positive control. Fibrosis, oxidative stress and lipid metabolism were evaluated. RESULTS: Nitrate treatment boosted the nitrate-nitrite-NO pathway, which ameliorated UUO-induced renal dysfunction and fibrosis in mice, represented by improved glomerular filtration and morphological structure and decreased renal collagen deposition, pro-fibrotic marker expression, and inflammation. In human proximal tubule epithelial cells (HK-2), inorganic nitrite treatment prevented transforming growth factor ß-induced pro-fibrotic changes. Mechanistically, boosting the nitrate-nitrite-NO pathway promoted AMP-activated protein kinase (AMPK) phosphorylation, improved AKT-mediated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) activity and restored mitochondrial function. Accordingly, treatment with nitrate (in vivo) or nitrite (in vitro) decreased lipid accumulation, which was associated with dampened NADPH oxidase activity and mitochondria-derived oxidative stress. CONCLUSIONS: Our findings indicate that inorganic nitrate and nitrite treatment attenuates the development of kidney fibrosis by targeting oxidative stress and lipid metabolism. Underlying mechanisms include modulation of AMPK and AKT-PGC1α pathways.

Red blood cells from patients with pre-eclampsia induce endothelial dysfunction.

RATIONALE: Pre-eclampsia is a multisystem disorder associated with systemic vascular dysfunction and decreased nitric oxide (NO) bioactivity. Arginase competes with NO synthase (NOS) for l-arginine, and its upregulation may reduce NOS-derived NO formation or induce production of reactive oxygen species (ROS) via uncoupling of NOS, resulting in endothelial dysfunction. Red blood cells (RBCs) have emerged as key players in NO homeostasis via their interactions with the endothelium. Studies have demonstrated that abnormal RBC arginase function in patients with diabetes contributes to oxidative stress and endothelial dysfunction. AIM: The aim of the study was to investigate if reduced NO bioavailability and increased ROS in pre-eclampsia is mediated via RBC-dependent mechanisms. METHODS: In this translational study, plasma and RBCs were isolated from gestationally matched pre-eclamptic and healthy pregnant women and co-incubated overnight with mouse aortas for vascular reactivity studies. NO bioactivity, that is, nitrate, nitrite and cGMP, was assessed in plasma. Arginase activity and expression were analysed in RBCs. RESULTS: Plasma markers of NO homeostasis and signalling were decreased in pre-eclamptic women vs. healthy pregnant women. Co-incubation of aorta with pre-eclamptic RBCs, but not healthy pregnant RBCs, induced endothelial dysfunction, which was ameliorated by pharmacological inhibition of arginase, scavenging of ROS, and by nitrite treatment. This pathological vascular phenotype was not observed following incubation with pre-eclamptic plasma. Arginase expression and activity in RBCs were increased in pre-eclamptic vs. healthy pregnant women and was associated with pre-eclampsia severity. Pre-eclamptic RBC-induced endothelial dysfunction was not because of increased haemolysis/cell-free haemoglobin. CONCLUSION: This study demonstrates a novel role of the RBC in mediating the endothelial dysfunction associated with pre-eclampsia through arginase-dependent and oxidative stress-dependent mechanisms. Targeting of RBC arginase may provide a novel treatment modality for pre-eclampsia.

Renovascular effects of inorganic nitrate following ischemia-reperfusion of the kidney.

BACKGROUND: Renal ischemia-reperfusion (IR) injury is a common cause of acute kidney injury (AKI), which is associated with oxidative stress and reduced nitric oxide (NO) bioactivity and increased risk of developing chronic kidney disease (CKD) and cardiovascular disease (CVD). New strategies that restore redox balance may have therapeutic implications during AKI and associated complications. AIM: To investigate the therapeutic value of boosting the nitrate-nitrite-NO pathway during development of IR-induced renal and cardiovascular dysfunction. METHODS: Male C57BL/6 J mice were given sodium nitrate (10 mg/kg, i. p) or vehicle 2 h prior to warm ischemia of the left kidney (45 min) followed by sodium nitrate supplementation in the drinking water (1 mmol/kg/day) for the following 2 weeks. Blood pressure and glomerular filtration rate were measured and blood and kidneys were collected and used for biochemical and histological analyses as well as renal vessel reactivity studies. Glomerular endothelial cells exposed to hypoxia-reoxygenation, with or without angiotensin II, were used for mechanistic studies. RESULTS: IR was associated with reduced renal function and slightly elevated blood pressure, in combination with renal injuries, inflammation, endothelial dysfunction, increased Ang II levels and Ang II-mediated vasoreactivity, which were all ameliorated by nitrate. Moreover, treatment with nitrate (in vivo) and nitrite (in vitro) restored NO bioactivity and reduced mitochondrial oxidative stress and injuries. CONCLUSIONS: Acute treatment with inorganic nitrate prior to renal ischemia may serve as a novel therapeutic approach to prevent AKI and CKD and associated risk of developing cardiovascular dysfunction.

The obligatory role of host microbiota in bioactivation of dietary nitrate.

Nitric oxide (NO) is a key signalling molecule in the regulation of cardiometabolic function and impaired bioactivity is considered to play an important role in the onset and progression of cardiovascular and metabolic disease. Research has revealed an alternative NO-generating pathway, independent of NO synthase (NOS), in which the inorganic anions nitrate (NO3-) and nitrite (NO2-) are serially reduced to form NO. This work specifically aimed at investigating the role of commensal bacteria in bioactivation of dietary nitrate and its protective effects in a model of cardiovascular and metabolic disease. In a two-hit model, germ-free and conventional male mice were fed a western diet and the NOS inhibitor l-NAME in combination with sodium nitrate (NaNO3) or placebo (NaCl) in the drinking water. Cardiometabolic parameters including blood pressure, glucose tolerance and body composition were measured after six weeks treatment. Mice in both placebo groups showed increased body weight and fat mass, reduced lean mass, impaired glucose tolerance and elevated blood pressure. In conventional mice, nitrate treatment partly prevented the cardiometabolic disturbances induced by a western diet and l-NAME. In contrast, in germ-free mice nitrate had no such beneficial effects. In separate cardiovascular experiments, using conventional and germ-free animals, we assessed NO-like signalling downstream of nitrate by administration of sodium nitrite (NaNO2) via gavage. In this acute experimental setting, nitrite lowered blood pressure to a similar degree in both groups. Likewise, isolated vessels from germ-free mice robustly dilated in response to the NO donor sodium nitroprusside. In conclusion, our findings demonstrate the obligatory role of host-microbiota in bioactivation of dietary nitrate, thus contributing to its favourable cardiometabolic effects.

Dietary Nitrate Reduces Blood Pressure in Rats With Angiotensin II-Induced Hypertension via Mechanisms That Involve Reduction of Sympathetic Hyperactivity.

Several experimental and clinical studies have shown that dietary nitrate supplementation can increase nitric oxide bioavailability. In the oral cavity, commensal bacteria reduce nitrate to nitrite, which is subsequently absorbed into the circulation where reduction to nitric oxide by enzymatic systems occur. Although it is well-known that boosting the nitrate-nitrite-nitric oxide pathway can improve cardiovascular, renal, and metabolic functions and that sympathoexcitation contributes to the development of the same disorders, the potential effects of dietary nitrate on sympathetic activity remain to be elucidated. In this study, we hypothesized that treatment with inorganic nitrate could prevent the increase in sympathetic nerve activity in an experimental model of Ang II (angiotensin II)-induced hypertension. Multiple in vivo approaches were combined, that is, Wistar rats orally treated with the nitric oxide synthase inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester, 0.5 g/L) and implanted with subcutaneous osmotic minipump for continuous delivery of Ang II (120 ng/kg per minute; 14 days). Simultaneously, rats were supplemented with sodium nitrate (10 mmol/L) or placebo (sodium chloride; 10 mmol/L) in the drinking water. Blood pressure, heart rate, and renal sympathetic nerve activity were recorded. In placebo-treated rats, Ang II+L-NAME treatment-induced arterial hypertension, which was linked with reduced spontaneous baroreflex sensitivity and increased renal sympathetic nerve activity, as well as upregulation of AT1Rs (Ang II type-1 receptors) in the rostral ventrolateral medulla. Supplementation with nitrate normalized the expression of AT1Rs in rostral ventrolateral medulla and reduced sympathetic nerve activity, which was associated with attenuated development of hypertension. In conclusion, chronic dietary nitrate supplementation blunted the development of hypertension via mechanisms that involve reduction of sympathetic outflow.

The novel organic mononitrate NDHP attenuates hypertension and endothelial dysfunction in hypertensive rats.

RATIONALE: Development and progression of cardiovascular diseases, including hypertension, are often associated with impaired nitric oxide synthase (NOS) function and nitric oxide (NO) deficiency. Current treatment strategies to restore NO bioavailability with organic nitrates are hampered by undesirable side effects and development of tolerance. In this study, we evaluated NO release capability and cardiovascular effects of the newly synthesized organic nitrate 1, 3-bis (hexyloxy) propan-2-yl nitrate (NDHP). METHODS: A combination of in vitro and in vivo approaches was utilized to assess acute effects of NDHP on NO release, vascular reactivity and blood pressure. The therapeutic value of chronic NDHP treatment was assessed in an experimental model of angiotensin II-induced hypertension in combination with NOS inhibition. RESULTS: NDHP mediates NO formation in both cell-free system and small resistance arteries, a process which is catalyzed by xanthine oxidoreductase. NDHP-induced vasorelaxation is endothelium independent and mediated by NO release and modulation of potassium channels. Reduction of blood pressure following acute intravenous infusion of NDHP was more pronounced in hypertensive rats (two-kidney-one-clip model) than in normotensive sham-operated rats. Toxicological tests did not reveal any harmful effects following treatment with high doses of NDHP. Finally, chronic treatment with NDHP significantly attenuated the development of hypertension and endothelial dysfunction in rats with chronic NOS inhibition and angiotensin II infusion. CONCLUSION: Acute treatment with the novel organic nitrate NDHP increases NO formation, which is associated with vasorelaxation and a significant reduction of blood pressure in hypertensive animals. Chronic NDHP treatment attenuates the progression of hypertension and endothelial dysfunction, suggesting a potential for therapeutic applications in cardiovascular disease.

Lesion of the tuberomammillary nucleus E2-region attenuates postictal seizure protection in rats.

Postictal seizure protection (PSP) is an endogenous anticonvulsant phenomenon that follows an epileptic seizure and inhibits the induction of further seizures. The tuberomammillary nucleus (TM), located in the posterior hypothalamus, consists of five subregions and is the sole source of histaminergic neurons in the brain. To determine whether the TM is involved in PSP in rats, we tested the effects of bilateral electrolytic lesions of the TM E2-region on seizures induced by intermittent maximal electroshock (MES). The TM E2-region lesions significantly attenuated PSP during the intermittent MES procedure. Furthermore, intracerebroventricular injection of alpha-fluoromethylhistidine (100 microg), a selective and irreversible histidine decarboxylase inhibitor, mimicked the attenuation of PSP induced by the lesion of TM E2-region. In addition, neurochemical experiments revealed that the TM E2-region lesions markedly decreased basal histamine levels in the cortex, hippocampus, brainstem and hypothalamus, but had no significant effect on basal glutamate and GABA levels. Moreover, intermittent MES induced a persistent decrease of brain histamine levels in both sham-operated and lesioned rats. These results indicate that through its intrinsic histaminergic system, the TM may exert powerful inhibitory function during the intermittent MES procedure and actively participate in the mechanisms of PSP.

Renal denervation attenuates hypertension and renal dysfunction in a model of cardiovascular and renal disease, which is associated with reduced NADPH and xanthine oxidase activity.

Oxidative stress is considered a central pathophysiological event in cardiovascular disease, including hypertension. Early age reduction in renal mass is associated with hypertension and oxidative stress in later life, which is aggravated by increased salt intake. The aim of the present study was to examine if renal sympathetic denervation can exert blood pressure lowering effects in uninephrectomized (UNX) rats (3-week old) fed with high salt (HS, 4%; w/w) diet for 4 weeks. Moreover, we investigated if renal denervation is associated with changes in NADPH and xanthine oxidase-derived reactive oxygen species. Rats with UNX + HS had reduced renal function, elevated systolic and diastolic arterial pressures, which was accompanied by increased heart weight, and cardiac superoxide production compared to sham operated Controls. UNX + HS was also associated with higher expression and activity of NADPH and xanthine oxidase in the kidney. Renal denervation in rats with UNX + HS attenuated the development of hypertension and cardiac hypertrophy, but also improved glomerular filtration rate and reduced proteinuria. Mechanistically, renal denervation was associated with lower expression and activity of both NADPH oxidase and xanthine oxidase in the kidney, but also reduced superoxide production in the heart. In conclusion, our study shows for the first time that renal denervation has anti-hypertensive, cardio- and reno-protective effects in the UNX + HS model, which can be associated with decreased NADPH oxidase- and xanthine oxidase-derived reactive oxygen species (i.e., superoxide and hydrogen peroxide) in the kidney.

Blood Pressure-Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor.

Inorganic nitrate and nitrite from dietary and endogenous sources are metabolized to NO and other bioactive nitrogen oxides that affect blood pressure. The mechanisms for nitrite bioactivation are unclear, but recent studies in rodents suggest that gastric acidity may influence the systemic effects of this anion. In a randomized, double-blind, placebo-controlled crossover study, we tested the effects of a proton pump inhibitor on the acute cardiovascular effects of nitrite. Fifteen healthy nonsmoking, normotensive subjects, aged 19 to 39 years, were pretreated with placebo or esomeprazole (3×40 mg) before ingesting sodium nitrite (0.3 mg kg-1), followed by blood pressure monitoring. Nitrite reduced systolic blood pressure by a maximum of 6±1.3 mm Hg when taken after placebo, whereas pretreatment with esomeprazole blunted this effect. Peak plasma nitrite, nitrate, and nitroso species levels after nitrite ingestion were similar in both interventions. In 8 healthy volunteers, we then infused increasing doses of sodium nitrite (1, 10, and 30 nmol kg-1 min-1) intravenously. Interestingly, although plasma nitrite peaked at similar levels as with orally ingested nitrite (≈1.8 µmol/L), no changes in blood pressure were observed. In rodents, esomeprazole did not affect the blood pressure response to the NO donor, DEA NONOate, or vascular relaxation to nitroprusside and acetylcholine, demonstrating an intact downstream NO-signaling pathway. We conclude that the acute blood pressure-lowering effect of nitrite requires an acidic gastric environment. Future studies will reveal if the cardiovascular complications associated with the use of proton pump inhibitors are linked to interference with the nitrate-nitrite-NO pathway.

Synthesis and characterization of a novel organic nitrate NDHP: Role of xanthine oxidoreductase-mediated nitric oxide formation.

In this report, we describe the synthesis and characterization of 1,3-bis(hexyloxy)propan-2-yl nitrate (NDHP), a novel organic mono nitrate. Using purified xanthine oxidoreductase (XOR), chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy, we found that XOR catalyzes nitric oxide (NO) generation from NDHP under anaerobic conditions, and that thiols are not involved or required in this process. Further mechanistic studies revealed that NDHP could be reduced to NO at both the FAD and the molybdenum sites of XOR, but that the FAD site required an unoccupied molybdenum site. Conversely, the molybdenum site was able to reduce NDHP independently of an active FAD site. Moreover, using isolated vessels in a myograph, we demonstrate that NDHP dilates pre-constricted mesenteric arteries from rats and mice. These effects were diminished when XOR was blocked using the selective inhibitor febuxostat. Finally, we demonstrate that NDHP, in contrast to glyceryl trinitrate (GTN), is not subject to development of tolerance in isolated mesenteric arteries.