AT1 receptors modulate ethanol-induced loss of anticontractile effect of perivascular adipose tissue.

Ethanol consumption activates renin-angiotensin-aldosterone system (RAAS), which plays a major role in the pro-contractile and hypertensive effects linked to ethanol. We hypothesized that ethanol consumption induces loss of the anticontractile effect of perivascular adipose tissue (PVAT)through RAAS-mediated mechanisms. We examined the contribution of angiotensin II type 1 receptors (AT1R) to ethanol-induced PVAT dysfunction. With this purpose, male Wistar Hannover rats were treated with ethanol 20 % (in volume ratio) and/or losartan (antagonist of AT1R; 10 mg/kg/day, gavage) for 9 weeks. Losartan prevented the increase in blood pressure and the loss of the anticontractile effect of PVAT induced by ethanol consumption. PVAT dysfunction occurred after 3 and 9 weeks of treatment with ethanol in an endothelium-dependent manner. Blockade of AT1R prevented ethanol-induced reduction of adiponectin levels in PVAT from ethanol-treated rats. Functional assays revealed that ethanol impaired the anticontractile effect of PVAT-derived angiotensin (1-7) and endothelial nitric oxide (NO). In conclusion, AT1R are implicated in ethanol-induced loss of the anticontractile effect of PVAT. In PVAT, AT1R activation decreases the production of adiponectin, a PVAT-derived factor that promotes vasorelaxation in an endothelium-dependent manner. In the endothelium, AT1R favors the production of superoxide (O2•-) leading to a reduction in NO bioavailability. These responses impair the vasodilator action induced by PVAT-derived angiotensin (1-7), which occurs via Mas receptors located in endothelial cells. Ethanol-induced PVAT dysfunction favors vascular hypercontractility, a response that could contribute to the hypertensive state associated with ethanol consumption.

Orally administered sodium nitrite prevents the increased α-1 adrenergic vasoconstriction induced by hypertension and promotes the S-nitrosylation of calcium/calmodulin-dependent protein kinase II.

The unsatisfactory rates of adequate blood pressure control among patients receiving antihypertensive treatment calls for new therapeutic strategies to treat hypertension. Several studies have shown that oral sodium nitrite exerts significant antihypertensive effects, but the mechanisms underlying these effects remain unclear. While these mechanisms may involve nitrite-derived S-nitrosothiols, their implication in important alterations associated with hypertension, such as aberrant α1-adrenergic vasoconstriction, has not yet been investigated. Here, we examined the effects of oral nitrite treatment on vascular responses to the α1-adrenergic agonist phenylephrine in two-kidney, one clip (2K1C) hypertensive rats and investigated the potential underlying mechanisms. Our results show that treatment with oral sodium nitrite decreases blood pressure and prevents the increased α1-adrenergic vasoconstriction in 2K1C hypertensive rats. Interestingly, we found that these effects require vascular protein S-nitrosylation, and to investigate the specific S-nitrosylated proteins we performed an unbiased nitrosoproteomic analysis of vascular smooth muscle cells (VSMCs) treated with the nitrosylating compound S-nitrosoglutathione (GSNO). This analysis revealed that GSNO markedly increases the nitrosylation of calcium/calmodulin-dependent protein kinase II γ (CaMKIIγ), a multifunctional protein that mediates the α1-adrenergic receptor signaling. This result was associated with reduced α1-adrenergic receptor-mediated CaMKIIγ activity in VSMCs. We further tested the relevance of these findings in vivo and found that treatment with oral nitrite increases CaMKIIγ S-nitrosylation and blunts the increased CaMKIIγ activity induced by phenylephrine in rat aortas. Collectively, these results are consistent with the idea that oral sodium nitrite treatment increases vascular protein S-nitrosylation, including CaMKIIγ as a target, which may ultimately prevent the increased α1-adrenergic vasoconstriction induced by hypertension. These mechanisms may help to explain the antihypertensive effects of oral nitrite and hold potential implications in the therapy of hypertension and other cardiovascular diseases associated with abnormal α1-adrenergic vasoconstriction.

TRPA1 Polymorphisms Modify the Hypotensive Responses to Propofol with No Change in Nitrite or Nitrate Levels.

Anesthesia with propofol is frequently associated with hypotension. The TRPA1 gene contributes to the vasodilator effect of propofol. Hypotension is crucial for anesthesiologists because it is deleterious in the perioperative period. We tested whether the TRPA1 gene polymorphisms or haplotypes interfere with the hypotensive responses to propofol. PCR-determined genotypes and haplotype frequencies were estimated. Nitrite, nitrates, and NOx levels were measured. Propofol induced a more expressive lowering of the blood pressure (BP) without changing nitrite or nitrate levels in patients carrying CG+GG genotypes for the rs16937976 TRPA1 polymorphism and AG+AA genotypes for the rs13218757 TRPA1 polymorphism. The CGA haplotype presented the most remarkable drop in BP. Heart rate values were not impacted. The present exploratory analysis suggests that TRPA1 genotypes and haplotypes influence the hypotensive responses to propofol. The mechanisms involved are probably other than those related to NO bioavailability. With better genetic knowledge, planning anesthesia with fewer side effects may be possible.

Gene-gene interactions in the protein kinase C/endothelial nitric oxide synthase axis impact the hypotensive effects of propofol.

Anaesthesia with propofol is frequently associated with hypotension, which is at least partially attributable to increased nitric oxide (NO) formation derived from the activation of protein kinase C (PKC)/endothelial NO synthase (NOS3) axis. In this cross-sectional study, we tested whether PRKCA (which encodes PKCα) polymorphisms, or haplotypes, and interactions among PRKCA and NOS3 polymorphisms affect the hypotensive responses to propofol. We collected venous blood samples from 164 patients before and 10 min after propofol administration. Genotypes were determined by PCR and haplotype frequencies were estimated. Nitrite and NOx (nitrites+nitrates) levels were measured by using an ozone-based chemiluminescence assay and the Griess reaction, respectively. We used multifactor dimensionality reduction to test interactions among PRKCA and NOS3 polymorphisms. Propofol promoted enhanced blood pressure-lowering effects and increased nitrite levels in subjects carrying GA + AA genotypes for the rs16960228 and TC + CC genotypes for the rs1010544 PRKCA polymorphisms, and the CCG haplotype. Moreover, genotypes for the rs1010544 PRKCA polymorphism were associated with higher or lower blood pressure decreases in response to propofol depending on the genotypes for the rs2070744 NOS3 polymorphism. Our findings suggest that PRKCA genotypes and haplotypes impact the hypotensive responses to propofol, possibly by modifying NO bioavailability, and that PRKCA-NOS3 interactions modify the blood pressure-lowering effects of propofol.

Antioxidant tempol modulates the increases in tissue nitric oxide metabolites concentrations after oral nitrite administration.

Nitric oxide (NO) metabolites have physiological and pharmacological importance and increasing their tissue concentrations may result in beneficial effects. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) has antioxidant properties that may improve NO bioavailability. Moreover, tempol increases oral nitrite-derived gastric formation of S-nitrosothiols (RSNO). We hypothesized that pretreatment with tempol may further increase tissue concentrations of NO-related species after oral nitrite administration and therefore we carried out a time-dependent analysis of how tempol affects the concentrations of NO metabolites in different tissues after oral nitrite administration to rats. NO metabolites (nitrate, nitrite and RSNO) were assessed by ozone-based reductive chemiluminescence assays in plasma, stomach, aorta, heart and liver samples obtained from anesthetized rats at baseline conditions and 15 min, 30 min, 2 h or 24 h after oral nitrite (15 mg/kg) was administered to rats pretreated with tempol (18 mg/kg) or vehicle 15 min prior to nitrite administration. Aortic protein nitrosation was assessed by resin-assited capture (SNO-RAC) method. We found that pretreatment with tempol transiently enhanced nitrite-induced increases in nitrite, RSNO and nitrate concentrations in the stomach and in the plasma (all P < 0.05), particularly for 15-30 min, without affecting aortic protein nitrosation. Pretreatment with tempol enhanced nitrite-induced increases in nitrite (but not RSNO or nitrate) concentrations in the heart (P < 0.05). In contrast, tempol attenuated nitrite-induced increases in nitrite, RSNO or nitrate concentrations in the liver. These findings show that pretreatment with tempol affects oral nitrite-induced changes in tissue concentrations of NO metabolites depending on tissue type and does not increase nitrite-induced vascular nitrosation. These results may indicate that oral nitrite therapy aiming at achieving increased nitrosation of cardiovascular targets requires appropriate doses of nitrite and is not optimized by tempol.

Antiseptic mouthwash inhibits antihypertensive and vascular protective effects of L-arginine.

L-arginine supplementation increases nitric oxide (NO) formation and bioavailability in hypertension. We tested the possibility that many effects of L-arginine are mediated by increased formation of NO and enhanced nitrite, nitrate and nitrosylated species concentrations, thus stimulating the enterosalivary cycle of nitrate. Those effects could be prevented by antiseptic mouthwash. We examined how the derangement of the enterosalivary cycle of nitrate affects the improvement of endothelial dysfunction (assessed with isolated aortic ring preparation), the antihypertensive (assessed by tail-cuff blood pressure measurement) and the antioxidant effects (assessed with the fluorescent dye DHE) of L-arginine in two-kidney, one-clip hypertension model in rats by using chlorhexidine to decrease the number of oral bacteria and to decrease nitrate reductase activity assessed from the tongue (by ozone-based chemiluminiscence assay). Nitrite, nitrate and nitrosylated species concentrations were assessed (ozone-based chemiluminiscence). Chlorhexidine mouthwash reduced the number of oral bacteria and tended to decrease the nitrate reductase activity from the tongue. Antiseptic mouthwash blunted the improvement of the endothelial dysfunction and the antihypertensive effects of L-arginine, impaired L-arginine-induced increases in plasma nitrite and nitrosylated species concentrations, and blunted L-arginine-induced increases in aortic nitrate concentrations and vascular antioxidant effects. Our results show for the first time that the vascular and antihypertensive effects of L-arginine are prevented by antiseptic mouthwash. These findings show an important new mechanism that should be taken into consideration to explain how the use of antibacterial mouth rinse may affect arterial blood pressure and the risk of developing cardiovascular and other diseases.

Omeprazole induces vascular remodeling by mechanisms involving xanthine oxidoreductase and matrix metalloproteinase activation.

Proton pump inhibitors (PPI) are commonly used drugs that may increase the cardiovascular risk by mechanisms not entirely known. We examined whether the PPI omeprazole promotes vascular oxidative stress mediated by xanthine oxidoreductase (XOR) leading to activation of matrix metalloproteinases (MMPs) and vascular remodeling. We studied Wistar rats treated with omeprazole (or vehicle) combined with the XOR inhibitor allopurinol (or vehicle) for four weeks. Systolic blood pressure (SBP) measured by tail-cuff plethysmography was not affected by treatments. Omeprazole treatment increased the aortic cross-sectional area and media/lumen ratio by 25% (P < 0.05). Omeprazole treatment decreased gastric pH and induced vascular remodeling accompanied by impaired endothelium-dependent aortic responses (assessed with isolated aortic ring preparation) to acetylcholine (P < 0.05). Omeprazole increased vascular active MMP-2 expression and activity assessed by gel zymography and in situ zymography, respectively (P < 0.05). Moreover, omeprazole enhanced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay (both P < 0.05). All these biochemical changes caused by omeprazole were associated with increased vascular XOR activity (but not XOR expression assessed by Western blot) and treatment with allopurinol fully prevented them (all P < 0.05). Importantly, treatment with allopurinol prevented the vascular dysfunction and remodeling caused by omeprazole. Our results suggest that the long-term use of omeprazole induces vascular dysfunction and remodeling by promoting XOR-derived reactive oxygen species formation and MMP activation. These findings provide evidence of a new mechanism that may underlie the unfavorable cardiovascular outcomes observed with PPI therapy. Clinical studies are warranted to validate our findings.

Arginase II polymorphisms modify the hypotensive responses to propofol by affecting nitric oxide bioavailability.

PURPOSE: Propofol anesthesia is usually accompanied by hypotensive responses, which are at least in part mediated by nitric oxide (NO). Arginase I (ARG1) and arginase II (ARG2) compete with NO synthases for their common substrate L-arginine, therefore influencing the NO formation. We examined here whether ARG1 and ARG2 genotypes and haplotypes affect the changes in blood pressure and NO bioavailability in response to propofol. METHODS: Venous blood samples were collected from 167 patients at baseline and after 10 min of anesthesia with propofol. Genotypes were determined by polymerase chain reaction. Nitrite concentrations were measured by using an ozone-based chemiluminescence assay, while NOx (nitrites + nitrates) levels were determined by using the Griess reaction. RESULTS: We found that patients carrying the AG + GG genotypes for the rs3742879 polymorphism in ARG2 gene and the ARG2 GC haplotype show lower increases in nitrite levels and lower decreases in blood pressure after propofol anesthesia. On the other hand, subjects carrying the variant genotypes for the rs10483801 polymorphism in ARG2 gene show more intense decreases in blood pressure (CA genotype) and/or higher increases in nitrite levels (CA and AA genotypes) in response to propofol. CONCLUSION: Our results suggest that ARG2 variants affect the hypotensive responses to propofol, possibly by modifying NO bioavailability. TRIAL REGISTRATION: NCT02442232.

Ethanol and cyclophosphamide induce similar nephrotoxic effects: possible role for Nox4 and superoxide.

We tested the hypothesis that ethanol consumption would aggravate the renal damage induced by cyclophosphamide (CYP). Male C57BL/6 J mice from control (n = 8) and CYP (n = 12) groups had free access to filtered water and standard rodent chow for 12 weeks. Then, 24 h before euthanasia mice received an intraperitoneal injection of saline or CYP (300 mg/kg). Mice from ethanol (n = 8) and CYP + ethanol (n = 12) groups had free access to increasing doses of ethanol for 12 weeks. Twenty-four hours before euthanasia, mice from ethanol and CYP + ethanol groups received an intraperitoneal injection of saline or CYP, respectively. Ethanol, CYP, or the association of both drugs augmented serum levels of creatinine and increased the levels of superoxide ([Formula: see text]) generation and thiobarbituric acid reactive substances in the renal cortex. Upregulation of Nox4 and increased activity of superoxide dismutase were detected in the renal cortex of mice treated with ethanol, CYP, or the combination of these drugs; however, these molecular alterations induced by CYP were not potentiated by ethanol consumption. Our findings revealed that chronic ethanol consumption had no potentiating effect on the nephrotoxic effects displayed by CYP. It is possible that the combination of these drugs showed no synergistic effect because they share the same molecular mechanisms of renal toxicity.

Oral nitrite treatment increases S-nitrosylation of vascular protein kinase C and attenuates the responses to angiotensin II.

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II.

Consistent gastric pH-dependent effects of suppressors of gastric acid secretion on the antihypertensive responses to oral nitrite.

Proton pump inhibitors (PPI) are suppressors of gastric acid secretion (SGAS) that decrease gastric nitric oxide (NO) formation from nitrite and increase the cardiovascular risk. However, H2 receptor antagonists (H2RA) are considered safer than PPIs. We challenged this notion and hypothesized that both omeprazole (PPI) and ranitidine (H2RA) attenuate the responses to oral nitrite because both drugs increase gastric pH and therefore could decrease nitrite-derived NO formation in the stomach. We examined the blood pressure responses to oral nitrite in hypertensive rats treated with omeprazole, ranitidine, or vehicle. Chemiluminensce-based assays were used to measure gastric NO formation, plasma and gastric concentrations of nitrite, nitrate, and nitrosylated species (RXNO) to clarify the mechanism involved in the effects of SGAS on the responses to oral nitrite. Both drugs increased gastric pH, impaired oral nitrite-induced hypotensive responses, gastric NO formation, and blunted the increases in circulating RXNO concentrations, but not in circulating nitrite and nitrate concentrations. These findings were reproduced in a second study using sodium acetate buffers at pH 3.5, 4.5, and 5.5 to mimic gastric pH found with vehicle, ranitidine, and omeprazole, respectively. Increasing gastric pH impaired oral nitrite-induced hypotensive responses, gastric NO formation, and blunted the increases in circulating RXNO concentrations, but not in circulating nitrite and nitrate concentrations. Our results clearly indicate that SGAS impair nitrite-induced gastric formation of NO and vasoactive RXNO in a pH-dependent manner, thus resulting in impaired responses to oral nitrite. These findings may have several clinical implications, particularly to patients with cardiovascular diseases.

A comprehensive time course study of tissue nitric oxide metabolites concentrations after oral nitrite administration.

Nitrite and nitrate are considered nitric oxide (NO) storage pools. The assessment of their tissue concentrations may improve our understanding of how they attenuate pathophysiological mechanisms promoting disease. We hypothesized that significant differences exist when the tissue concentrations of nitrite, nitrate, and nitrosylated species (RXNO) are compared among different tissues, particularly when nitrite is administered orally because nitrite generates various NO-related species in the stomach. We studied the different time-dependent changes in plasma and tissue concentrations of nitrite, nitrate, and RXNO after oral nitrite 15 mg/kg was administered rats, which were euthanized 15, 30, 60, 120, 240, 480 or 1440 min after nitrite administration. A control group received water. Arterial blood samples were collected and the rats were perfused with a PBS solution containing NEM/DTPA to prevent the destruction of RXNO. After perfusion, heart, aorta, mesenteric artery, brain, stomach, liver and femoral muscle were harvested and immediately stored at -70°C until analyzed for their nitrite, nitrate and RXNO contents using an ozone-based reductive chemiluminescence assay. While nitrite administration did not increase aortic nitrite or nitrate concentrations for at least 60 min, both aorta and mesenteric vessels stored nitrite from 8 to 24 h after its administration and their tissue concentrations increased from 10 to 40-fold those found in plasma. In contrast, the other studied tissues showed only transient increases in the concentrations of these NO metabolites, including RXNO. The differences among tissues may reflect differences in mechanisms regulating cellular influx of nitrite. These findings have important pharmacological and clinical implications.

Sources and Effects of Oxidative Stress in Hypertension.

BACKGROUND: Disruption of redox signaling is a common pathophysiological mechanism observed in several diseases. In hypertension, oxidative stress, resulted either from enhances in Reactive Oxygen Species (ROS) production or decreases in antioxidant defenses, is associated with increase in blood pressure, endothelial dysfunction and vascular remodeling. Although the role of oxidative stress in the development of hypertension is well known, it is still unclear if this process is a cause or a consequence of tissue changes in hypertension. Indeed, unbalanced ROS formation results in several detrimental effects that contribute to hypertension, including reduction in nitric oxide bioavailability and activation of metalloproteinases. Additionally, ROS may also directly react with lipids, proteins and DNA, thereby contributing to tissue damage associated with hypertension. Therefore, a deep understanding of the role of oxidative stress in hypertension is essential to comprehend its pathophysiology and to identify new therapeutic targets. CONCLUSION: This mini-review discusses the main enzymatic sources of oxidants and the major antioxidant defenses in the vasculature, followed by the effects of oxidative stress in hypertension, highlighting endothelial dysfunction, vascular remodeling and tissue damage.

Treatment with nitrite prevents reactive oxygen species generation in the corpora cavernosa and restores intracavernosal pressure in hypertensive rats.

Hypertension is a risk factor for erectile dysfunction (ED) and both conditions are associated with oxidative stress. Given that nitrite is described to display antioxidant effects, we hypothesized that treatment with nitrite would exert antioxidant effects attenuating both reactive oxygen species (ROS) generation in the corpora cavernosa (CC) and ED induced by hypertension. Two kidney, one clip (2K1C) hypertension was induced in male Wistar rats. Treatment with sodium nitrite (15 mg/kg/day, p.o., gavage) was initiated two weeks after surgery to induce hypertension and maintained for four weeks. Nitrite abrogated both the decrease in intracavernosal pressure and endothelial dysfunction of the CC induced by hypertension. Treatment with nitrite decreased hypertension-induced ROS generation in the CC assessed in situ using the fluorescent dye dihidroethidium (DHE) and with the lucigenin assay. Western immunoblotting analysis revealed that nitrite prevented the increase in Nox1 expression in the CC from 2K1C rats. Decreased concentrations of hydrogen peroxide (H2O2) were found in the CC from hypertensive rats and treatment with nitrite prevented this response. Treatment with nitrite increased the fluorescence of DAF-2DA in the CC from sham-operated rats and restored nitric oxide (NO) levels in the CC from 2K1C rats. In summary, we found novel evidence that nitrite reversed the decrease in intracavernosal pressure induced by 2K1C hypertension. This response was partially attributed to the antioxidant effect of nitrite that blunted ROS generation and endothelial dysfunction in the CC. In addition, nitrite-derived NO may have promoted direct protective actions against hypertension-induced CC dysfunction.

Antioxidant and antihypertensive responses to oral nitrite involves activation of the Nrf2 pathway.

Impaired redox balance contributes to the cardiovascular alterations of hypertension and activation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway may counteract these alterations. While nitrite recycles back to NO and exerts antioxidant and antihypertensive effects, the mechanisms involved in these responses are not fully understood. We hypothesized that nitrite treatment of two-kidney, one-clip (2K1C) hypertensive rats activates the Nrf2 pathway, promotes the transcription of antioxidant genes, and improves the vascular redox imbalance and dysfunction in this model. Two doses of oral nitrite were studied: 15 mg/kg and the sub-antihypertensive dose of 1 mg/kg. Nitrite 15 mg/kg (but not 1 mg/kg) decreased blood pressure and increased circulating plasma nitrite and nitrate. Both doses blunted hypertension-induced increases in mesenteric artery reactive oxygen species concentrations assessed by DHE technique and restored the impaired mesenteric artery responses to acetylcholine. While 2K1C hypertension decreased nuclear Nrf2 accumulation, both doses of nitrite increased nuclear Nrf2 accumulation and mRNA expression of Nrf2-regulated genes including superoxide dismutase-1 (SOD1), catalase (CAT), glutathione peroxidase (GPX), thioredoxin-1(TRDX-1) and -2 (TRDX-2). To further confirm nitrite-mediated antioxidant effects, we measured vascular SOD and GPX activity and we found that nitrite at 1 or 15 mg/kg increased the activity of both enzymes (P < 0.05). These results suggest that activation of the Nrf2 pathway promotes antioxidant effects of nitrite, which may improve the vascular dysfunction in hypertension, even when nitrite is given at a sub-antihypertensive dose. These findings may have many clinical implications, particularly in the therapy of hypertension and other cardiovascular diseases.

Sodium nitrite improves hypertension-induced myocardial dysfunction by mechanisms involving cardiac S-nitrosylation.

Although nitrite improves vascular function and lowers blood pressure, its cardiac effects are not completely known. We investigated whether nitrite improves the cardiac function in normotensive and in hypertensive rats. Two-kidney, one-clip hypertension model (2K1C) was induced in Wistar rats. Blood pressure was evaluated by tail-cuff plethysmography over 6 weeks. By the end of week 2, hypertensive and normotensive rats received nitrite (daily dose of 1 or 15 mg/kg) by gavage for 4 weeks. Cardiac morphology and function were performed by transthoracic echocardiography. Intrinsic heart function was evaluated using the isolated heart model (Langendorff's preparation). Starling curves were generated under nitrite (1 µmol/L) and/or ascorbate (1 mmol/L) or vehicle. Cardiac tissue was collected and snap frozen for biochemical analysis. Nitrite treatment (15 mg/kg) lowered both systolic blood pressure and the increases in left ventricular (LV) mass found in 2K1C rats (P < .05). In addition, nitrite treatment restored the decreased cardiac output in 2K1C rats (P < .05) and improved the cardiac function. These findings were associated with increased nitrite, S-nitrosothiols, and protein S-nitrosylation (all P < .05) assessed in heart tissue. The cardiac effects of nitrite were further investigated in the isolated heart model, and nitrite infusion (1 µmol/L) enhanced cardiac contractility and relaxation. This infusion increased S-nitrosothiols concentrations and protein S-nitrosylation in the heart. Ascorbate completely blunted all nitrite-induced effects. These findings show that treatment with oral nitrite improves cardiac function by mechanisms involving increased S-nitrosothiols generation and S-nitrosylation of cardiac proteins. Pharmacological strategies promoting cardiac S-nitrosylation may be useful to improve myocardial function in heart diseases.

Nitric oxide in the dorsal periaqueductal gray mediates the panic-like escape response evoked by exposure to hypoxia.

Exposure of rats to an environment with low O2 levels evokes a panic-like escape behavior and recruits the dorsal periaqueductal gray (dPAG), which is considered to be a key region in the pathophysiology of panic disorder. The neurochemical basis of this response is, however, currently unknown. We here investigated the role played by nitric oxide (NO) within the dPAG in mediation of the escape reaction induced by hypoxia exposure. The results showed that exposure of male Wistar rats to 7% O2 increased nitrite levels, a NO metabolite, in the dPAG but not in the amygdala or hypothalamus. Nitrite levels in the dPAG were correlated with the number of escape attempts during the hypoxia challenge. Injections of the NO synthesis inhibitor NPA, the NO-scavenger c- PTIO, or the NMDA receptor antagonist AP-7 into the dorsolateral column of the periaqueductal gray (dlPAG) inhibited escape expression during hypoxia, without affecting the rats' locomotion. Intra-dlPAG administration of c-PTIO had no effect on the escape response evoked by the elevated-T maze, a defensive behavior that has also been associated with panic attacks. Altogether, our results suggest that NO plays a critical role in mediation of the panic-like defensive response evoked by exposure to low O2 concentrations.

Mechanisms impairing blood pressure responses to nitrite and nitrate.

Hypertension is a multifactorial disease associated with impaired nitric oxide (NO) production and bioavailability. In this respect, restoring NO activity by using nitrite and nitrate has been considered a potential therapeutic strategy to treat hypertension. This possibility is justified by the understanding that both nitrite and nitrate may be recycled back to NO and also promote the generation of other bioactive species. This process involves a complex biological circuit known as the enterosalivary cycle of nitrate, where this anion is actively taken up by the salivary glands and converted to nitrite by nitrate-reducing bacteria in the oral cavity. Nitrite is then ingested and reduced to NO and other nitroso species under the acid conditions of the stomach, whereas reminiscent nitrite that escapes gastric reduction is absorbed systemically and can be converted into NO by nitrite-reductases in tissues. While there is no doubt that nitrite and nitrate exert antihypertensive effects, several agents can impair the blood pressure responses to these anions by disrupting the enterosalivary cycle of nitrate. These agents include dietary and smoking-derived thiocyanate, antiseptic mouthwash, proton pump inhibitors, ascorbate at high concentrations, and xanthine oxidoreductase inhibitors. In this article, we provide an overview of the physiological aspects of nitrite and nitrate bioactivation and the therapeutic potential of these anions in hypertension. We also discuss mechanisms by which agents counteracting the antihypertensive responses to nitrite and nitrate mediate their effects. These critical aspects should be taken into consideration when suggesting nitrate or nitrite-based therapies to patients.

Nitrite treatment downregulates vascular MMP-2 activity and inhibits vascular remodeling in hypertension independently of its antihypertensive effects.

Hypertension is associated with cardiovascular remodeling. Given that impaired redox state activates matrix metalloproteinase (MMP)- 2 and promotes vascular remodeling, we hypothesized that nitrite treatment at a non-antihypertensive dose exerts antioxidant effects and attenuates both MMP-2 activation and vascular remodeling of hypertension. We examined the effects of oral sodium nitrite at antihypertensive (15 mg/kg) or non-antihypertensive (1 mg/kg) daily dose in hypertensive rats (two kidney, one clip; 2K1C model). Sham-operated and 2K1C hypertensive rats received vehicle or nitrite by gavage for four weeks. Systolic blood pressure decreased only in hypertensive rats treated with nitrite 15 mg/Kg/day. Both low and high nitrite doses decreased 2K1C-induced vascular remodeling assessed by measuring aortic cross-sectional area, media/lumen ratio, and number of vascular smooth muscle cells/aortic length. Both low and high nitrite doses decreased 2K1C-induced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay. Vascular MMP-2 expression and activity were assessed by gel zymography, Western blot, and in situ zymography increased with hypertension. While MMP-2 levels did not change in response to both doses of nitrite, both doses completely prevented hypertension-induced increases in vascular MMP activity. Moreover, incubation of aortas from hypertensive rats with nitrite at 1-20 µmol/L reduced gelatinolytic activity by 20-30%. This effect was fully inhibited by the xanthine oxidase (XOR) inhibitor febuxostat, suggesting XOR-mediated generation of nitric oxide (NO) from nitrite as a mechanism explaining the responses to nitrite. In vitro incubation of aortic extracts with nitrite 20 µmol/L did not affect MMP-2 activity. These results show that nitrite reverses the vascular structural alterations of hypertension, independently of anti-hypertensive effects. This response is mediated, at least in part, by XOR and is attributable to antioxidant effects of nitrite blunting vascular MMP-2 activation. Our findings suggest nitrite therapy to reverse structural alterations of hypertension.