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.

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.

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.

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.

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.

Contrasting effects of low versus high ascorbate doses on blood pressure responses to oral nitrite in L-NAME-induced hypertension.

Nitrite reduces blood pressure (BP) in both clinical and experimental hypertension. This effect is attributable to the formation of nitric oxide (NO) and other NO-related species, which may be improved by ascorbate or other antioxidants. However, the BP responses to oral nitrite result, at least in part, of increased gastric S-nitrosothiol formation. This study tested the hypothesis that ascorbate may destroy S-nitrosothiols and therefore not all doses of ascorbate enhance the BP responses to oral nitrite. We assessed the BP responses to oral sodim nitrite (0.2 mmol/kg) in L-NAME hypertensive rats pretreated with ascorbate (0, 0.02, 0.2, or 2 mmol/kg). Plasma and gastric wall concentrations of nitrite and nitroso compounds concentrations were determined using an ozone-based reductive chemiluminescence assay. Nitrate concentrations were determined using the Griess reaction. Free thiol concentrations were determined by a colorimetric assay. The BP responses to nitrite exhibited a bell-shape profile as they were not modified by ascorbate 0.02 mmol/l, whereas the 0.2 mmol/kg dose enhanced and the 2 mmol/kg dose attenuated BP responses. In parallel with BP responses, nitrite-induced increases in plasma nitrite and RSNO species were not modified by ascorbate 0.02 mmol/l, whereas the 0.2 mmol/kg dose enhanced and the 2 mmol/kg dose attenuated them. Similar experiments were carried out with an equimolar dose of S-nitrosogluthathione. Ascorbate dose-dependently impaired the BP responses to S-nitrosogluthathione, and the corresponding increases in plasma RSNO, but not in plasma nitrite concentrations. This is the first study to show that while ascorbate dose-dependently impairs the BP responses to oral S-nitrosogluthathione, there are contrasting effects when low versus high ascorbate doses are compared with respect to its effects on the blood pressure responses to oral nitrite administration. Our findings may have special implications to patients taking ascorbate, as high doses of this vitamin may impair protective mechanisms associated with nitrite or nitrate from dietary sources.

Endothelial nitric oxide synthase polymorphisms affect the changes in blood pressure and nitric oxide bioavailability induced by propofol.

Propofol anesthesia is usually accompanied by hypotension, which is at least in part related to enhanced endothelial nitric oxide synthase (NOS3)-derived NO bioavailability. We examined here whether NOS3 polymorphisms (rs2070744, 4b/4a VNTR, rs3918226 and rs1799983) and haplotypes affect the changes in blood pressure and NO bioavailability induced by propofol. Venous blood samples were collected from 168 patients at baseline and after 10 min of anesthesia with propofol 2 mg/kg administered intravenously by bolus injection. Genotypes were determined by polymerase chain reaction and haplotype frequencies were estimated. Nitrite concentrations were measured by using an ozone-based chemiluminescence assay, while NOx (nitrites + nitrates) levels were determined by using the Griess reaction. We found that CT + TT genotypes for the rs3918226 polymorphism, the ba + aa genotypes for the 4b/4a VNTR and the CTbT haplotype were associated with lower decreases in blood pressure and lower increases in nitrite levels after propofol anesthesia. On the other hand, the TCbT and CCbT haplotypes were associated with more intense decreases in blood pressure and higher increases in nitrite levels in response to propofol. Our results suggest that NOS3 polymorphisms and haplotypes influence the hypotensive responses to propofol, possibly by affecting NO bioavailability.

The antihypertensive effects of sodium nitrite are not associated with circulating angiotensin converting enzyme inhibition.

Nitrite-derived nitric oxide (NO) formation exerts antihypertensive effects. Because NO inhibits angiotensin converting enzyme (ACE) activity, we carried a comprehensive series of experiments in rats to test the hypothesis that sodium nitrite exerts antihypertensive effects by inhibiting ACE. We examined whether sodium nitrite (15 mg/kg; or vehicle; by gavage): (I) attenuates the pressor responses to angiotensin I at doses of 0.03, 0.1, 0.3, 1, 3, and 10 µg/kg intravenously; (II) attenuates the acute hypertension induced by L-NAME (100 mg/kg; or vehicle; by gavage); (III) attenuates the chronic hypertension induced by L-NAME (1 g/L in drinking water; or vehicle) administered for 6 weeks; (IV) attenuates the hypertension in the 2 kidney-1 clip (2K1C) chronic hypertension model. Blood samples were collected at the end of each study and plasma angiotensin converting enzyme (ACE) activity was measured with a fluorimetric assay using Hippuryl-His-Leu as substrate. ACE inhibitors were used as positive controls. Plasma nitrite concentrations were measured by ozone-based reductive chemiluminescence. The in vitro effects of sodium nitrite (0, 1, 3, 10, 30, 100 µmol/L) on plasma ACE activity were also determined. We found that sodium nitrite did not affect the pressor responses to angiotensin I. Moreover, while sodium nitrite exerted significant antihypertensive effects in acute and chronic hypertension models, no significant effects on plasma ACE activity were found. In vitro experiments showed no effects of sodium nitrite on plasma ACE activity. This is the first study to demonstrate that the acute and chronic antihypertensive effects of sodium nitrite are not associated with significant inhibition of circulating ACE activity.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre-eclampsia.

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

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.

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.

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.

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.

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.

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.

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.

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.