TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Free Radical Biology and Medicine 65 (2013) 446–455

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Original Contribution

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation Jefferson H. Amaral a, Marcelo F. Montenegro a, Lucas C. Pinheiro a, Graziele C. Ferreira a, Rafael P. Barroso b, Antonio J. Costa-Filho b, Jose E. Tanus-Santos a,n a b

Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirao Preto, SP, Brazil Department of Physics, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto. Av. Bandeirantes, 3900, 14040-901, Ribeirao Preto, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 27 May 2013 Received in revised form 4 July 2013 Accepted 18 July 2013 Available online 23 July 2013

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (Nω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite. & 2013 Elsevier Inc. All rights reserved.

Keywords: Nitrite TEMPOL Nitric oxide Hypertension L-NAME

Introduction Nitric oxide (NO) is oxidized to form the anions nitrite (NO2
) and nitrate (NO3
) [1]. While those anions were considered as Abbreviations: C-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide; DHE, dihydroethidium; EPR, electron paramagnetic resonance; L-NAME, Nω -nitro-L-arginine methyl ester; MAP, mean arterial pressure; NO, nitric oxide; ROS, reactive oxygen species; SGJ, simulated gastric juice; SHR, spontaneously hypertensive rats; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl. n Corresponding author. Fax: +55 16 3602 0220. E-mail addresses: [email protected], [email protected] (J.E. Tanus-Santos). 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.032

relatively inactive oxidation products of NO in the past, it is now widely acknowledged that they can recycle back to NO, and that a nitrate-nitrite-NO pathway plays a major role in physiological and pathological NO formation independent of NO synthases [2–4]. These findings have now brought increased attention to the relevance of dietary nitrite or nitrate, which directly, or after reduction of nitrate to nitrite by oral bacteria, promote NO formation under the acidic conditions of the stomach [5,6]. In a seminal study showing that ingestion of lettuce increased intragastric nonenzymatic NO production, Lundberg et al. clearly verified the major importance of low gastric pH for NO formation [7]. While the hypotensive effects of nitrite likely involve bioactivation of plasma nitrite, the mechanisms explaining how orally

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administered nitrite exerts antihypertensive effects are not entirely known and may depend, at least in part, on gastric NO formation [8]. Indeed, we have recently shown that treatment with omeprazole (a gastric proton pump inhibitor) increased gastric pH and attenuated the hypotensive effects of oral nitrite administration [9], thus aligning with those initial fundamental findings [7] and showing that the acidic conditions of the stomach are very important for the hypotensive effects of oral nitrite. In this respect, factors other than the gastric pH may affect NO formation from nitrite [10,11]. Indeed, reducing agents may facilitate NO formation from nitrite [12], and it is possible that antioxidant drugs with reducing properties may improve the responses to orally administered nitrite. However, few studies have examined the cardiovascular implications of interactions between antioxidant drugs with reducing properties and nitrite, particularly under the acidic conditions of the stomach [13,14]. Here, we hypothesized that TEMPOL, a water-soluble, paramagnetic nitroxyl radical with superoxide dismutase (SOD) mimetic activity [15], could enhance nitrite-derived NO generation under acidic conditions of the stomach and therefore enhance the hypotensive effects of orally administered sodium nitrite. This hypothesis is supported by a recent study showing that dietary polyphenols may favor NO formation from nitrite under low pH conditions [14]. In addition, we also hypothesized that the antioxidant effects of TEMPOL could increase NO bioavailability and enhance the responses to intravenous nitrite. To test these hypotheses, we examined the hypotensive effects of sodium nitrite (both intravenously and orally) in unanesthetized freely moving rats with L-NAME (Nω -nitro-L-arginine methyl ester)-induced hypertension that were treated with TEMPOL or vehicle. While this antioxidant did not enhance the effects of intravenous nitrite, TEMPOL enhanced orally administered nitriteinduced hypotension and increases in plasma nitrite concentrations. These effects were critically dependent on gastric acidic conditions. In vitro experiments confirmed in vivo findings, and TEMPOL enhanced nitrite-derived NO only under acidic conditions.

Materials and methods Animals and treatments This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, and the animals were handled according to the guiding principles published by the National Institutes of Health. Male Wistar rats (200–250 g) obtained from the colony at University of São Paulo (Ribeirao Preto Campus, Brazil) were maintained on a 12-h light/dark cycle at a room temperature (22–25 1C) with free access to standard rat chow and water. Assessment of mean arterial pressure (MAP) in unanesthetized freely moving rats One day before the experiments, the animals were anesthetized with tribromoethanol (250 mg/kg, ip) and the femoral artery was cannulated (3 cm segment of a PE-10 tube connected to 14 cm of a PE-50 tubing; Clay Adams, Parsippany, NJ, USA). The catheter was then tunneled subcutaneously and exteriorized through the back of the neck. In some experiments, the femoral vein also was cannulated following the same procedures for drug infusions. After surgery, the nonsteroidal anti-inflammatory flunixine meglumine (2.5 mg/kg, sc, Banamine, Schering Plough, Brazil) was administered for postoperation analgesia. Following 12 h of fasting, the arterial cannula was connected to a pressure

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transducer and the MAP in freely moving rats was recorded using a data acquisition system (MP150CE; Biopac Systems Inc., CA, USA) connected to a computer (Acknowledge 3.2, for Windows). Before collecting data, we allowed at least 15 min of stabilization before or after each oral gavage. Effects of TEMPOL on the hypotensive responses to intravenously administered sodium nitrite Because TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl) exerts antioxidant effects, we examined whether TEMPOL enhances the hypotensive effects of intravenously administered sodium nitrite in rats with L-NAME (Nω -nitro-L-arginine methyl ester hydrochloride)-induced hypertension. Therefore, the rats received a single gavage of L-NAME (100 mg/kg) to increase the MAP. This dose of L-NAME was chosen based on pilot studies showing that it increases MAP by approximately 40 mm Hg. Thirty minutes after L-NAME treatment, the rats received vehicle or TEMPOL (18 mg/kg) by gavage, and 15 min later the rats received vehicle or cumulative doses of sodium nitrite (0, 0.16, 0.5, 1.6, 5, and 15 mg/kg) [9] intravenously. Each dose was injected every 15 min and the maximum changes in MAP were analyzed. Measurment of 8-isoprostane levels in plasma and vascular reactive oxygen species (ROS) production to assess antioxidant effects of TEMPOL To examine antioxidant effects exerted by TEMPOL, plasma samples from rats were used to measure 8-isoprostanes (8isoPGF2α) concentrations. The measurements were carried out as previously described [16], with commercially available enzymelinked immunosorbent assay kits (Cayman Chemical Co., Ann Arbor, MI, USA). To assess vascular oxidative stress, ROS production in the aorta from animals was measured by dihydroethidium (DHE), a ROSsensitive fluorescent dye, as previously described [17]. Aortic cryosections (5 μm thick) were incubated at room temperature with DHE (10 μmol/L) for 30 min. Sections were examined by fluorescence microscopy (Leica Imaging Systems Ltd., Cambridge, UK) and the image was captured at  400. Red fluorescence from 20 fields around the vessels was evaluated using ImageJ software (http://rsbweb.nih.gov/ij/). Effects of TEMPOL on orally administered sodium nitrite-induced hypotension Because TEMPOL did not enhance the hypotensive effects of intravenously administered sodium nitrite, we examined whether pretreatment with TEMPOL enhances the hypotensive effects of orally administered sodium nitrite in rats with L-NAME-induced hypertension. Therefore, the rats received the same oral dose of L-NAME and TEMPOL described above, and 15 min later the rats received vehicle or sodium nitrite (0, 1.6, 5, or 15 mg/kg, by gavage) [9], and the changes in MAP were evaluated for 10 min. Then the animals were sacrificed to collect their aorta and blood samples. At the end of experiments, arterial blood samples were collected into tubes containing heparin and immediately centrifuged at 1000 g for 3 min. The plasma samples were stored at
70 1C until used to measure plasma nitrite and nitrate concentrations as described below. Measurement of plasma nitrite and plasma nitrate concentrations Plasma aliquots were analyzed in duplicate for their nitrite content using an ozone-based reductive chemiluminescence assay

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as previously described [18]. Briefly, to measure nitrite concentrations in plasma, 50 ml of plasma samples was injected into a solution of acidified tri-iodide, purging with nitrogen in-line with a gas-phase chemiluminescence NO analyzer (Sievers Model 280 NO analyzer, Boulder, CO, USA). Approximately 8 ml of tri-iodide solution (2 g potassium iodide and 1.3 g iodine dissolved in 40 ml water with 140 ml acetic acid) was placed in the purge vessel into which plasma samples were injected. The data were analyzed using the software Origin Lab 6.1. We measured plasma nitrite + nitrate concentrations in duplicate by using the Griess reaction as described previously [19]. Briefly, 40 μl of plasma was incubated with the same volume of nitrate reductase buffer (0.1 M potassium phosphate, pH 7.5, containing 1 mM beta nicotinamide adenine dinucleotide phosphate, and 2 units of nitrate reductase/ml) in individual wells of a 96-well plate. Samples were allowed to incubate overnight at 37 1C in the dark. Eighty microliters of freshly prepared Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride in 5% phosphoric acid) was added to each well and the plate was incubated for an additional 15 min at room temperature. A standard nitrate curve was obtained by incubating sodium nitrate (0.2–200 mM) with the same reductase buffer. To calculated plasma nitrite concentrations, we subtracted plasma nitrite concentrations measured by chemiluminescence plasma nitrite + nitrate concentrations measured by Griess reaction. Assessment of in vitro effects of TEMPOL on nitric oxide formation from sodium nitrite in a simulated gastric juice solution (electrochemical and chemiluminescence NO detection) To examine whether TEMPOL affects nitric oxide formation from sodium nitrite in vitro, we used a Clark-type electrochemical NO sensor amiNO-700 (inNO-T Analyzer, Innovative Instruments Inc., Tampa, FL, USA), which was inserted into a simulated gastric juice (SGJ) solution, as previously described [14]. The SGJ solution contained HCl 7 mM and NaCl 50 mM in distilled water, and the pH was adjusted to 2.0 by adding HCl [14]. NO formation was detected by using 2 ml of SGJ solution containing NaNO2 (20 mM) in the absence or presence of TEMPOL (0, 2, 20, and 200 mM) actively mixed with a magnetic stirrer. To further confirm the findings obtained with the Clark-type electrochemical NO sensor, we carried out experiments with NO detection by chemiluminescence using the Sievers NO analyzer. Briefly, we examined whether TEMPOL increases NO formation from sodium nitrite. Therefore, we prepared the same SGJ described above and sodium nitrite (0, 1, and 10 mM) was added to the SGJ solution containing TEMPOL 20 mM. The maximum chemiluminescence signal (mV) was measured after each sodium nitrite injection. Effects of sodium nitrite concentrations on TEMPOL signal detected by electron paramagnetic resonance (EPR) TEMPOL may be converted into other compounds and therefore EPR spectroscopy was carried out to assess TEMPOL concentrations in solutions containing sodium nitrite. EPR measurements were performed in a JEOL FA-200 spectrometer equipped with a rectangular cavity. Data acquisition parameters included field modulation amplitude, 0.6 G; time constant, 0.03 s; sweep time, 60 s; sweep width, 75 G; and microwave power 10 mW. These parameters were set to maximize signal-to-noise ratio and to avoid distortion and/or saturation of EPR signal [20]. EPR spectra were measured at room temperature. An aliquot of 50 ml of an aqueous solution (water or SGJ, as described above) containing TEMPOL (100 mM) and sodium nitrite (0, 3, 10, 30, 100, or 300 mM) was transferred to a glass capillary

and taken into an EPR cylindrical quartz tube. EPR signal intensity was measured in triplicate as the peak-to-peak height of the central-field EPR lines, using the software provided with the JEOL spectrometer. Effects of TEMPOL on pH-dependent nitric oxide formation from sodium nitrite in a simulated gastric juice solution To examine how pH modifies the effects of TEMPOL on sodium nitrite-derived NO formation, we measured NO formation using the Clark-type electrochemical NO sensor as described above. SGJ solutions with pH varying from 2.0 to 7.0 were prepared as described above. Vehicle or TEMPOL 20 mM was added to the SGJ solutions, and NO formation was measured after sodium nitrite 20 mM was added to the solutions. Effects of increased gastric pH on the changes in the hypotensive effects of sodium nitrite induced by TEMPOL To examine how gastric pH affects the TEMPOL-induced changes in the hypotensive effects of nitrite, we examined whether pretreatment with omeprazole (a proton pump inhibitor) modifies the effects of TEMPOL on sodium nitrite (15 mg/kg)induced hypotension found with protocol 1. Therefore, rats received a single gavage of omeprazole (30 mg/kg) or vehicle (1 ml/kg of 2% Tween 80), and the procedures described in first protocol were repeated 1 h after the administration of vehicle or omeprazole. The dose of omeprazole was chosen based on previous findings showing that this dose attenuated the effects of nitrite [9]. To assess the effects of omeprazole on gastric pH, we measured gastric washing pH as previously detailed [9]. In addition, to examine how gastric pH affects the increases in plasma nitrite and nitrate concentrations in rats treated with TEMPOL and nitrite, we measured the concentrations of both products of NO oxidation as described above. Effects of NO scavenger C-PTIO on the hypotensive effects of sodium nitrite with or without TEMPOL To examine whether the hypotensive effects of sodium nitrite in the presence or absence of TEMPOL are mediated by NO formation from nitrite, we repeated the experiments described in first protocol in rats infused with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (0.2 mg/kg/min) or saline (50 ml/min). In addition, as a positive control, we examined the effects of the same infusion of C-PTIO in hypertensive rats treated with DETANONOate (17 mg/kg). Drugs and solutions All drugs and reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA) and all solutions were prepared immediately before use. Statistical analysis The results are expressed as means 7 SEM. The comparisons between groups were assessed by two-way analysis of variance using Bonferroni correction or one-way analysis of variance followed by Dunnett's multiple comparison tests. A probability value o0.05 was considered significant. Statistical analyses were carried out using the GraphPad PRISM software (GraphPad Prism, Software Inc., San Diego, CA, USA).

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Results Antioxidant effects of TEMPOL are not associated with increased hypotensive effects to intravenously administered sodium nitrite in hypertensive animals To test the possibility that the antioxidant TEMPOL enhances the responses to intravenously administered sodium nitrite, we

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examined the responses to increasing doses of sodium nitrite in rats with L-NAME-induced hypertension that were pretreated with TEMPOL (or vehicle). While sodium nitrite induced dosedependent hypotensive responses (P o0.05; Fig. 1), pretreatment with TEMPOL did not enhance the responses to any dose of intravenous nitrite (P4 0.05; Fig. 1). While TEMPOL had no effects on the responses to intravenous nitrite, this antioxidant clearly decreased markers of oxidative stress. Whereas L-NAME hypertensive animals showed higher 8isoprostane levels in plasma and increased aortic ROS levels than normotensive controls (both P o0.05; Fig. 2A–C), treatment with TEMPOL reduced 8-isoprostane levels and blunted the increases in aortic ROS levels associated with hypertension (both P o0.05; Fig. 2A–C). TEMPOL enhances the acute hypotensive effects of oral sodium nitrite and the increases in circulating nitrite concentrations We examined the possibility that TEMPOL could enhance the hypotensive effects of oral sodium nitrite in hypertensive animals. Whereas no significant changes in MAP were seen in the groups treated with vehicle or TEMPOL only (data not shown), treatment of L-NAME hypertensive rats with sodium nitrite induced dosedependent decreases in MAP (Fig. 3). Treatment with TEMPOL significantly increased the hypotensive effects of sodium nitrite at 1.6, 5, and 15 mg/kg (all Po 0.05; Fig. 3). The effects of TEMPOL on the circulating levels of nitrite or nitrate were examined in hypertensive animals treated with oral sodium nitrite. While treatment with sodium nitrite increased plasma nitrite and nitrate concentrations in a dose-dependent manner, pretreatment with TEMPOL was associated with higher increases in plasma nitrite concentrations and with lower increases in plasma nitrate concentrations in animals treated sodium nitrite at 5 or 15 mg/kg (all Po 0.05; Fig. 4A and B).

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DHE (Fluorescense Intensity)

Plasma 8-Isooprostanes (pg/mL)

Fig. 1. Changes in mean arterial pressure (MAP) measured in unanesthetized freemoving hypertensive rats in response to increasing doses of intravenously administered NaNO2. The rats received L-NAME (100 mg/kg, by gavage) to increase MAP by approximately 40 mm Hg, followed 30 min later by saline or TEMPOL (18 mg/kg; by gavage). The maximum changes in MAP in response to cumulative doses of NaNO2 (0.16, 0.5, 1.6, 5, and 15 mg/kg; injected intravenously every 15 min) were not modified by pretreatment with TEMPOL. The upper panel shows the MAP results and the lower panel shows the changes in MAP after each dose of NaNO2. Data are shown as mean 7 SEM (n¼ 5 per group). nPo 0.05 for TEMPOL versus Vehicle.

20 15 10 5 0 Control Control L-NAME L-NAME + + + + Vehicle Tempol Vehicle Tempol

TEMPOL facilitates nitrite-derived NO formation in vitro in a simulated gastric juice solution and its EPR signal decreases with increasing nitrite concentrations To examine whether TEMPOL enhances sodium nitrite-induced hypotension in vivo through mechanisms facilitating nitritederived NO, we used two independent methods to study the in vitro effects of TEMPOL on NO formation from sodium nitrite

Control

Control + Tempol

L-NAME

L-NAME+ Tempol

40 30 20 10 0 Control Control L-NAME L-NAME + + + + Vehicle Tempol Vehicle Tempol

Fig. 2. Plasma 8-isoprostanes concentrations and in situ vascular O2
production assessed by dihydroethidium (DHE) fluorescence. Panel A shows 8-isoprostanes concentrations (pg/ml). Panel B shows the fluorescence intensity in each experimental group, and panel C shows representative photomicrographs (  400) of aortic arteries incubated with DHE, which produces a red fluorescence when oxidized to hydroxyethidium by O2
. Data are shown as mean 7 SEM (n¼ 5 per group). nPo 0.05 versus Control + Vehicle. # Po 0.05 versus L-NAME + Vehicle.

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Fig. 3. Changes in mean arterial pressure (MAP) measured in unanesthetized freemoving hypertensive rats in response to variable doses of NaNO2. The rats received L-NAME (100 mg/kg, by gavage) to increase MAP by approximately 40 mm Hg, followed 30 min later by saline or TEMPOL (18 mg/kg; by gavage). Ten minutes later, the changes in MAP in response to NaNO2 (1.6, 5, or 15 mg/kg, by gavage) were recorded. The upper panel shows the baseline and MAP after each dose of NaNO2, whereas the lower panel shows the changes in MAP after each dose of NaNO2. Data are shown as mean 7 SEM (n¼ 4–6 per group). nPo 0.05 for TEMPOL versus Vehicle.

under the acidic conditions of a SGJ solution (pH 2.0). The results obtained with a Clark-type electrochemical NO sensor show that TEMPOL facilitates NO formation from sodium nitrite in a concentration-dependent manner (Fig. 5; Po 0.05). In line with these findings, the chemiluminescence results confirmed that TEMPOL increases sodium nitrite-derived NO at different concentrations (Po 0.05; Fig. 6A–C). Because TEMPOL may be converted into other compounds, we used EPR to assess TEMPOL concentrations in solutions (water or SGJ) containing sodium nitrite. We found no significant differences in the EPR signal when TEMPOL was incubated at pH 7.2 (SGJ) or 2.0 (30530 77089 versus 2735076213 arbitrary units, respectively; P 40.05). While the EPR signal was not modified when TEMPOL was incubated with sodium nitrite (0–300 mM) in water (P 40.05; Fig. 6D), TEMPOL EPR signal decreased in a concentration-dependent manner when TEMPOL was incubated with the same concentrations of sodium nitrite in pH 2.0 SGJ solutions (Po0.05; Fig. 6D and E).

TEMPOL enhances nitrite-derived in vitro NO formation in a pH-dependent manner We determined whether pH modifies the effects of TEMPOL on sodium nitrite-derived NO formation using a Clark-type electrochemical NO sensor. As expected, the amounts of nitrite-derived NO were inversely associated with the pH of SGJ solutions, and

Fig. 4. Plasma nitrite concentrations (mmol/l; upper panel) and plasma nitrate concentrations (mmol/L; lower panel) in rats that received vehicle (saline) or TEMPOL (18 mg/kg) followed 10 min later by NaNO2 (0, 1.6, 5, and 15 mg/kg, by gavage). Blood samples were collected 20 after nitrite gavage. Data are shown as mean 7 SEM (n¼ 4–6 per group). nPo 0.05 for TEMPOL versus Vehicle.

TEMPOL significantly enhanced NO formation at pH 2.0 to 4.0 (P o0.05; Fig. 7), but not at higher pH values. Pretreatment with omeprazole attenuates the hypotension induced by sodium nitrite and blunts the effects of TEMPOL Because TEMPOL enhanced nitrite-derived NO formation only under acidic conditions, we examined the effects of pretreatment with omeprazole on TEMPOL-induced changes in the hypotensive effects of nitrite in hypertensive rats. While TEMPOL enhanced the hypotensive effect of sodium nitrite, pretreatment with omeprazole attenuated such effects (Fig. 8A; Po 0.05). Importantly, pretreatment with omeprazole completely prevented TEMPOL from enhancing the hypotensive effect of sodium nitrite (Fig. 8A). To verify the effects of omeprazole on gastric pH, we measured gastric washing pH, and we found that omeprazole increased the gastric washing pH (Fig. 8B; P o0.05), and this effect was not modified by TEMPOL or nitrite. Because TEMPOL increased nitrite-induced hypotension, and pretreatment with omeprazole blunted this effect, we examined whether both drugs affect plasma nitrite and nitrate concentrations. While the increases in plasma nitrite concentrations more than doubled after nitrite administration in the presence of TEMPOL (Fig. 8C; Po0.05), omeprazole had no effects on the increases in nitrite concentrations (Fig. 8C; P40.05). Interestingly, omeprazole completely blunted the enhancement in plasma nitrite concentrations associated with TEMPOL (Fig. 8C; P40.05).

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Tempol 200 μM Tempol 20 μM Tempol 2 μM Tempol 0 μM

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Fig. 5. Effects of different concentrations of TEMPOL on NO production assessed electrochemically by using a Clark-type electrode. Panel A shows increased NO production from NaNO2 (20 mM) with increasing concentrations of TEMPOL (0–200 mM) added to a simulated gastric juice solution (SGJ, pH 2.0). Panel B shows representative tracings of experiments summarized in panel A. The arrow indicates when TEMPOL was added to the simulated gastric juice solution. Data are shown as mean 7 SEM (n¼ 4–5 per group). nP o0.05 versus Vehicle.

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Fig. 6. Effect of TEMPOL on NO production evaluated by reductive chemiluminescence and effects of NaNO2 on TEMPOL EPR spectrum. Panel A shows the maximum chemiluminescence values obtained after nitrite (0, 1, and 10 mM) was added to a simulated gastric juice solution (SGJ, pH 2.0) containing vehicle or TEMPOL (20 mM). Panel B shows representative tracings of experiments summarized in panel A. It also shows that TEMPOL alone does not change the baseline chemiluminescence signal. Panel C shows a representative tracing of experiments very similar to those shown in panel B, except for the fact that TEMPOL (closed arrow) is added to the solution after nitrite (open arrow) had been previously added. TEMPOL increases nitrite-derived-NO. Panel D shows that sodium nitrite (0–300 mM) does not affect the EPR spectrum of TEMPOL at pH 7.2 (Control), whereas the EPR signal corresponding to TEMPOL decreases with increasing sodium nitrite concentrations (0–300 mM) when at low pH 2.0 conditions of simulated gastric juice (SGJ) solution. Panel D shows representative EPR spectra of TEMPOL at variable sodium nitrite concentrations (0–300 mM). Data are shown as mean 7 SEM (n¼ 3–4 per group). nPo 0.05 for TEMPOL + Nitrite versus Vehicle + Nitrite.

While lower increases in plasma nitrate concentrations were found when nitrite was administered to animals pretreated with TEMPOL (Fig. 8D; Po0.05), omeprazol alone had no effects (Fig. 8D; P40.05) and it blunted the effects of TEMPOL (Fig. 8D; Po0.05).

NO scavenger C-PTIO almost completely blunted the hypotensive effects of sodium nitrite and its enhanced effects in the presence of TEMPOL The hypotensive effects of oral sodium nitrite were previously blunted by C-PTIO, thus indicating NO formation [9], which could

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be increased by TEMPOL treatment. Fig. 9 shows hypotensive effects of oral sodium nitrite (15 mg/kg), very similar to those observed with DETANONOate (7.5 mg/kg; a positive control). Pretreatment with TEMPOL (18 mg/kg) increased the effects of

Nitric Oxide (nM)

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2300 1300 300 200 100 0 2.0 3.0 4.0 5.0 6.0 7.0 pH of Simulated Gastric Juice solution

Fig. 7. Effect of simulated gastric juice (SGJ) solution pH on sodium nitrite-derived NO production in the absence (open bars) or presence (closed bars) of TEMPOL. The bars show NO production (as detected electrochemically by a Clark-type electrode) from sodium nitrite (20 mM) and TEMPOL (20 mM) added to SGJ solutions with pH varying from 2.0 to 7.0. Reduced NO if formed as pH increases from 2.0 to 7.0. TEMPOL significantly increased NO formation at pH 2.0, 3.0, and 4.0. Data are shown as mean 7 SEM (n¼ 4–5 per group). nP o0.05 for TEMPOL + NaNO2 versus Vehicle + NaNO2.

sodium nitrite (P o0.05; Fig. 9). However, C-PTIO attenuated the hypotensive effects of sodium nitrite and DETANONOate, and completely blunted the TEMPOL-enhanced hypotension (all Po 0.05; Fig. 9).

Discussion The main novelty reported here is that the antioxidant TEMPOL promotes nitrite reduction to NO in the stomach, and enhances plasma nitrite concentrations and its hypotensive effects after oral nitrite administration, but not after intravenous administration. We show that this mechanism is critically dependent on gastric pH. Antihypertensive effects of oral nitrite were demonstrated in spontaneously hypertensive rats (SHR) more than two decades ago [21]. More recently, we and others showed consistent antihypertensive effects of nitrite in different animal models, including LNAME-induced [22] and two-kidney, one-clip hypertension [23]. While vasodilatory effects of nitrite occur in various different vascular beds, probably explaining its effects on blood pressure [2,24,25], little is known about mechanisms converting nitrite into NO in hypertension. In this respect, increased erythrocytic xantine oxidoreductase activity leading to increased NO formation from nitrite was shown in SHR [26]. Moreover, we showed that nitrite downregulates vascular NADPH oxidase activity [23], and thus may exert antioxidant effects. While the precise mechanisms explaining vasodilatory and antihypertensive effects of nitrite in

Fig. 8. Effects of omeprazole (30 mg/kg) and/or TEMPOL (18 mg/kg) on sodium nitrite (15 mg/kg)-induced decreases in mean arterial pressure (MAP; panel A), on gastric washing pH (panel B), and on plasma nitrite (panel C) and nitrate (pane D) concentrations. Vehicle (saline), TEMPOL, or omeprazole alone had no effects on MAP or nitrite and nitrate concentrations. After the same oral dose of nitrite, TEMPOL enhanced the hypotensive effect of nitrite, and the increases in plasma nitrite concentrations, whereas it attenuated the increases in plasma nitrate concentrations. Treatment with omeprazol attenuated the hypotensive effects of nitrite, and prevented TEMPOL from enhancing both nitrite-induced hypotensive effects and the increases in plasma nitrite concentrations. Data are shown as mean 7 SEM (n¼ 7–8 per group). nP o 0.05 versus NaNO2. # P o 0.05 versus TEMPOL + NaNO2.

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hypertension are not entirely clear, upregulation of enzymatic and nonenzymatic nitrite conversion into NO in the vascular wall [27,28] may play a role, and possibly explain consistent dosedependent antihypertensive effects that we found in the present study. TEMPOL is a water-soluble, paramagnetic nitroxyl radical with superoxide dismutase mimetic activity [15]. This drug is widely used to study the role of oxidative stress in cardiovascular diseases including hypertension [29,30], and its antihypertensive effects have been attributed, at least in part, to its antioxidant properties, which may improve NO bioavailability [30]. While our 8-isoprostanes and aortic ROS results clearly show antioxidant effects of TEMPOL, we found no hypotensive effects with the dose of TEMPOL used in the present study. Indeed, antihypertensive effects of TEMPOL are dose-dependent, and the dose we used is not expected to exert antihypertensive effects [29,30]. In fact, we aimed at examining whether TEMPOL enhances the hypotensive effects of sodium nitrite, and therefore we decided to use a dose of TEMPOL without significant antihypertensive effects. In contrast to enhanced responses to orally administered nitrite after pretreatment with TEMPOL, this antioxidant did not enhance the effects of intravenous nitrite, thus suggesting that systemic or vascular antioxidant mechanisms are probably not involved in the enhanced responses to orally administered nitrite in rats pretreated with TEMPOL.

Saline infusion

C-PTIO infusion

Change in MAP (mmHg)

0

-10

-20 Vehicle Tempol DETANONOate NaNO2 TEMPOL + NaNO 2

-30

#

-40

Fig. 9. Infusion of NO scavenger carboxy-PTIO (C-PTIO) blunts the changes in mean arterial pressure (MAP) induced by sodium nitrite. The figure shows the changes in MAP induced by vehicle, TEMPOL (18 mg/kg), DETANONOate (7.5 mg/kg), sodium nitrite (15mg/kg), or TEMPOL + sodium nitrite administered during saline or C-PTIO infusion (0.2 mg/kg/min). Data are shown as mean 7 SEM (n¼5–7 per group). #P o0.05 versus NaNO2. nP o0.05 for C-PTIO infusion group versus respective saline infusion group.

blood NO2-

Gastric wall

NO2-

To study how TEMPOL affects nitrite-derived NO formation, we carried out in vitro experiments at variable pH. We used two different methods (electrochemical and chemiluminescence NO detection) to clearly show that TEMPOL facilitates NO formation from nitrite in a concentration-dependent manner, and this effect increased with pH lowering. These results show that TEMPOL facilitates nitrite reduction to NO, especially at low pH. While nitrite disproportionation into NO has been known for a long time [12], no previous study has examined whether TEMPOL affects this reaction. Interestingly, we found that the EPR signal of TEMPOL decreased in a nitrite concentration-dependent manner only at low pH. The in vitro effects of TEMPOL may explain how TEMPOL facilitates the antihypertensive effects of oral nitrite. Interestingly, we found that TEMPOL enhanced the increases in plasma nitrite concentrations after all doses of oral sodium nitrite. Because nitrite reduces blood pressure in a dose-dependent manner, it is reasonable to believe that TEMPOL enhances the hypotensive effects of oral nitrite simply by promoting conditions leading to higher plasma nitrite concentrations (Fig. 10). This is valid for each and every dose of oral sodium nitrite tested here. Although simplistic, our in vitro experiments are consistent with the idea that TEMPOL allows a more reducing environment in the stomach and enhances gastric nitrite-derived NO formation in vivo. This effect results in higher plasma nitrite levels and in more hypotension in the presence of TEMPOL, possibly because nitrite is entering blood circulation as nitrite or as another nitrite-derived species more diffusible than nitrite, which may be oxidized back to nitrite in the circulation. This effect may be strikingly potentiated by TEMPOL, as our in vitro data suggest, thus resulting in higher plasma nitrite concentrations. Indeed, under acidic conditions, nitrite is protonated to form nitrous acid (HNO2) [5,7], and the undissociated form of nitrous acid is found at a much higher fraction than its dissociated form at low pH (nitrous acid pKa ¼ 3.14) [31]. Therefore increased amounts of undissociated nitrous acid will easily diffuse accross cell membranes and across the gastric wall into blood circulation, where physiological pH causes the dissociated form of nitrous acid to predominate. This important pHdependent partitioning of nitrous acid may result in fast increases in plasma nitrite concentrations, as we found in the present study. In support of this mechanism, nitrite was experimentally shown to penetrate through biological membranes in the form of undissociated nitrous acid and accumulate in areas with high pH [31]. Other mechanisms possibly explaining how TEMPOL enhances plasma nitrite levels may include the formation of chemical adduct facilitating nitrite uptake, or it is possible that nitrite-derived NO in the stomach is oxidized to nitrite by stomach oxidases as

blood NO2

Gastric wall

NO2-

blood NO2-

+

453

blood NO2-

+

H+

H+

TEMPOL HNO2

HNO2

.NO

.NO

blood NO2-

blood NO2-

blood NO2-

.NO

blood NO2.NO

blood NO3

-

blood NO3-

Fig. 10. Mechanisms possibly explaining how TEMPOL facilitates nitrite-derived gastric nitric oxide formation and enhances the antihypertensive effects of nitrite.

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formed NO diffuses across the gastric wall into gastric vessels, thus resulting in higher nitrite concentrations than expected. This suggestion is based on recent studies showing that plasma ceruplasmin [32] or lung epithelial cells [33] have NO oxidase activity and therefore facilitate nitrite synthesis from NO. However, this possibility remains to be examined. In parallel with the effects that we found with TEMPOL, reducing agents such as polyphenols caused nitrite to generate predominantly NO [14,34]. We found that treatment with TEMPOL was associated with greater increases in plasma nitrite and lower increases in plasma nitrate after 5 or 15 mg of sodium nitrite, possibly as a result of a more reductive gastric environment in the presence of TEMPOL. In fact, nitrite delivery (15 mg/kg) in the stomach results in a plasma nitrite:nitrate ratio of 0.2:1, whereas TEMPOL delivery with nitrite reduces the nitrate levels in plasma, relative to nitrite alone, and increases the nitrite levels in plasma, for a nitrite:nitrate ratio of 1.7:1. This suggests that TEMPOL treatment increases nitrite bioavailability and reduces nitrite oxidation to nitrate, possibly by enhanced nitrite uptake mechanisms. More convincingly, we showed that treatment with omeprazole completely blunted the effects of TEMPOL, both on plasma nitrite concentrations and on blood pressure. These findings, both in vitro and in vivo, consistently show that acidic conditions of the stomach are critical for TEMPOL to enhance nitrite-derived NO formation. As expected, the hypotensive effects of nitrite were almost completely blunted by the NO scavenger C-PTIO, independent of the enhancing effects of TEMPOL, and again indicate nitrite-derived NO formation. Our results align with previous findings showing that TEMPOL and other nitroxides increase NO bioavailability [35], and this effect may underlie the cardiovascular protective effects of antioxidants and nitrate/nitrite-rich diets [36,37]. Moreover, our findings may help to explain inconsistencies between studies showing antihypertensive effects of vegetable-rich diets in patients with hypertension [38,39], whereas antioxidants apparently exert no significant protective effects [40,41]. It is possible that the amounts of nitrate/ nitrite may interact with antioxidants found in the diet, especially in vegetable-rich diets and potentiate their antihypertensive effects. Simply taking antioxidants may not be of real help, whereas increasing nitrite/nitrate intake may result in antihypertensive effects and lower the incidence of cardiovascular events in patients. An important limitation of our results is that ascorbic acid is actively secreted into the gastric juice in rats [42], and therefore ascorbic acid may affect the ability of TEMPOL to alter NO formation from nitrite in the stomach. Although we have not added ascorbic acid to our in vitro experiments, this limitation is probably not relevant to our in vivo findings, which we believe are the most important findings in the present study. Important implications of our findings deserve some comments. TEMPOL increased NO formation from nitrite, particularly under the acidic conditions of the stomach, and this improved the hypotensive effects of oral nitrite. Therefore, it is possible that TEMPOL may enhance NO formation from nitrite under other relevant acidic conditions, for example, during tissue ischemia [43]. While a previous study reports that reducing substances such as thiols or ascorbate failed to increase NO formation at pH 5.5 [12], the effects of TEMPOL under such conditions are unknown and should be tested. However, it is important to note that that coinfusion of ascorbate with nitrite modestly potentiated the vasodilatory effects of nitrite in the human forearm, possibly as a result of ascorbate-induced decreases in redox potential [44]. If our in vitro results are confirmed in vivo, it is possible that TEMPOL facilitates NO formation from nitrite in proportion with tissue acidosis and cellular ischemic stress [43,45,46]. However, this suggestion requires experimental validation. If proved true, it may have important clinical applications.

In conclusion, we found that TEMPOL enhances nitrite-derived NO formation under the acidic conditions of the stomach, and this effect increases orally administered nitrite-induced increases in circulating nitrite concentrations and hypotension. The effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite. Our findings may suggest a pharmacological approach to maximize NO formation from nitrite under acidic conditions.

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