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primary nitrite reductase within this cell type under physiological conditions. In addition, our previous studies have shown that purified RBCs collected from healthy volunteers facilitate nitrite reduction that is dependent upon activity of endothelial nitric oxide synthase (eNOS) at pH 7.4, and xanthine oxidoreductase (XOR) at more acidic pH (6.8) (Webb et al., Circ Res 103 (2008) 957–964). Whether such a profile might also be evident in hypertension is unknown. We investigated RBC nitrite reductase activity in the spontaneously hypertensive rat (SHR) and Wistar Kyoto (WKY) strain controls and in patients with hypertension. Methods: Male SHR (SBP 153.4 ± 3.9, DBP 101.1 ± 2.7 mm Hg) and WKY (SBP 119.3 ± 2.6, DBP 74.4 ± 3.8 mm Hg) (14–16 weeks of age, Charles River, UK) were anaesthetized for blood collection. Hypertensive patients (SBP 151.5 ± 1.8, DBP 89.7 ± 2.2 mm Hg) were recruited and blood samples collected. Plasma and erythrocyte nitrite and nitrate concentration were measured by liquid phase ozone chemiluminescence. The nitrite reductase activity of blood vessel homogenates or purified RBCs was determined using gas phase ozone chemiluminescence. To ascertain the role of XOR in any activity seen samples were incubated with the XOR inhibitor allopurinol (100 lmol/L) for 30 min. XOR protein expression was quantified using Western blotting. Results: There were no differences in nitrite and nitrate concentrations in the plasma (n = 18) and aortic homogenates (n = 12–15) between SHRs and WKY controls. Levels of RBC nitrite (WKY 53.4 ± 10.0 nmol/g; SHR 7.8 ± 1.3 nmol/g, p < 0.001) and nitrate (WKY 706.0 ± 187.2 nmol/g; SHR 268.4 ± 45.1 nmol/g, p < 0.05) were significantly lower in SHR (n = 8) compared to WKY (n = 4), respectively. Nitrite-derived (300 lmol/L) NO formation was similar in blood vessel homogenates of SHR (n = 5) and WKY (n = 5) at pH 7.4 and 6.8, however erythrocytic nitrite reductase activity of SHRs (n = 8) was substantially enhanced compared to WKY (9i = 4) at pH 7.4 (p < 0.05). Furthermore, allopurinol (100 lmol/L) virtually abolished this activity in SHRs at pH 7.4 (n = 8, p < 0.001) and pH 6.8 (n = 8, p < 0.001). In contrast, no effect of allopurinol was evident in erythrocytic nitrite reductase activity of WKY (n = 4). Western blotting for erthrocytic XOR demonstrated a doubling of expression in SHRs. Erythrocytes isolated from the blood of grade 1 hypertensives (n = 11) caused concentration-dependent NO generation that was attenuated by allopurinol at pH 7.4 (p < 0.001) and pH 6.8 (p < 0.001). This enhanced activity in hypertensive volunteers was associated with an approximate doubling of erythrocytic XOR expression. Conclusion: Our findings suggest that in hypertension the RBC is an important site for nitrite reduction within the circulation and that XOR acts as a major nitrite reductase. SMG is funded by an MRC MRes Ph.D. and this work was supported by the BHF. Disclosure: MRC MRes PhD students hiPBHF support.

http://dx.doi.org/10.1016/j.niox.2013.02.030

P29 Nitrite influences proliferation of myoblasts via its reduction to nitric oxide in skeletal muscle tissue Ulrike B. Hendgen-Cotta, Andreas Schicho, Matthias Totzeck, Christos Rammos, Malte Kelm, Tienush Rassaf University Hospital Düsseldorf, Düsseldorf, Germany Background: Skeletal muscle regeneration is governed by complex cellular signaling processes. Proliferation of skeletal myoblasts is a key initial event in muscle regeneration and has been largely related

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to mammalian target of rapamycin (mTOR) signal transduction mechanisms. Although the precise underlying signaling remains incompletely understood, the role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, the oxidation product of NO, is regarded as a stable source for NO-like bioactivity in cases when the oxygen-dependent NO-synthases become dysfunctional. Whether nitrite itself has the potential to affect myoblast proliferation and metabolic activity under normoxic conditions with or without activation of the NO/cyclic guanosine monophosphate (cGMP) pathway is not known. Methods: The influence of nitrite and NO on C2C12 myoblast proliferation was observed in cultures grown in DMEM + 10% FCS at 37 °C/ 5% CO2, with or without sodium nitrite, NO-donor DETA-NONOate, NO-scavenger cPTIO, soluble guanylatecyclase (sGC) inhibitor ODQ, or mTOR inhibitor rapamycin. Neutral red staining, propodium iodide staining for cell cycle analysis, WST-8 viability assay, mTOR phosphorylation activity assay and intracellular cGMP measuerements were performed according to modified manufacturer’s protocols. Results: We here show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently in normoxia. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and does not affect intracellular cGMP levels, implicating a cGMPindependent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR phosphorylation activity. Conclusion: Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.031

P30 Effects of nitric oxide on photosynthesis rate of Phaeodactylum tricornutum Xiaoyan Wang a, Yangcang Xu a,b, Yunshan Zhong a, Bolin Jing a a Tianjin University of Science & Technology, Tianjin, China b Tianjin Key Laboratory of Marine Resources and Chemistry, Tianjin, China Background: Nitric oxide (NO) is a bioactive molecule that exerts a number of diverse actives in physiological processes in plants. In this paper we studied the effect of NO on photosynthesis rate of Phaeodactylum tricornumtum. Methods: P. tricornutum was cultured in f/2 medium (Gulllard, 1962) which the salinity was 25‰, pH 8.0. The growth environment was set at day/night temperatures of 20/16 °C, 14 h photoperiod supplied by fluorescent lamps with tungsten supplement. The photosynthesis rate was represented by the release of oxygen which was determined by oxygenlab method (Shi, 1987). Exogenous NO was supplied by sodium nitroprusside (SNP). Membrance lipid superoxide level was represented by MDA content which was determined by TBA method (Wilbur, 1949). Results and discussion: When P. tricornutum cultured in the environment of 100 lmol m 2 s 1 light intensity, the photosynthesis rate was increased by 14.4% by 150 nM NO. It is possible that NO increase photosynthesis rate by increase Chlorophyll content which plays an important role in photosynthesis. Therefore, the chlorophyll content was determined, and it was found that NO has no significant effect on chlorophyll content. Then NO probably affected photosyn-

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Abstracts / Nitric Oxide 31 (2013) S13–S48

thesis rate by reducing photodamage. Therefore,the effect of NO on photosynthesis rate under different light intensity was studied. When light intensity was 150 lmol m 2 s 1, 200 lmol m 2 s 1, 250 lmol m 2 s 1 and 300 lmol m 2 s 1, the photosynthesis rate of P. tricornutum was increased by 150 nM NO by 15.0%, 15.6%, 88.3% and 66.5%, respectively. It was clear that the increase level of photosynthesis rate was larger under high light than that under low light. It had been shown that chloroplast easily produced superoxide oxygen under high light (Miskza, 1995), and NO could decrease superoxide oxygen content by increasing SOD and POD activity (Delledonne, 2001). Therefore,The increase of photosynthesis rate of P. tricornutum by NO probably ascribed to its protective role to cell structure. In order to prove this view, the membrance lipid superoxide test was conducted. And it was found when P. tricornutum cultured in medium contained 150 nM NO under 300 lmol m 2 s 1 light intensity, the MDA content was significant lower than that of control. Conclusion: Exogenous NO increased photosynthesis rate of P. tricornutum, especially under high light environment. And one of the reason was NO protected cell structure from high light damage. Disclosure: This work was supported by the Foundation (No. 201102) of Tianjin Key Laboratory of Marine Resources and Chemistry (Tianjin University of Science & Technology), PR China.

rates of NO2 oxidation, which was not however associated with any deficit in NO2 -dependent vasodilation. Transfusion of fresh or stored RBC into mice after trauma-hemorrhage led to significant acute lung injury in the latter. Conclusion: Collectively, these data suggest that endogenous NOsignaling will be modulated by the relative distribution of young vs. older RBC. Also the storage of RBC under current blood banking conditions results in cells with higher NO-scavenging activities that are predicted to complement increased hemolysis in contributing to pro-inflammatory stresses associated with transfusion. In addition, we posit that increased oxidation of nitrite, which would decrease an endogenous substrate for NO-formation during hypoxia, also contributes to storage lesion pathology and raises the concept of nitritebased therapy to replete NO-signaling during trauma-hemorrhage. Disclosure: Supported by NIH T32 HL007918.

http://dx.doi.org/10.1016/j.niox.2013.02.033

P32 Potential role for 8-Nitro-cGMP in nitrite-mediated hypoxic signaling

http://dx.doi.org/10.1016/j.niox.2013.02.032

P31 The effects of red blood cell age on nitric oxide and nitritedependent signaling: Implications for transfusion related toxicity Ryan Stapley, Ben Owusu, Cilina Rodriguez, Marisa Marques, Rakesh Patel University of Alabama at Birmingham, Birmingham, AL, United States Background: RBC are critical hubs for regulated NO-homeostasis, affecting both inhibition (NO and nitrite oxidation) and stimulatory (nitrite-reduction to NO) pathways. How RBC affect this balance, and whether this changes in disease remains unclear. Importantly, biophysical and biochemical properties of the RBC are key parameters, and which change as a function of RBC age in vivo and storage time ex vivo. Transfusion with older RBC is associated with increased morbidity and mortality in critically ill patients with dysfunction in NObioavailability underlying, in part, increased inflammation (e.g., in the lung leading to acute lung injury) and circulatory dysfunction. We hypothesized that changes in RBC morphology/ biochemical composition that occur during aging/ storage results in a shift in the balance presented above towards a greater inhibition of NO-signaling both by accelerated rates of NO-and nitrite oxidation. Method: Comparisons of NO dioxygenation and nitrite oxidation kinetics were made between fresh and stored RBC as well as RBC of different ages. Functional assessment of changes in NO/nitrite reaction kinetics were evaluated by testing effects of RBC on NO-or nitrite-dependent vasodilation of aortic rings ex vivo and in vivo using a murine model of storage lesion in the context of traumahemorrhage and acute lung injury. Results: NO-scavenging kinetics by intact oxygenated RBC were 40-fold higher with RBC stored for 42 days compared to freshly isolated RBC; an effect that translated to a greater inhibition of NOdependent vasodilation. Similarly, RBC isolated from freshly drawn blood and separated as a function of endogenous age, showed that older RBC scavenged NO 2-fold faster compared to younger RBC, which also translated to a greater degree of inhibition of NO-dependent vasodilation. Both stored and older RBC exhibited increased

Benjamin Y. Owusu a, Ryan Stapley a, Takaaki Akaike b, Rakesh P. Patel a a University of Alabama at Birmingham, Birmingham, AL, United States b Kumamoto University, Kumamoto, Japan Background: Nitrite has been shown to elicit both acute and chronic nitric oxide (NO)-dependent effects, especially at lower oxygen tensions.In this paradigm, nitrite reduction by deoxyhemoglobin and deoxymyoglobin activates NO-signaling to mediate several functions including hypoxic vasodilation.The mechanism underlying this however remains unclear, with a major challenge being how to reconcile nitrite-derived NO-signaling in an NO-scavenging environment, considering the high heme concentrations (10 mM) and rapid reaction rates between heme and NO (k  107 M 1 s 1).Recent studies also implicate the derivative of cGMP, 8-nitro-cGMP, as a novel second messenger in NO-dependent signaling.Both cGMP and 8-nitro-cGMP activates PKG, however, unlike cGMP, 8-nitro-cGMP is resistant to PDE5 degradation and has the potential to elicit NO-induced signal transduction via a new chemical modification of proteins referred to as S-guanylation.To date, nitrite-dependent NO formation and 8-nitro-cGMPdependent activation of NO-signaling have been considered independent of each other.In this study, we proposed a link between these two signaling paradigms.We hypothesized that 8-nitro-cGMP is a critical mediator of nitrite-dependent activation of hypoxic NO-signaling and tested if nitrite promotes hypoxic NO-signaling via 8-nitro-cGMP. Methods: First, 8-nitro-cGMP formation in rat aorta exposed to nitrite at high O2 (21%) and low O2 (1%) tensions was assessed by immunostaining.The role of 8-nitro-cGMP in nitrite-mediated vasodilation was assessed using ex vivo aortic vessel dilation as an index of nitrite-dependent NO-signaling at high O2 (21%) and low O2 (1%) tensions, with and without the PDE5 inhibitors, sildenafil or zaprinast. Results: The highest intensity of staining for 8-nitro-cGMP was observed in the vessels treated with nitrite at low O2 (1%) tension and in the presence of RBC, indicating a nitrite-induced formation of 8-nitro-cGMP under hypoxic conditions.Nitrite stimulated vasodilation at 1% O2 tension in a dose-dependent manner and this response was significantly improved in the presence of RBCs.Treatment of the aortic vessel segments with the PDE5 inhibitors, sildenafil or zaprinast improved RBC-dependent nitrite-mediated vasodilation by 2-fold at 21% O2.However, PDE5 inhibition had no effect on nitrite-dependent vasodilation at 1% O2. Moreover,