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Disclosure: NIH Grants R01HL098032, RO1HL096973, and PO1HL103455; the German Sciency Foundation (DFG) and the Fonds der Chemischen Industrie. http://dx.doi.org/10.1016/j.niox.2013.02.064

P63 The antidotal action of nitrites toward cyanide intoxication: An example of medicine disguised as quackery Jim Peterson, Linda L. Pearce University of Pittsburgh, Pittsburgh, PA, United States Sodium nitrite ameliorates sub-lethal cyanide toxicity in mice when given from 1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. In most experiments nitrite was given intraperitoneally, but may also be administered as an inhaled aqueous vapor-contrary to recommended EMR protocols, it is not necessary to infuse the antidote intravenously and the coadministration of adjuvants (specifically thiosulfate) is unnecessary. Intraperitoneally administered nitrite rapidly produces NO in the bloodstream as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. However, the commonly held belief that the antidotal mechanism of nitrites involves the scavenging of cyanide anion by methemoglobin is firmly contraindicated by recent evidence. Intriguingly, the FDAapproved and clinically tested cyanide scavenger cobalamin has, in fact, rather indifferent binding affinity for cyanide anion. It follows that the clinical usefulness of cobalamin may be dependent on endogenous nitric oxide displacing the cyanide from the active site of cytochrome c oxidase to facilitate the cyanide-scavenging action of cobalamin. Antagonism of cyanide inhibition of cytochrome c oxidase by NO appears to be the crucial antidotal activity rather than any methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood results in the production of sulfidomethemoblobin as detected by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase; the effects of the two inhibitors being essentially additive under standard assay conditions, rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication. Isoamyl nitrite, given intraperitoneally in equimolar amounts to sodium nitrite, is comparatively less effective at restoring the righting recovery of cyanide-challenged mice. The results of investigations with the hydrolysis products of isoamyl nitrite, isoamyl alcohol and nitrite anion, suggest that the toxicity of isoamyl alcohol renders the organic nitrite a less desirable antidotal agent than sodium nitrite. Disclosure: Supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grant No. NS063732 to J.P and L.L.P and Bruce R. Pitt. http://dx.doi.org/10.1016/j.niox.2013.02.065

P64 Biochemical and structural characterization of the soluble guanylate cyclase catalytic domain

Elsa D. Garcin, Franziska Seeger University of Maryland, Baltimore County, Baltimore, MD, United States Background: Soluble guanylate cyclase (sGC) is a key enzyme in the NO–sGC–cGMP signaling cascade and is crucial to cardiovascular system regulation. NO binding to the sGC regulatory domain enhances its basal catalytic activity to convert GTP to cGMP The second messenger cGMP modulates downstream targets leading to vasodilation. Low output of the NO–sGC–cGMP system results in hypertension and acute heart failure. Nitrite delivery has been shown to enhance hypoxic NO signaling via cGMP production. sGC is a heterodimer of two homologous subunits, a and b, which both contain three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiled-coil (CC) domain, and a C-terminal catalytic domain (GC). The sGC enzyme is basally active in the absence of NO, but NO binding to the heme group of the b-subunit HNOX domain enhances catalytic output several hundred fold. The mechanism by which the HNOX domain inhibits activity in the basal state and relays the NO activation signal to the catalytic domain remains elusive. Methods and results: Here, we report the first crystal structure of wild type human heterodimeric abGC at 1.9 Å resolution as well as activity data comparing full-length sGC to our truncated catalytic constructs. The cGMP activity measurements show that abCC–GC exhibits higher levels of catalytic activity than both abGC and basally active full-length sGC. Conclusions: Activity levels of abCC–GC compared to basal and activated full-length sGC and abGC hint at structural differences between the two constructs and full-length sGC. Our results also suggest that additional sGC domains are necessary for full enzymatic activity. These differences will be further characterized by structural studies using protein Xray crystallography. Disclosure: Supported by American Heart Association Scientist Development Grant. http://dx.doi.org/10.1016/j.niox.2013.02.066

P65 Nitrite after cardiac arrest has antioxidant effects reducing reperfusion cerebral hyperemia and subsequent caspase activation Cameron Dezfulian, Amalea Misse, Jason P. Stezoski, Patrick M. Kochanek, Hulya Bayir, Robert S. Clark University of Pittsburgh, Pittsburgh, PA, United States Background: We previously demonstrated nitrite mediated neuroprotection after asphyxial cardiac arrest (ACA). Reperfusion injury after successful return of spontaneous circulation (ROSC) has been linked to reactive oxygen species generation resulting in loss of cerebral autoregulation with early hyperemia, activation of caspases and delayed neuronal death. Having previously demonstrated nitrite’s cardiac antioxidant effects after cardiac arrest, we hypothesized that a similar mechanism may prevent loss of autoregulation and subsequent caspase activation resulting in neuroprotection. Methods: Adult male Sprague–Dawley rats were anesthetized, intubated and catheterized (arterial and venous). An ultrasonic flow probe was placed around the ascending aorta to continuously measure cardiac output (CO) and cerebral cortex blood flow (CBF) was measured using laser speckle analysis through a 5 mm craniotomy. ACA was induced with paralysis and cessation of mechanical ventilation for 8 min prior to manual cardiopulmonary resuscitation with

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Fig. 1.

epinephrine and sodium bicarbonate. Animals were randomized 5 min post-ROSC to a 10 min 500 ll infusion of 8 lM nitrite or plasmalyte placebo. Temperature was maintained at 37 °C while heart rate (HR), blood pressure (BP), CO and CBF were measured for one hour and results are reported normalized to baseline values. At 1 h animals were perfused with saline, decapitated and brains homogenized for ascorbate and caspase measurements. In separate experiments without CO/CBF monitoring, animals were survived 24 h before brain homogenization for caspase analysis. Caspase 3/7 activity was measured using a commercial luminescent assay and ascorbate measured using a fluorescent assay with acridine TEMPO. All measurements were normalized to protein content of samples. Comparisons are by repeated measures ANOVA, 1 way ANOVA and Mann–Whitney U tests and mean (physiology, caspase) or median (ascorbate) values are reported below. Results: Animals in both treatment arms had baseline HR, MAP, CO and CBF similar to each other and non-arrested shams and consistent with reported normal values (Fig. 1). Sham physiologic values did not change significantly over 1 h of observation under isoflurane anesthesia. ACA resulted in complete loss of CO and CBF. Ten min after ROSC (5 min after treatment was initiated), CO was diminished (80%) in both treatment groups yet BP was significantly (138%) increased. In placebo animals, this resulted in a significant increase in CBF (187%) compared to nitrite treated animals (136%; p < 0.01). This difference persisted until 20 min after ROSC at which point groups converged. Nitrite treated animals had significantly higher levels of ascorbate (33.09 nmol/mg protein) compared to placebo treated animals (26.17; p = 0.027) 1 h after ROSC. At 24 h, ACA resulted in significant(p < 0.01) increases in caspase-3/7 activity compared to shams (1381 RLU/mg protein) with higher levels in placebo (5383) compared to nitrite (4340) treated animals. Conclusion: Nitrite acts as antioxidant when administered after ROSC blunting subsequent loss of autoregulation and hyperemia, preserving antioxidant reserve and reducing subsequent caspase-3/ 7 activation. This provides a mechanistic insight into nitrite medi-

ated neuroprotection. Ongoing studies are examining potential subcellular targets for nitrite therapy which may explain these effects. Disclosure: Supported by NINDS K08.

http://dx.doi.org/10.1016/j.niox.2013.02.067

P66 Nebulized inhaled nitrite (AIR001) for pulmonary arterial hypertension: Studies to determine safety, pharmacokinetics, and maximum tolerated dose, lack of pharmacodynamic interaction with sildenafil and optimal nebulizer device Ed L. Parsley a, Hiroko Masamune a, Will L. Hoye a, Lewis J. Rubin b, Mark T. Gladwin c a Aires Pharmaceuticals, Inc., San Diego, CA, United States b University of California, San Diego, San Diego, CA, United States c University of Pittsburgh, Pittsburgh, PA, United States Background: Prior work by Bradley et al. [1] demonstrated single doses of inhaled sodium nitrite up to 125 mg (dose loaded into the nebulizer) were well tolerated and resulted in increased plasma nitrite concentrations, increased exhaled NO, and minimal methemoglobin increases. Thus further clinical development of nebulized sodium nitrite (AIR001) for treatment of pulmonary arterial hypertension (PAH) was planned. Nitrite-generated NO, by acting upon guanylate cyclase is likely to be synergistic with phosphodiesterase type 5 inhibitors (PDE-5i’s), as both increase cGMP and likely would be used concomitantly in PAH patients, thus an interaction study was performed. Clinical trials utilizing AIR001 require a portable, highly efficient nebulizer capable of precise dosing as well as monitoring adherence and compliance which necessitated careful device characterization and tolerance studies.