Potential therapeutic action of nitrite in sickle cell disease.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically.

Reactions between nitrosopersulfide and heme proteins.

When nitrosothiols react with excess hydrogen sulfide, H2S, they form several intermediates including nitrosopersulfide (SSNO-). The stability and importance of this species has been debated. While some data suggest SSNO- can be a relatively stable source of NO activity, others suggest that the species degrades too quickly. We find the species to be relatively stable in isolation. Due to the abundance and prominence of iron-containing proteins throughout the human body, it is important to establish the interaction of ferrous- and ferric-iron containing proteins with SSNO-. Study of the reactions of SSNO- with heme proteins can also provide information about the potential in vivo stability and spontaneous reactivity of this species. We have used time-resolved electron paramagnetic resonance and UV-Vis absorption spectroscopy to study the reactions of SSNO- with heme proteins. Iron-nitrosyl hemoglobin is formed when SSNO- is reacted with deoxyhemoglobin and deoxygenated methemoglobin, suggesting NO formation from SSNO-. However, the yields of nitrosyl hemoglobin in reactions of SSNO- with deoxyhemoglobin are much less than when SSNO- is reacted with deoxygenated methemoglobin. Very little to no nitrosyl hemoglobin is formed when SSNO- is reacted carboxyhemoglobin, HbCO, and when SSNO- is reacted with oxygenated hemoglobin, minimal methemoglobin is formed Taken together, these data confirm the release of NO, but indicate a vacant heme is necessary to facilitate a direct heme-SSNO- reaction to form substantial NO. These data also suggest that the ferric iron in methemoglobin potentiates SSNO- reactivity. These results could potentially impact NO and sulfide bioavailability and reactivity.

The relationship between plasma and salivary NOx.

Several studies have shown that fasting plasma nitrite (NO2(-)) is an indicator of endothelial nitric oxide synthase (NOS) activity while plasma nitrate (NO3(-)) or the sum of NO2(-) and NO3(-) (NOx) does not reflect NOS function. Plasma NO2(-) can also be elevated through dietary NO3(-) where the NO3(-) is partially reduced to NO2(-) by oral bacteria and enters the plasma through the digestive system. NO3(-) is taken up from plasma by salivary glands and the cycle repeats itself. Thus, one may propose that salivary NO2(-) is an indicator of plasma NO2(-) and consequently of NO production. Many brands of nitric oxide (NO) saliva test strips have been developed that suggest that their product is indicative of circulatory NO availability. However, data supporting a relationship between salivary and plasma NO2(-) or NO bioavailability are lacking. Here we have measured basal salivary and plasma NO2(-) and NO3(-) to determine if any correlation exists between these in 13 adult volunteers. We found no significant correlation between basal salivary and plasma NO2(-). Also no correlation exists between salivary NO3(-) and plasma NO2(-). However, we did see a correlation between salivary NO3(-) and plasma NO3(-), and between salivary NO2(-) and plasma NO3(-). In a separate study, we compared the efficiency of salivary NO3(-) reduction with the efficacy of increasing plasma NO3(-) and NO2(-) after drinking beet juice, a high NO3(-)-containing beverage, in 10 adult volunteers. No significant correlation was observed between the ex vivo salivary reduction of NO3(-) to NO2(-) and plasma increases in NO3(-) or NO2(-). These results suggest that measures of salivary NO3(-), NO2(-) or NOx are not good indicators of endothelial function. In addition, the efficiency of saliva to reduce NO3(-) to NO2(-)ex-vivo does not demonstrate one's ability to increase plasma NO2(-) following consumption of dietary NO3(-).

Prenatal and early-life exposure to high-level diesel exhaust particles leads to increased locomotor activity and repetitive behaviors in mice.

Abundant evidence indicates that both genetic and environmental factors contribute to the etiology of autism spectrum disorders (ASDs). However, limited knowledge is available concerning these contributing factors. An epidemiology study reported a link between increased incidence of autism and living closely to major highways, suggesting a possible role for pollutants from highway traffic. We investigated whether maternal exposure to diesel exhaust particles (DEP) negatively affects fetal development leading to autism-like phenotype in mice. Female mice and their offspring were exposed to DEP during pregnancy and nursing. Adult male offspring were then tested for behaviors reflecting the typical symptoms of ASD patients. Compared to control mice, DEP-exposed offspring exhibited higher locomotor activity, elevated levels of self-grooming in the presence of an unfamiliar mouse, and increased rearing behaviors, which may be relevant to the restricted and repetitive behaviors seen in ASD patients. However, the DEP-exposed mice did not exhibit deficits in social interactions or social communication which are the key features of ASD. These results suggest that early life exposure to DEP could have an impact on mouse development leading to observable changes in animal behaviors. Further studies are needed to reveal other environmental insults and genetic factors that would lead to animal models expressing key phenotypes of the autism spectrum disorders.