Determination of Nitric Oxide and Its Metabolites in Biological Tissues Using Ozone-Based Chemiluminescence Detection: A State-of-the-Art Review.

Ozone-based chemiluminescence detection (CLD) has been widely applied for determining nitric oxide (•NO) and its derived species in many different fields, such as environmental monitoring and biomedical research. In humans and animals, CLD has been applied to determine exhaled •NO and •NO metabolites in plasma and tissues. The main advantages of CLD are high sensitivity and selectivity for quantitative analysis in a wide dynamic range. Combining CLD with analytical separation techniques like chromatography allows for the analytes to be quantified with less disturbance from matrix components or impurities. Sampling techniques like microdialysis and flow injection analysis may be coupled to CLD with the possibility of real-time monitoring of •NO. However, details and precautions in experimental practice need to be addressed and clarified to avoid wrong estimations. Therefore, using CLD as a detection tool requires a deep understanding of the sample preparation procedure and chemical reactions used for liberating •NO from its derived species. In this review, we discuss the advantages and pitfalls of CLD for determining •NO species, list the different applications and combinations with other analytical techniques, and provide general practical notes for sample preparation. These guidelines are designed to assist researchers in comprehending CLD data and in selecting the most appropriate method for measuring •NO species.

Red blood cell endothelial nitric oxide synthase: A major player in regulating cardiovascular health.

Red blood cells (RBCs) have traditionally been seen as simple carriers of gases and nutrients in the body. One important non-canonical function of RBCs in the cardiovascular system is the regulation of nitric oxide (NO) metabolism. It has been shown that RBCs can scavenge NO, transport NO metabolites and produce NO in hypoxic conditions, thereby inducing hypoxic vasodilation. RBCs also express endothelial nitric oxide synthase (eNOS). However, the physiological significance of RBC eNOS has been controversial for many years. This review article provides a comprehensive overview of the experimental research on RBC eNOS signalling in vivo. The data show that RBC eNOS signalling modulates intracellular NO production and NO-haem levels, as well as participating in extracellular paracrine NO metabolite signalling, which contributes to regulating peripheral vascular resistance, blood pressure and cardioprotection. Additionally, this article explores the molecular mechanisms of sytemic regulation mediated by RBC eNOS and the implications of RBC eNOS in cardiovascular health and disease.

Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation.

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized ß-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of ß-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.

Red blood cell eNOS is cardioprotective in acute myocardial infarction.

Red blood cells (RBCs) were shown to transport and release nitric oxide (NO) bioactivity and carry an endothelial NO synthase (eNOS). However, the pathophysiological significance of RBC eNOS for cardioprotection in vivo is unknown. Here we aimed to analyze the role of RBC eNOS in the regulation of coronary blood flow, cardiac performance, and acute myocardial infarction (AMI) in vivo. To specifically distinguish the role of RBC eNOS from the endothelial cell (EC) eNOS, we generated RBC- and EC-specific knock-out (KO) and knock-in (KI) mice by Cre-induced inactivation or reactivation of eNOS. We found that RBC eNOS KO mice had fully preserved coronary dilatory responses and LV function. Instead, EC eNOS KO mice had a decreased coronary flow response in isolated perfused hearts and an increased LV developed pressure in response to elevated arterial pressure, while stroke volume was preserved. Interestingly, RBC eNOS KO showed a significantly increased infarct size and aggravated LV dysfunction with decreased stroke volume and cardiac output. This is consistent with reduced NO bioavailability and oxygen delivery capacity in RBC eNOS KOs. Crucially, RBC eNOS KI mice had decreased infarct size and preserved LV function after AMI. In contrast, EC eNOS KO and EC eNOS KI had no differences in infarct size or LV dysfunction after AMI, as compared to the controls. These data demonstrate that EC eNOS controls coronary vasodilator function, but does not directly affect infarct size, while RBC eNOS limits infarct size in AMI. Therefore, RBC eNOS signaling may represent a novel target for interventions in ischemia/reperfusion after myocardial infarction.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice.

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Using diaminofluoresceins (DAFs) in nitric oxide research.

Diaminofluoresceins (DAFs) are fluorescent probes widely applied to measure nitric oxide (NO) formation in cells and tissues. The main advantages of these compounds are their availability and low cost, and the general availability of instruments able to detect green fluorescence in all laboratories; these include fluorimeters, flow cytometers, and fluorescent microscopes. What made these molecules particularly interesting for many scientists approaching the NO field is that they are apparently very easy to use, as compared with other techniques requiring specific instrumentation and knowledge like chemiluminescence and electron paramagnetic resonance. However, the reactivity and biological chemistry of these probes in the cellular environment is rather complex and still not fully understood. Moreover, secondary reactions with ascorbate, or interference with thiols occur in cells. Therefore, the use of DAFs requires specific experimental planning and a careful interpretation of the results obtained. In this methodological review, we described in detail what is known about the reactivity of DAFs, their application in biological assays, list some principles to help experimental planning, including the necessary controls, and list the caveats concerning result interpretation. These guiding principles will help to understand the "Method behind our DAF-madness".