Dennis J. Stuehr

Mammalian nitric oxide synthases

The nitric oxide (NO) synthase family of enzymes generate NO from L-arginine, which acts as a biologic effector molecule in a broad number of settings. This report summarizes some of the current information regarding NO synthase structure-function, reaction mechanism, control of catalysis, and protein interactions.

Function of Mitochondrial Stat3 in Cellular Respiration

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3–/– cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the proteins function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.

Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes.

Modulation of nitrogen oxide synthesis in vivo: NG-monomethyl-L-arginine inhibits endotoxin-induced nitrate/nitrate biosynthesis while promoting hepatic damage.

Attempts were made to promote or inhibit nitric oxide (·N=O) synthesis in a murine model of hepatic damage (Corynebacterium parvum followed by lipopolysaccharide; LPS) to determine the role of N=O in the liver injury. Moderate hepatic damage and increases in circulating NO2 ‐/NO3 ‐ levels were detectable after C. parvum alone. Administration of LPS to these mice resulted in severe hepatic damage and acute elevations in circulating nitrogen oxide levels. L‐arg had no influence on the C. parvum or LPS‐induced changes. NG‐monomethyl‐L‐arginine (NMA) had no effect in the absence of LPS, but when given with LPS, a dose‐dependent suppression in plasma NO2 ‐/NO3 ‐ levels and an increase in liver injury were seen. The NMA‐induced changes were partially reversed by the simultaneous administration of L‐arg. These findings suggest a protective role for · N=O in this model.

Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism.

In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO2-, NO3-, and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at end-expiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O2 concentration in the hypoxic range (KmO2 190 microM). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels (KmO2 135 microM), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its KmO2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range.

Macrophage and endothelial cell nitric oxide synthesis: Cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine

Many cell types are known to synthesize nitric oxide (NO.) from L-arginine. There appear to be at least two forms of NO. synthase: an inducible, tetrahydrobiopterin- and flavin-dependent activity exemplified by the macrophage enzyme and a constitutive, Ca+(+)-dependent activity exemplified by the endothelial cell enzyme. L-NG-methylarginine inhibits NO. synthesis by both cell types. We now report that L-NG-aminoarginine and L-NG-nitroarginine are about 100-fold more potent than NG-methylarginine in blocking endothelial cell NO. synthesis. In contrast, NG-aminoarginine and NG-methylarginine are about equipotent with macrophages whereas NG-nitroarginine is much less potent. Since macrophage and endothelial cell NO. synthesis are differentially sensitive to the inhibitors, the panel of inhibitors can be used in complex biological systems to determine if macrophage-like or endothelial-like cells are the predominant source of NO.. Indeed, all three inhibitors elicit a strong pressor response in the anesthetized guinea pig, a result consistent with the view that endothelial cells continually produce vasodilatory NO(.).

Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis.

The etiology and mechanisms by which severe trauma or sepsis induce hepatic failure are unknown. Previously we showed that Kupffer cells (KC), the fixed macrophages of the liver, induce a profound decrease in hepatocyte (HC) total-protein synthesis when exposed to endotoxin. Furthermore we demonstrated that endotoxin-activated KCs induce these changes in HC protein synthesis through the induction of a novel L-arginine-dependent biochemical pathway within the HC. In this pathway, the guanido nitrogen of L-arginine is converted to the highly reactive molecule nitric oxide (NO.). To identify the KC factors that act as signals for induction of HC NO. biosynthesis, recombinant cytokines were added to HC cultures and HC nitrogen oxide production and protein synthesis levels were determined. We found that no single cytokine, but rather a specific combination of tumor necrosis factor, interleukin-1, interferon-gamma, and endotoxin, were required for maximal induction of HC nitrogen oxide production. This specific combination of cytokines induced a 248.8 +/- 26.0 mumol/L (micromolar) increase in HC nitrogen oxide production and simultaneously inhibited HC total protein synthesis by 36.1% +/- 3.1%. These data demonstrate that multiple cytokines, produced by endotoxin-activated KC, induce the production of NO. within HC, which in turn leads to the inhibition of HC total-protein synthesis.

Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans

The low barometric pressure at high altitude causes lower arterial oxygen content among Tibetan highlanders, who maintain normal levels of oxygen use as indicated by basal and maximal oxygen consumption levels that are consistent with sea level predictions. This study tested the hypothesis that Tibetans resident at 4,200 m offset physiological hypoxia and achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of bioactive forms of NO, the main endothelial factor regulating blood flow and vascular resistance. The natural experimental study design compared Tibetans at 4,200 m and U.S. residents at 206 m. Eighty-eight Tibetan and 50 U.S. resident volunteers (18–56 years of age, healthy, nonsmoking, nonhypertensive, not pregnant, with normal pulmonary function) participated. Forearm blood flow, an indicator of systemic blood flow, was measured noninvasively by using plethysmography at rest, after breathing supplemental oxygen, and after exercise. The Tibetans had more than double the forearm blood flow of low-altitude residents, resulting in greater than sea level oxygen delivery to tissues. In comparison to sea level controls, Tibetans had >10-fold-higher circulating concentrations of bioactive NO products, including plasma and red blood cell nitrate and nitroso proteins and plasma nitrite, but lower concentrations of iron nitrosyl complexes (HbFeIINO) in red blood cells. This suggests that NO production is increased and that metabolic pathways controlling formation of NO products are regulated differently among Tibetans. These findings shift attention from the traditional focus on pulmonary and hematological systems to vascular factors contributing to adaptation to high-altitude hypoxia.

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Protein tyrosine nitration in the mitochondria from diabetic mouse heart: Implications to dysfunctional mitochondria in diabetes

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.