The chemical biology of nitric oxide: Implications in cellular signaling

Abstract

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO] < 1–30 nM), Akt phosphorylation ([NO] = 30–100 nM), stabilization of HIF-1α ([NO] = 100–300 nM), phosphorylation of p53 ([NO] > 400 nM), and nitrosative stress (1 μM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Introduction

Over the past few decades, free radicals and other reactive small molecules have emerged as important players in a multitude of physiologic and pathologic conditions. From the beginning of this field of research, many of the short-lived reactive molecules being studied were found to have opposing effects under seemingly similar circumstances [1]. Initially, reactive oxygen species (ROS), reactive nitrogen species (RNS), carbon monoxide (CO), and hydrogen sulfide (H₂S) were thought to be primarily cytotoxic species that increased tissue injury [2], [3], [4], [5], [6]. After the discovery that they were endogenously produced, their role in pathophysiology was reevaluated. We now know that these “toxic” species not only are endogenously generated but are an essential part of the immune response and many physiological signal transduction pathways [7], [8], [9], [10]. Although their chemical reactivity can lead to toxicity, the biological properties of these same reactive species can be beneficial and explain their apparently dichotomous actions.

The free radical nitric oxide (NO) is the best example of a reactive molecule demonstrating both cytotoxic and cytoprotective properties [10]. Two lines of research led to its discovery. NO was identified in the 1980s as endothelium-derived relaxation factor, a substance generated by the endothelium that caused vascular relaxation and was also the active component of nitrovasodilators [7], [11], [12], [13]. Conversely, NO was found to be generated by macrophages participating in the anti-tumor and anti-pathogen response [14], [15], [16]. These few initial observations sparked a new field of investigation and led to an explosion of NO research, which has revealed the importance of this diatomic molecule in nearly every tissue in the body.

Contradictory results, however, soon began to emerge regarding the participation of NO in pathophysiological responses [17]. Whereas a number of studies implied that endogenous NO was toxic, others showed that NO was protective [18], [19], [20]. The NO-mediated toxicity was attributed to the generation of reactive nitrogen oxide species that mediated cell death, whereas the protective effects were proposed to be through antioxidant mechanisms [19]. Over the past couple of decades, there was much debate as to the mechanism of this dichotomy and whether NO is a deleterious or a beneficial agent.

Unlike most small signaling molecules, the biological effects of nitric oxide are determined by their chemical reactions, such as binding to the regulatory heme in soluble guanylate cyclase (sGC), rather than traditional protein receptor–ligand interactions. The unique chemistry of NO allows it to participate in numerous reactions [19]. During the mid-1990s, the concept of the chemical biology of NO was introduced to help explain this complexity in the context of biological conditions. The purpose of this thesis was to discern the physiologically relevant chemical reactivity of NO. For instance, various reactions of nitrogen oxides occur over many days at elevated temperature and pressure, which makes them kinetically and thermodynamically unlikely and incompatible with human physiology. On the other hand, some reactions are sufficiently fast to occur under achievable biological conditions.

The chemical biology of NO divides these potential reactions into two categories: direct and indirect [18]. The direct effects of NO are those chemical reactions that occur fast enough to allow NO to react directly with a biological target molecule. In contrast, the indirect effects require that NO reacts with oxygen or superoxide to generate RNS, which subsequently react with the biological targets. One advantage of dividing the chemistry of NO in these two categories is that direct effects generally occur at low concentrations, whereas indirect effects occur at much higher concentrations.

Indirect effects can be further subdivided into two categories based on RNS chemistry: nitrosative and oxidative stress [21]. Oxidative chemistry refers to a process in which the oxidation state of the target molecule is increased. There are several main types of oxidative reactions, electron transfer (i.e., radical formation), hydrogen atom abstraction, and oxygen atom transfer (oxygen atom insertion, addition, transfer, or hydroxylation reactions). Nitrosative stress implies the addition of a nitrosonium (NO⁺) equivalent to a thiol or secondary amine or hydroxy groups (although this reaction also represents a formal oxidation of a thiol or amine, we make a distinction here because the modifications occur via a nitrosation reaction). Reactive oxygen species (OH radical, O₂⁻) such as those produced by the Fenton reaction are most often associated with oxidative stress. However, peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂), which can be formed from the reaction of NO with superoxide (O₂⁻), are also potent oxidants (> 1.0 V NHE) [22]. In contrast, N₂O₃ formed from the reaction of NO with O₂ (autoxidation), as well as the NO/O₂⁻ reaction, is a mild oxidant and prefers to nitrosate nucleophiles such as amines and thiols [23], [24], [25]. The balance between oxidation and nitrosation chemistry as it was found depends largely on the flux of NO (Fig. 1). In the case of the autoxidation in hydrophobic environments, NO₂ is first generated but as NO levels increase there is rapid formation of N₂O₃ (Eqs. (1) and (2)), ultimately forming nitrite in water (Eq. 3):

2NO + O₂ → 2NO₂ (1)

NO₂ + NON₂O₃ (2)

H₂O + N₂O₃ → 2NO₂⁻ + 2H⁺ (3)

An equilibrium is formed between NO and NO₂ and the proportion of oxidation vs nitrosation is determined by the amount of substrate present. The same applies to ONOO⁻ formation from the NO/O₂⁻ reaction. When ONOO⁻ decomposes in the presence or absence of CO₂, powerful oxidants are formed [26], [27]. However, the oxidative balance can be tipped toward nitrosation when these oxidizing intermediates are converted to N₂O₃ through further reaction with NO [28]. Nucleophiles are preferentially nitrosated rather than oxidized under these conditions.

O₂⁻ + NO → ONOO⁻ (4)

$$\begin{array}{l}{\text{H}}^{+}\\ {\text{ONOO}}^{-}+2\text{NO}\to {\text{N}}_2{\text{O}}_3+{\text{NO}}_2^{-}\end{array}$$

$$\begin{array}{l}{\text{CO}}_2\\ {\text{ONOO}}^{-}+2\text{NO}\to {\text{N}}_2{\text{O}}_3+{\text{NO}}_2^{-}\end{array}$$

The reaction rates of NO with these RNS are nearly diffusion controlled, facilitating the rapid conversion from an oxidative to a nitrosation profile (Eqs. (5), (6)). Therefore, a balance is formed between nitrosative and oxidative chemistry depending on the relative concentration of NO. At low NO fluxes, these reactions would tend to lead to oxidation of substrate, whereas at higher levels of NO they will preferentially nitrosate. In mechanisms of toxicity, there is a critical balance between the stoichiometric amount of RNS generated to participate in oxidative chemistry, which would tend to be low, and the conversion of these intermediates to nitrosating species. This indicates that high NO levels at which the indirect effects are predicted, in particular those that can be measured in a biological system, will favor nitrosative stress.

The chemical biology of NO gives us a framework to understand how this simple diatomic molecule could have numerous biological properties based simply on its concentration in terms of the chemical toxicology. Low concentrations of NO such as those that occur in vascular and stromal cells (i.e., from endothelial (eNOS) and neuronal (nNOS) NO synthase) regulate normal physiological processes, and the high levels such as those expected in activated macrophages (via inducible (i) NOS) are thought to serve a cytotoxic/cytostatic function [17], [29], [30]. However, at these higher concentrations, it is not always clear that cell death is the ultimate outcome. Nitrosative stress has a protective side, where nitrosation of caspase-3 and -8 as well as poly(ADP-ribose) polymerase (PARP) leads to protection against apoptosis [31], [32], [33]. Nitrosation and other oxidants close NMDA channels, preventing calcium influx [34], [35], [36], [37], [38]. Oxidative mechanisms such as nitration also have been shown to be biological signals of protection against apoptosis [39]. Nitration of the transferrin receptor leads to proteasomal degradation, which limits iron uptake, reducing apoptosis in endothelial cells. These examples suggest that tissues have adapted to conditions of inflammation and that biological mechanisms use this chemistry to mediate protective signals.