Review ArticleSubcellular and cellular locations of nitric oxide synthase isoforms as determinants of health and disease
Introduction
Since 1987, when the endothelium-derived relaxing factor was identified as nitric oxide [1], [2], numerous reports have indicated that this small gaseous molecule, nitric oxide, is a ubiquitous mediator of many different biological processes, such as vasodilation [3], neurotransmission [4], [5], macrophage-mediated cytotoxicity [6], gastrointestinal smooth muscle relaxation [5], and bronchodilation [7], through a variety of downstream pathways (Fig. 1). According to Fick's laws of diffusion, the diffusion coefficient of nitric oxide is 4.8 × 10− 5 cm2 in water at 37 °C [8], [9], similar to that of oxygen under comparable conditions [10]. It has been estimated that the half-life of nitric oxide varies from about 1 s in blood-free perfused guinea pig heart to 30 s in physiological buffers [8]. Based on these half-life values, the diffusion distances are expected to be in the 120–700 μm range [8]. Nitric oxide has been detected at a distance of 100 to 500 μm from RAW 264.7 macrophages stimulated with interferon, yielding a diffusion radius of 10 to 50 cells (assuming an average macrophage diameter of 10 μm) [11]. Thus, theoretically, based on its diffusion coefficient and the assumption that cells in culture are a representative model for the diffusion of nitric oxide in vivo, the effects of this gaseous molecule could extend to many cells beyond its production site.
However, estimations of the half-life of nitric oxide based on its diffusion radius do not apply to complex biological systems. Factors that limit nitric oxide diffusion and therefore its half-life in biological systems include its interactions with soluble guanylyl cyclase and other proteins (e.g., hemoglobin), lipids, and free radicals [11], [12], [13]. When measured in isolated rat aorta, for example, its diffusion radius was shown to be fourfold smaller in an aortic wall than in a homogeneous medium such as water [14]. It has also recently been reported that the cholesterol content in membranes decreases nitric oxide diffusion by 20 to 40% [12]. This decrease was attributed to changes in membrane fluidity caused by cholesterol. Nitric oxide efflux produced by activated macrophages was also reduced by 41% in the presence of albumin and by 53 to 70% in the presence of liposomes, indicating that intracellular structures or biomolecules could also limit nitric oxide diffusion [11], thereby establishing compartmentalized effects of nitric oxide within the cells.
Depending on the environment, other factors can affect the half-life of nitric oxide and therefore its diffusion. When high concentrations of nitric oxide are produced by activated inducible nitric oxide synthase (NOS2)1, and superoxide anion is present, the formation of peroxynitrite will limit the diffusion of nitric oxide [8]. In addition, depending on the oxygen gradient near mitochondria, cytochrome c oxidase can become a target of nitric oxide, resulting in the inhibition of mitochondrial respiration [13].
In addition to the diffusion of nitric oxide from its production site, the partitioning of nitric oxide between polar and apolar media could play a major role in terms of localized effects [15]. Nitric oxide and oxygen have similar partition coefficients in apolar media, being 70 times more soluble in hydrophobic than in hydrophilic media [16]. Therefore, both molecules are more concentrated in a hydrophobic milieu, such as liposomes, lipoproteins, or biomembranes or within the hydrophobic pockets of proteins, than in polar-based environments [16], [17]. Higher concentrations of nitric oxide and oxygen in an apolar environment may result in chemical reactions favoring the formation of nitrogen oxides with chemical properties different from those of nitric oxide [18], [19].
The fact that nitric oxide encounters diffusion barriers throughout the body to find its targets and that nitric oxide-mediated responses are cell/tissue-specific, the existence of nitric oxide synthase (NOS) isoforms and their fine regulation at the pre- and posttranslational levels constitute a sine qua non condition to accomplish its specific yet diverse functions.
In view of these arguments, we propose that the systemic effects of nitric oxide derive from the cumulative effects of the autocrine and paracrine levels in specific organs.
Section snippets
Isoforms of nitric oxide synthase and their cell specificity
Nitric oxide is synthesized by NOS. The enzymatic synthesis of nitric oxide is accomplished by three NOS isoforms: the neuronal NOS (NOS1), the endothelial NOS (NOS3), and the inducible NOS (NOS2) (see Table 1). The activation of the first two enzymes depends on calcium, whereas NOS2 is independent of calcium [20]. It has been reported that NOS1 and NOS3 are constitutively expressed, whereas NOS2 is induced only during the immune response [21], [22]. However, more recently, it has been shown
Compartmentalization of nitric oxide synthases in various organs
In this section, we present and discuss how differential compartmentalization of the NOS isoforms in various organs relates to clinical examples based on the current literature and some recent results from our group.
Subcellular compartmentalization of nitric oxide synthases
Given the short half-life of nitric oxide in biological systems (see Introduction) and the need for nitric oxide at specific sites in the cell, intracellular compartmentalization of this compound is crucial for its signal transduction activities [92], [93]. In addition, as previously mentioned, nitric oxide diffusion is limited by its interactions with various molecules within the cells, and therefore the subcellular location of the NOS isoforms affects nitric oxide diffusion. Spatially
Translocation of nitric oxide synthases among cellular compartments
As indicated above, the NOS isoforms are localized in various subcellular compartments. However, after a given stimulus, some of them have been known to change location, suggesting the occurrence of posttranslational modifications, such as phosphorylation, or the activation of translocation, a more complex process that occurs with the aid of specific protein–protein interactions, such as what has been described for NOS3 [43], [44]. If nitric oxide were as diffusible in a biological setting
Regulation of gene transcription
Several lines of evidence indicate that the regulation of nitric oxide production can also occur at the level of gene transcription. In one study, for example, NOS1 was localized in the cytosol of rat cortical astrocytes during the first 6 days of culture; however, at the 7th day, NOS1 was mainly present in the nuclei of these cells, concomitant with the nuclear production of nitric oxide and the decrease of NOS2 protein expression [104]. These results suggest that the presence of NOS1 in nuclei
Activation/inhibition of signal transduction pathways
NOS3 is primarily expressed in the Golgi apparatus and plasmalemmal caveolae of endothelial cells [114], [115]. It has been shown that serum starvation increases the perinuclear location of the NOS3/caveolin complex in cultured endothelial cells isolated from bovine aorta [116]. If insulin is added to the culture, NOS3 is phosphorylated through the Akt pathway, and in response to the palmitoylation of caveolin, the complex is translocated to the caveolae; this NOS3/caveolin association inhibits
Modulation of enzymatic activity
Here, we present two examples from the literature of the modulation of enzymatic activity: one of the lipoxygenase activity modulation by NOS, the other of the NOS activity modulation by NOSIP. NOS3 has been detected primarily in the nuclei of human mast cells, whereas NOS1 is localized primarily in the cytosol of these cells [99]. After activation of mast cells with A23187 (a calcium ionophore) or IgE/anti-IgE, cytosolic NOS3 was phosphorylated and translocated to nuclei, and nitric oxide was
Conclusions
Even though nitric oxide is a small molecule produced in confined compartments within various types of cells, its site-specific effects are sensed throughout the entire organism. The effects of this molecule at the organism level are not the result of a long half-life, high stability, or free diffusion, but the consequence of localized effects of nitric oxide at various cellular levels and in various cell types, modulating and orchestrating complex responses requiring cross talk among organs
Acknowledgments
This study was supported by funds provided by the University of California–Mexus/Consejo Nacional de Ciencia y Tecnología (Collaborative Grant 2008) to C.G. and C.V. Additional support was provided by NIH (RC1DK087307) to C.G. Dr. Villanueva was a Visitor Scholar supported by a fellowship from the University of California–Mexus/Consejo Nacional de Ciencia y Tecnología. The intellectual contributions of Dr. Bulent Mutus (Visiting Professor, University of Windsor, ON, Canada) to this study are
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