Subcellular and cellular locations of nitric oxide synthase isoforms as determinants of health and disease

Abstract

The effects of nitric oxide in biological systems depend on its steady-state concentration and where it is being produced. The organ where nitric oxide is produced is relevant, and within the organ, which types of cells are actually contributing to this production seem to play a major determinant of its effect. Subcellular compartmentalization of specific nitric oxide synthase enzymes has been shown to play a major role in health and disease. Pathophysiological conditions affect the cellular expression and localization of nitric oxide synthases, which in turn alter organ cross talk. In this study, we describe the compartmentalization of nitric oxide in organs, cells, and subcellular organelles and how its localization relates to several relevant clinical conditions. Understanding the complexity of the compartmentalization of nitric oxide production and the implications of this compartmentalization in terms of cellular targets and downstream effects will eventually contribute toward the development of better strategies for treating or preventing pathological events associated with the increase, inhibition, or mislocalization of nitric oxide production.

Figure 1. Nitric oxide generation and signaling

Nitric oxide, generated by NOS, activates soluble guanylate cyclase (sGC) and particulate guanylate cyclase (pGC), and inhibits cytochrome c oxidase. cGMP activates cGMP-dependent protein kinases (PKG). As shown in the figure, some downstream pathways and cellular functions (grey boxes) are involved in the effects of endogenous cGMP. The concentration of cGMP can be controlled by the action of phosphodiesterases (PDE). In addition, nitric oxide can affect other pathways through protein modifications (nitric oxide-metal adduct formation, S-nitrosation, nitration). For instance, the nitration of specific Tyrosine residues in the beta-subunit of Complex V results in lower ATPase activity during nitrative stress or aging [137, 138]. ANP, atrial natriuretic peptide; PK, protein kinases (letter indicates the type of kinase); PDEs, phosphodiesterases; sGC, soluble guanylyl cyclase; IRAG, IP₃ receptor-associated cGKIβ substrate; MLC phosphatase (MLCP); RhoA, a substrate for cGMP-dependent protein kinases (PKG); large-conductance Ca²⁺-activated K⁺ (BKCa) channels. Other details were previously described by Hofmann et al. [139].

Figure 2. Metabolite-controlled production of NO

Nitric oxide (NO) is produced by nitric oxide synthases (NOS) from L-arginine. Nitric oxide can interact with specific targets, such as soluble guanylate cyclase (sGC) and cytochrome c oxidase (CCO), or with other molecules, such as superoxide anion, to trigger nitrative stress. High levels of citrulline (mM) inhibit N^G,N^G-dimethylarginine dimethylaminohydrolase (Ddah), resulting in an increase of N^G,N^G-dimethyl-L-arginine (ADMA). ADMA, in turn, is a potent NOS inhibitor. Arginine concentrations can be modulated by the activity of arginases, which catalyze the formation of L-ornithine (Orn) and urea from L-arginine. Another abbreviation: DMA, dimethylarginine. Enzyme names are in italic.

Figure 3. Location of NOS in liver, liver cells and subcellular compartments

NOS1, NOS2 and NOS3 are present in liver. NOS1 has been shown in rat hepatocytes ([140] and Villanueva et al., 2010, submitted manuscript). NOS2 and NOS3 have been demonstrated in normal human hepatocytes [26]. NOS2 is present in Kupffer cells, whereas NO3 is present at the endothelial cells of the hepatic sinusoids [26, 141]. Within the cells, the NOS isoforms are located in different subcellular compartments such as the Golgi apparatus, caveolae or mitochondria [94]. NOS1 and NOS2 can be translocated from cytoplasm to nuclei in pathological conditions such as diabetes (see Figure 4) and cirrhosis [26], respectively. Within the cell, nitric oxide produced by NOS can interact with various specific targets (soluble guanylate cyclase, cytochrome c oxidase) and other biomolecules such as lipids, proteins and carbohydrates.

Figure 4. Immunohistochemistry of NOS1 in liver from control, Type-1 diabetic and endotoxin-treated rats

NOS1 distribution was evaluated in the perivenous area, as shown by immunohistochemistry (at 20X; shown in a red-brown color) (Villanueva et al., 2010, submitted manuscript). Arrows show the distribution of NOS1 in endothelium (black), hepatocytes (white) and Kupffer cells (grey). A rabbit NOS1 polyclonal primary antibody (from Cayman Chemical Co.) was used in the immunostaining procedure that is followed by a diaminobenzidine-based development. In control animals, NOS1 expression was visible only at the endothelium (Control). Using the same conditions in Type-1 diabetic rats, NOS1 was present in hepatocytes, endothelium and Kupffer cells. Nuclear localization of NOS1 was only seen in hepatocytes of Type-1 diabetic rats (compare cells from the three groups). In endotoxin animals (5 h after lipopolysaccharide injection), the number of NOS1-positive cells was higher than that in control animals.

Figure 5. Role of caveolin in modulating NOS3 activity

Caveolin-1 (CAV1), by anchoring proteins to the membrane, inhibits the activity of certain proteins, for example, NOS3. Following ligand- or mechano-stimulation, NOS3 is dissociated from CAV1, allowing its accessibility to calmodulin (CaM) and 90-kD heat-shock protein (HSP90) to produce nitric oxide (modified from Carver and Schnitzer, [142]).