After the discovery of nitric oxide as a biological mediator, many researchers have focused on the importance of nitric oxide in the physiology of the nervous system. NO, as the smallest signaling molecule, is produced by three types of NO synthase that arise from three different genes called neuronal nitric oxide synthase (also known as nNOS, Type I, NOS-I, and NOS-1) found in tissue neurons, inducible nitric oxide synthase (also known as iNOS, Type II, NOS-II, and NOS-2) that is synthesized after the formation of pro-inflammatory cytokines or endotoxin and endothelial nitric oxide synthase (also known as eNOS, Type III, NOS ‐III and NOS – 3) found in endothelial cells.
NOS and eNOS are constitutively expressed and considered calcium-dependent, but when iNOS activity is fully activated at baseline intracellular calcium concentration, it would be independent of calcium. The main difference between the three NOS isoforms with respect to the reactions achieved lies in the rate of oxidation of nicotinamide-adenine-dinucleotide phosphate (NADPH), called the uncoupled reaction. Furthermore, nNOS continues to transfer electrons to heme and therefore NADPH oxidase at a high rate, whereas in eNOS and iNOS this reaction can occur at a much slower rate.
NOS is the main source of NO in different populations of neurons and synaptic spines in the brain and peripheral nervous system, whereas eNOS can occur in some neurons and iNOS can exist in microglia and astrocytes, but generally not in neurons. The nNOS-expressing interneurons are involved in physiological procedures such as neurovascular coupling to regulate neocortical blood flow, homeostatic sleep control, synaptic integration of adult neurons, and balance of excitatory and inhibitory signaling in the brain.
The nNOS monomer with a molecular weight of 160.8 kDa and 1434 amino acids is inactive and can be activated after dimerization by the binding of tetrahydrobiopterin (BH4), heme, and l-arginine (L-Arg). Each nNOS monomer has two domains, including a reductase domain (C-terminal) and an oxygenase domain (N-terminal) that can be separated by a calmodulin-binding motif. The reductase domain that binds the NADPH substrate comprises a binding site for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). An autoinhibitory loop within the FMN-binding domain regulates the activity of nNOS.
All forms of NOS use l-arginine and molecular oxygen as a substrate and reduced nicotinamide-adenine-dinucleotide phosphate (NADPH) as cosubstrates to produce citrulline NO. FAD and FMN with BH4 are cofactors of all isoenzymes. All NOS proteins are homodimers. A functional NOS transfers electrons from NADPH, via FAD and FMN in the carboxy-terminal reductase domain to heme in the amino-terminal oxygenase domain.
This electron flow (NADPH → FAD → FMN → heme) can be facilitated by Ca2 + / CaM binding. The oxygenase domain also binds to the cofactor BH4, molecular oxygen, and l-arginine. At the heme site, electrons are used to activate O2 to oxidize l-arginine to l-citrulline and NO. Sequences close to the heme cysteine ligand are also involved in the binding of L-arginine and BH4. The NOS enzyme is monooxygenases, which generate NO and citrulline from L-arginine (L-Arg), NADPH, and O2.
NOS has been found in neurons, astrocytes, adventitia of blood vessels in the brain, and cardiac myocytes. In addition to brain tissue, nNOS has been distinguished by immunohistochemistry in the spinal cord, sympathetic ganglia and adrenal glands, peripheral nitrogen nerves, skeletal and cardiac myocytes, epithelial cells of different organs, cells of the renal macula densa, cells of the pancreatic islets Non-adrenergic, non-adrenergic, non-cholinergic peripheral autonomic nerve fibers and vascular smooth muscle and endothelial cells. In mammals, the most important source of nNOS with respect to tissue mass is skeletal muscle.
Since NO can be stored in cells, a new synthesis is necessary for it to have its activities. Therefore, nNOS must bind to the plasma membrane directly or through adapter proteins. Fractionation studies have shown that brain nNOS is particulate and soluble in the cytosol away from patch-like membranes. Furthermore, during the first six days in cultured rat cerebral cortical astrocytes, nNOS immunoreactivity appeared primarily in the cytoplasm.
However, on day 7, nNOS immunoreactivity was predominantly expressed in the nucleus, and this nuclear localization continued for around 10 h. So, the nNOS immunoreactivity was mainly expressed in the cytoplasm again. Recently, some investigators showed the nuclear localization of nNOS without nNOS cytoplasmic staining in some parts of neural and glial cells. Therefore, various functions of nNOS in the cell may arise from differential subcellular localization.