**3. Nitric oxide biochemistry**

NO is a highly reactive molecule with other free radical species and possesses an extremely short half-life (Rubbo, Darley-Usmar, & Freeman, 1996). NO is produced endogenously or delivered exogenously where it can react with a variety of cellular targets resulting in vasorelaxation, enhanced neuronal transmission, reduced apoptosis, inhibition of neutrophil aggregation and adhesion, and modulation of vascular smooth muscle proliferation.

NO synthesis is dependent on the enzyme nitric oxide synthase (NOS). NOS catalyzes the net reaction:

$$\text{Li-Arginine} + \text{NADPH} + \text{O}\_2 = \text{Citrulline} + \text{Nitric oxide} + \text{NADP\*} \tag{1}$$

(adapted from Alderton, Cooper, & Knowles, 2001)

This complex enzyme system generates NO from the terminal nitrogen atom of L-arginine in the presence of NADPH and dioxygen. NOS binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and calmodulin from L-arginine and oxygen by a family of three NO synthases (NOS), all of which are expressed in a variety of cell types.

Three distinct isoforms are known: 1) Neuronal NOS (NOS I), is produced in central and peripheral nerves and is pivotal in neuronal transmission and cell-to-cell communication

micro-envirnoment) may have different effects on these two phases. Early injury is mediated by a rapid change in the biochemical redox state of the tissue to a more oxidative one. It occurs within 5 minutes, and is not associated with leukocyte infiltration. Following the acute state is an increase in endothelial cell adhesion molecules, chemokines and cytokines. These molecules then herald the late phase characterized by a significant infiltration of polymorphonuclear neutrophils, further release of a reactive oxygen species (ROS) and

NO plays a significant role in the acute phase of IRI, as this phase is associated with a rapid decrease in available NO. This decrease occurs either by depressed production by eNOS in sinusoidal endothelial cells (SECs), increased degradation by ROS, or both. The ROS implicated are chiefly O2•- (superoxide, see next paragraph), but also include hydrogen peroxide (H2O2). In the last few years, the implicated enzyme responsible for production of ROS has shifted from hepatoctye xanthine oxidase to NADPH oxidase in Kuppfer cells or

The term "reactive oxygen species" in the context of hepatic IRI primarily refers to superoxide. Two studies that incorporated manganese superoxide dismutase (MnSOD) – an enzyme which degrades superoxide – into liver tissue showed attenuation of IRI (He et al., 2006; Zwacka et al., 1998a). Therefore superoxide itself seems important in IRI. The mechanism by which superoxide imparts its damage is somewhat unclear, but it is known that membrane lipid peroxidation is associated with oxidative damage. Perhaps more importantly, damage by superoxide to mitochondrial membrane proteins and therefore ATP generating capacity and may a more important mechanism in IRI(Madesh & Hajnóczky,

NO is a highly reactive molecule with other free radical species and possesses an extremely short half-life (Rubbo, Darley-Usmar, & Freeman, 1996). NO is produced endogenously or delivered exogenously where it can react with a variety of cellular targets resulting in vasorelaxation, enhanced neuronal transmission, reduced apoptosis, inhibition of neutrophil

NO synthesis is dependent on the enzyme nitric oxide synthase (NOS). NOS catalyzes the

This complex enzyme system generates NO from the terminal nitrogen atom of L-arginine in the presence of NADPH and dioxygen. NOS binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and calmodulin from L-arginine and oxygen by a family of three NO synthases (NOS), all of which are expressed

Three distinct isoforms are known: 1) Neuronal NOS (NOS I), is produced in central and peripheral nerves and is pivotal in neuronal transmission and cell-to-cell communication

L-Arginine + NADPH + O2 = Citrulline + Nitric oxide + NADP+ (1)

aggregation and adhesion, and modulation of vascular smooth muscle proliferation.

extensive inflammation and tissue injury.

2001; Moon et al., 2008).

net reaction:

in a variety of cell types.

**3. Nitric oxide biochemistry** 

(adapted from Alderton, Cooper, & Knowles, 2001)

mitochondrial sources of ROS (Hines & Grisham, 2011).

within the central nervous system. 2) Inducible NOS (NOS II is induced by an inflammatory stimulus such as a microbe (Parul Tripathi, Prashant Tripathi, Kashyap, & Singh, 2007). Unlike the other types of NOS (I and III), NOS II is not constitutive and is independent of calcium regulation. While NOS II is expressed by immune cells such as neutrophils and macrophages, it is also present in other cell lines including hepatocytes. Endothelial NOS (NOS III), is constitutively expressed by endothelial cells and is critical for the regulation of vascular function, more specifically vasorelaxation.

(Modified, with permission from http://www.wiley.com/college/boyer)

Fig. 1. Mechanisms of smooth muscle relaxation. NO diffuses across the muscle cell membrane and binds to guanylyl cyclase. Guanylyl cyclase catalyzes the synthesis of cyclic GMP from GTP. cGMP activates a cGMP-dependent protein kinase which stimulates the uptake of calcium by the endoplasmic reticulum of the muscle cell. The reduced levels of cytoplasmic calcium cause the muscle cell to relax. As a consequence of muscle cell relaxation, vasodilation occurs. PKG – protein kinase G

The generation of NO leads to several actions that promote smooth muscle relaxation. First, activation of guanylate cyclase raises the level of intracellular cGMP which in turn inhibits the entry of calcium into the cell thereby inducing smooth muscle relaxation. Second, activation of K+ channels leads to cellular hyperpolarization and relaxation. Finally, stimulation of cGMP-dependent protein kinase activates of myosin light chain phosphatase leading to dephosphorylation of myosin light chains resulting in smooth muscle relaxation. NOSs are related but encoded by distinct genes. Classically, the ability of NO to elicit vasorelaxation is due to its ability to increase intracellular levels of cyclic guanosine monophosphate (cGMP) through the activation of soluble guanylate cyclase (sGC). cGMP– dependent protein kinases in turn decrease the sensitivity of myosin to calcium-induced contraction and lower intracellular calcium by activation of calcium-sensitive potassium channels and inhibits the release of calcium from the sacroplasmic reticulum. Mechanisms of smooth muscle relaxation are shown on Figure 1.
