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Endothelial Nitric Oxide Synthase and NO Production

Endothelial Nitric Oxide Synthase (eNOS) is one of the three Nitric Oxide Synthases (NOSs) enzymes which were initially characterised in 1989 (Palmer and Moncada, 1989). The three different isoforms of NOSs are; neuronal Nitric Oxide Synthase (nNOS), inducible Nitric Oxide Synthase (iNOS) and endothelial Nitric Oxide Synthase (eNOS) and were all cloned and purified between 1991 and 1994. These NOSs were named after the type of tissue they were first identified in, and are also denoted as type I or NOS-I or NOS-1 (nNOS), type II or NOS-II or NOS-2 (iNOS) and type III or NOS-III or NOS-3 (eNOS). These NOSs have also been differentiated on the basis of their constitutive – nNOS and eNOS, and inducible – iNOS expression; as well as on their calcium-dependence – eNOS and nNOS, and - independence – iNOS.

Initially identified in 1989, eNOS was cloned in 1992 (Janssens et al., 1992, Marsden et al., 1992). eNOS is phosphorylated and dephosphorylated on a number

of different sites, however, eNOS phosphorylation at Ser1177, and

dephosphorylation at Thr495 are the two significant sites that primarily regulate the activity of eNOS (Dimmeler et al., 1999, Fleming et al., 2001). Additionally, eNOS phosphorylation at Ser116 and Ser617 is also reported to regulate the enzyme activity and functions (Bauer et al., 2003, Boo et al., 2003). Over-expression of eNOS reduces blood pressure and plays an important role in blood pressure regulation (Ohashi et al., 1998). Calmodulin (CaM) is another protein that interacts with eNOS (Bredt and Snyder, 1990), and is required for the activity of all three

different NOSs by increasing the rate of electron transfer from Nicotinamide Adenine Dinucleotide Phosphate-hydroxylase (NADPH) to the reductase domain.

In 1987, Palmer and colleagues demonstrated the release of NO from the vascular endothelium for the very first time (Palmer et al., 1987) and characterised L-arginine as a substrate molecule for NO synthesis (Palmer et al., 1988). Initially, NO was characterised as a Endothelium-Derived Relaxing Factor (EDRF) by Furchgott and Zawadzki (Furchgott and Zawadzki, 1980). NO generated by the vascular endothelium is a major regulator of vascular homeostasis, and altered NO levels are implicated in the development of a number of diseases of the vascular system. NO also acts as an inhibitor of platelet aggregation and retains the anti- thrombotic properties of endothelium (Radomski et al., 1987). NO modulates leukocyte adhesion to the vascular wall and is also implicated in the down-regulation of expression of CAMs such as P-selectin (Davenpeck et al., 1994), E-selectin (De Caterina et al., 1995), VCAM-1 (Khan et al., 1996) and ICAM-1 (Biffl et al., 1996). NO is also suggested to play an important role in maintaining vascular integrity, as the absence of eNOS enzyme, which is implicated in NO production, results in increased fluid and protein flux (Kubes, 1995). NO increases vascular permeability by increasing VEGF protein expression, which then activates eNOS in a NO- dependent manner (Feng et al., 1999). eNOS-derived NO is reported to be involved in EC angiogenesis (Jenkins et al., 1995, Kroll and Waltenberger, 1998, Ziche and Morbidelli, 2000), as well as in capillary tube organisation (Papapetropoulos et al., 1997). VEGF, the most potent angiogenic factor increases the NO production via up- regulating eNOS activity (van der Zee et al., 1997, Hood et al., 1998), which then mediates the migratory and proliferatory actions of VEGF (Papapetropoulos et al.,

most important activator of eNOS that results in the activation of a number of pathways; especially PI3K pathway which activates Akt1 at Ser473 phosphorylation site, which then phosphorylates eNOS at Ser1177 (Ayajiki et al., 1996, Go et al., 1998, Dimmeler et al., 1999, Fulton et al., 1999), finally leading to NO production which is also known to promote angiogenesis (Liu et al., 2002). Angiogenesis induced via pathways independent of VEGF are also known to be modulated by angiogenic properties of NO (Ziche et al., 1994, Leibovich et al., 1994, Vodovotz et al., 1999).

Caveolae and NO Production

Caveolae are small cell-surface invagination of approximately 50 to 100nm (Yamada, 1955, Rothberg et al., 1992), and are characterised as ‘Ω-shaped’ structures in the cell membrane. Caveolin-1 is highly expressed in ECs and constitutes the largest amount in the caveolae (Lisanti et al., 1994). eNOS enzyme is reported to be localised in a caveolae, and interacts with caveolin-1 and caveolin-3 – the coat proteins of a caveolae – via a caveolin-binding motif in the eNOS (Garcia- Cardena et al., 1996). In resting ECs, both caveolin-1 and caveolin-3 are reported to bind to eNOS, and this interaction inhibits the activity of eNOS enzyme, hence, interfering with NO production (Bucci et al., 2000). Caveolae are also known to increase vascular permeability, as VEGF treatments are reported to induce caveolae clustering, and results in the formation of vesiculovacuolar organelles (VVOc) (Vasile et al., 1999); whereas, prolonged VEGF treatments resulted in the formation of fenestrae (Chen et al., 2002). Caveolin-1 is also reported to inhibit the activity of eNOS which may further contribute to the regulation of vascular permeability. These findings suggest an important role for caveolin-1 in cell-to-cell interactions, and also with extracellular matrix and many other different proteins (Wary et al., 1998).

Calcium/Calmodulin binding to eNOS competitively disrupts the eNOS-caveolin-1 binding, and dissociates eNOS from caveolin-1 protein, hence, increasing the activity of eNOS enzyme. Caveolin-1 also plays an important role in the process of angiogenesis; as caveolin-1 protein expression is reported to be down-regulated during EC proliferation, however, is markedly increased during EC differentiation and vessel formation (Liu et al., 2002).

Heat Shock Protein 90 and NO Production

Heat Shock Proteins (HSPs) are ubiquitously expressed proteins and are essential for maintaining cellular homeostasis, and are known to trigger various cellular responses after exposure to external stress stimuli. There are different classes of HSPs, however, HSP90 is the most important signalling HSP and is a key organiser of several cytoplasmic complexes. Five different isoforms of HSP90 have been identified, however, HSP90α and HSP90β are the major cytosolic isoforms. Both HSP90 isoforms share approximately 85% sequence homology, contain an ATP-binding domain in NH2-terminal region, a dimerised COOH-terminal, and a

highly charged mid-domain which encourages substrate-protein interactions (Hainzl et al., 2009, Csermely et al., 1998). Both these isoforms are known to regulate eNOS enzyme activity, NO and superoxide production (Cortes-Gonzalez et al.).

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Chemerin, a Novel Adipochemokine