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Cell Signalling Biology Michael J. Berridge Module 2 Cell Signalling Pathways 2 68

Module 2: Figure eNOS activation

Myristic
eNOS acid
Inactive
Caveolin Palmitic
CAM FAD BH 4
FMN Heme acid

Calcium-induced
NOS activation

eNOS
Active
CAM BH
Caveolin FAD 4
FMN Haem

Arginine Citrulline
NADPH NADP
2+ O
Ca 2 NO

Ca 2 + -dependent activation of endothelial nitric oxide synthase (eNOS).
An important feature of eNOS is its location on the membrane of caveolae (Module 6: Figure caveolae molecular organization). Its membrane
localization is facilitated by an N-terminal myristic acid and by two palmitic acid residues attached to two cysteine residues (Cys-15 and Cys-26),
whereas its association with the caveolae depends upon its attachment to caveolin, which is responsible for keeping the enzyme inactive under resting
conditions. There appears to be a competition between caveolin and calmodulin (CaM) for the caveolin-binding site (amino acids 350--358) on eNOS.
In the absence of Ca 2 + , caveolin dominates, but when Ca 2 + increases and binds to CaM, the latter relieves the inhibitory effect of caveolin, and the
enzyme is activated to generate NO from arginine using oxygen and NADPH as co-substrates.

calmodulin (CaM) to stimulate the enzyme to release NO rophages, where its expression is up-regulated by inflam-
(Module 2: Figure eNOS activation). matory mediators. Although macrophages are the main
The NO released from endothelial cells diffuses out to cells that express iNOS, it is also found in other cell types
regulate smooth muscle cell contraction and hence controls (cardiac cells, vascular smooth muscle cells and glial cells).
blood pressure, smooth muscle cell proliferation, aggrega- Unlike the other isoforms, the activation of iNOS does
tion of blood platelets and leucocyte adhesion. Studies on not require an elevation of Ca 2 + . However, iNOS does
NO/cyclic GMP and cardiac hypertrophy have revealed bind calmodulin (CaM), which is essential for its activa-
that expression of eNOS in endothelial cells can inhibit tion. Since the enzyme is constitutively active, its primary
hypertrophy in neighbouring cardiac cells. Given its cent- regulation depends upon its induction by inflammatory re-
ral role in regulating so many cellular processes, alterations ceptors such as those that respond to interferon-γ (IFN-γ)
in the endothelial production of NO have been implicated or lipopolysaccharide (LPS). As large amounts of enzyme
in many disease states, such as hypertension, diabetes and are produced, this is a high-output pathway capable of de-
hypercholesterolaemia. livering NO for prolonged periods as part of the cells de-
fence against invading micro-organisms. As such, it does
Inducible nitric oxide synthase (iNOS) not strictly function as a messenger. However, the large
Inducible nitric oxide synthase (iNOS), which is also production of NO at the sites of inflammation will spill
known as immunocyte NOS, was first described in mac- over to affect neighbouring cells. The large up-regulation

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