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The enzyme of this second pathway - guanosine triphosphate cyclohydrolase I - is in turn
affected by certain cytokines and inflammatory products. So while this operates as a control
for the isoforms, in fact the limiting effect may be more important for the iNOS enzyme
which is present in higher amounts, particularly in infection and inflammation when much
greater levels of these cytokines are around and when the production of NO is higher (Stuehr
1999; Werner-Felmayer, Golderer et al.2002). This will be discussed in more depth below in
Section 3.4.3 (ii).
The rates of formation of NO and L-citrulline are non linear for all isoenzyme forms which
demonstrates there is negative feedback inhibition and this is also a mechanism to control
overproduction (Rogers and Ignarro 19921, Assreuy, Cunha et al. 1993; Bastian and Hibbs
r9e4).
In addition, there are some differing control mechanisms operating for each of the constitutive
isoenzymes. Firstly, the nNOS form can be regulated by alternative splicing to give multiple
transcripts and molecular diversity. This means that the protein can be spliced in different
places giving different molecular weights which possibly fulfil a differing role. There are two
major transcriptional clusters identified within the human nNOS gene, with one form lacking
approximately 315 base pairs, or 105 amino acids and this smaller version is seen
predominantly in the peripheral nervous system, renal tract and in the male reproductive tract
(Papapetropoulos, Rudic et al. 1999).
Secondly, regarding the eNOS form, it was noted that some physiological situations led to
increased eNOS expression such as shear stress and exercise training (Nishida, Harrison et al.
1992; Sessa, Ftitchard et al. 1994; Uematsu, Ohara et al. 1995), and situations of local
hypoxia (Marsden, Schappert et al. 1992). While both iNOS and nNOS exist in soluble forms,
eNOS exists in a particulate form (Nathan 1992). The importance of this is that the proper
localisation of eNOS is a pre-requisite to enable it to interact with specific proteins that will
allow full activity. Post translation, cNOS undergoes a process of acylation (Shaul, Smart et
al. 1996) which appears necessary to anchor the enzyme to the membrane in the Golgi
apparatus, and in the plasmalemmal vesicles (the caveolae). There are some regulatory
proteins that play a role in promoting this within the cells (C-protein coupled receptors,
caveolin or Hsp90), that contribute to the correct localisation and that can also be individually
influenced therefore contributing to more or less NO being produced. For example, Hsp90
increases with increased histamine or fluid shear stress (Garcia-Cardena, Fan et al. 1998).
Changes in phosphorylation of eNOS have been observed with shear stress (Corson, James et
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