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al. 1996; Eng, Morton et al. 1996; Fleming, Bauersachs et al. 1998), secondary to calcium
mobilising agents (Michel, Li et al. 1993), tyrosine phosphatase inhibitors (Garcia-Cardena,
Fan et al. 1996), and a number of protein kinases (Hirata, Kuroda et al. 1995; Chen,
Mitchelhill et al. 1999). The phosphorylation of the enzyme makes it more sensitive to
calcium necessary for the active pathway.
The most potent activator is lysophosphotidylcholine (IlC), a major phospholipid found in
oxidised low density lipoproteins and this allows up to an 11 fold increase in mRNA
induction and eNOS production, although there is a varying discrepancy between the levels of
the mRNA and both the absolute protein levels and the levels of activity pointing to continued
control later in the pathway (Papapetropoulos, Rudic et al. 1999). Interestingly, the content of
LPC in atherosclerotic vessels is higher than normal vessels. Statin-based cholesterol
lowering drugs result in a modest increase in the eNOS mRNA levels by a post transcriptional
mechanism involving stabilisation (Laufs and Liao 1998). Oestrogens are capable of modestly
increasing eNOS expression (Wiener, Itokazu et al. 1995; Kleinert, Wallerath et al. 1998).
The activity of the enzyme is reduced by the presence of TNFo which destabilises the eNOS
mRNA and reduces the half life of the enzyme from 48 to 3 hours (Yoshizumi, Perrella et al.
le93).
3.4.3 (ii) The inducible form
Control of the iNOS isoenzyme can also operate at pre-transcriptional, post transcriptional
and post translational levels, and drugs also interact to affect enzyme generation. However,
different to the constitutive forms, control, particularly of gross overproduction, is extremely
important to host survival.
Firstly, Iooking at the pre-transcriptional level; the activation of the iNOS promoter region is
the main control for this isoenzyme. Most of the work looking at effects at a transcriptional
level has been done in explanted mouse macrophages or macrophage cell lines (MacMicking,
Xieet al. 1997; Papapetropoulos,Rudic etal. 1999).Thishasrevealedanumberof binding
sequences including interferon gamma (INFI), TNFo, TNFp, interferon alpha (IFNa), NF-KB,
gamma activated sites, interleukin 6 (IL6), activating protein sites and a basal transcription
site. However the importance of only 2 of these has been clearly documented. The NF-rB site
is important for iNOS induction (Nunokawa, Ishida et al. 1994) and LPS induction also
operates through this site (Nathan 1992), and a cluster of four sites for IFNI are important for
NOS transcription (Xie, Kashiwabara et al. 1994). The need for second signal as well as IFNy
or an IFNy-like substance to activate iNOS is probably an important control to prevent
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