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inappropriate production from commencing (Lorsbach, Murphy et al. 1993). Another possible
protective mechanism is, while INF1 induces iNOS, it also induces production of ILl0 an
anti-inflammatory cytokine. Pre-treatment of macrophages with IL10 inhibits their ability to
express iNOS or to produce NO, although once iNOS exists then it has no effect (Cunha,
Moncada et al. 1992). IL,l added with ILl0 acts even more strongly in inhibiting the
production of the enzyme (Oswald, Gazzinelli et al. 1992).
Secondly, examining the post transcriptional control, certain cytokines can have either
stabilising or destabilising effects on the mRNA for iNOS, thus greatly altering its half life.
For example, IFNI stabilises the mRNA prolonging its action while transforming growth
factor beta (TGFp) can destabilise the mRNA and reduce its transcription as well as
accelerating its breakdown (Vodovotz, Bogdan et al. 1993; Imai, Hirata et al. 1994; Sirsjo,
Soderkvist et al. 1994; Perrella, Patterson et al. 1996; MacMicking, Xie et al. 1997). While
this inhibits the production of iNOS in this way, it does not effect cNOS production (Ding,
Nathan et al. 1990).
Thirdly, post translational control, as mentioned above, includes the availability of the
substrate and cofactors. However the inducible form is independent from some of the
regulations described for the constitutive forms. While all the isoenzymes require calmodulin
as a cofactor, it is tightly bound to this form of the enzyme (Xie, Cho et al. 1992). In addition,
the constitutive enzymes require calcium at levels of 200 nanomoles, whereas the inducible
form can activate with calcium at much lower levels down to 39 nanomoles and notably the
cellular basal level of calcium is nearer 70 to 100 nanomoles. This also explains why this
form of the enzyme can keep producing NO when the constitutive enzymes have stopped
production once the calcium falls below a certain level @astian and Hibbs 1994). A low
amount of the substrate L-arginine acts as a control mechanism, there are three possibilities as
mentioned above. There may be an absolute low availability of L-arginine. The uptake of
arginine into the cell occurs through a sodium and pH dependent pathway, mediated by a
family of cationic amino acid proteins (CATI, CATZ, CAT2B, CAT3), (Closs, Scheld et al.
2000; Nicholson, Manner et al. 2001), but this can be up-regulated by the presence of LPS.
External to the cell, the arginine levels are controlled by the enzyme arginase which degrades
arginine to urea and ornithine (Gotoh and Mori L999; Munder, Eichmann et al. 1999;
Rutschman, Lang et al. 2001). Macrophages contribute to this, possibly also developed as a
protective mechanism, as they release an abundance of arginase. However macrophages and
vascular smooth muscle cells can also regenerate arginine from citrulline and therefore re-
utilize the citrulline amino acid. This is with the use of another enzyme arginosuccinate
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