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cyclase does not produce significant levels of NO, but the relaxant factor, EDRF, was thought
to alter the binding in so much that it increased the activation of the enzyme by 400 fold.
However it is now known that it is nitrosylation of the guanylate cyclase that results in high
levels of cGMP which both prevents entry and promotes movement out of the cell of calcium
resulting in vasodilatation. The Moncada research goup then identified that the substrate for
NO synthesis was L-arginine and also went on to synthesise an inhibitor of NO formation by
producing a false competitive inhibitor of L-arginine; N* monomethyl L-arginine (L-
NMMA), (Gardiner, Compton et al. 1990). It was by using this inhibitor that further
clarification of the role of NO was possible.
2.3.2 Immunefunction
At the same time that EDRF was being identified as NO, other research groups were looking
at NO in a different biological system -
that of host immune defence.
Macrophages were known to be able to inhibit tumour growth and induce tumour cell death
(Nathan 1992). In 1987 (Hibbs, Vavrin et al. 1987) it was suggested that the macrophage
cytotoxicity might in part be due to the ability of the macrophages to synthesise NO, nitrite
and nitrates from L-arginine. At the tissue level, NO was known specifically to be toxic to
cells. A number of bacteria were also known to be able to generate reactive species of nitro-
oxygen suspected as occasionally causing lung disease. However the main interest in these
bacteria was not in the medical arena but centred on the importance of bacteria using these
pathways causing meat to degenerate. Organisms that had demonstrated the production of
nitro-oxygen compounds included Achromobacter cycloclastes, Alcaligenes faecalds, and
Bacillus halodenitrificans (Godden, Turley et al. 1991).
The suggestion that the use of NO in this way could occur within living animals came from
the demonstration that germ free rats excreted more nitrate than they ingested (Green,
Tannenbaum et al. 1981). This was also found in humans in a stable state but further
investigations showed that the amounts increased dramatically during intercurrent infection
(Green, Ruiz de Luzuriaga et al. l98l). Other researchers confirmed this by injecting rats and
mice with pro-inflammatory or infectious agents and showing that there was an increase in the
amount of nitrates that were excreted (Wagner, Young et al. 1983, Stuehr, 1985 #580). In part
this was described by using macrophages from these rats in vitro and showing that these cells
could produce nitrite and nitrate by oxidizing L-arginine (Iyengar, Stuehr et al. 1987) to yield
citrulline and a compound with the ability to react with amines and generate nitrosamines
(Miwa, Stuehr et al. 1987). It was thought that a compound with such ability had to be an
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