The endogenous free radical signalling molecule, nitric oxide (NO), is the
mediator of many physiological processes including control of vasomotor tone, and
the inhibition of smooth muscle cell (SMC) proliferation and platelet activation.
Additionally, NO plays an important role in the regulation of the inflammatory
response through its ability to influence apoptosis in a variety of cells. This
fundamental process governing cell survival is cmcial to ensuring the successful
resolution of the inflammatory response as apoptotic inflammatory cells are removed
from a site of tissue injury by non-inflammatory phagocytosis. NO has both pro- and
anti-apoptotic properties depending on its concentration, the NO-related species
generated and the cell type in question. Pharmacological manipulation of apoptosis
by NO may aid the resolution of inflammation during chronic inflammatory
disorders such as atherosclerosis.
NO is often reported as having paradoxical effects in many biological settings.
This may be due to the formation of NO-related species in vivo: NO combines
rapidly with superoxide anion to generate peroxynitrite (ONOO ) and with
endogenous thiols to form S-nitrosothiols (RS-N=0). Both ONOO" and RS-N=0
have biological properties that are independent of the liberation of NO radical per se.
Characterisation of the NO-related species generated by the NO donor
compounds 1,2,3,4,-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-chloride (GEA3162), diethylamine diazeniumdiolate (DEA/NO), (Z)-l-[2-(2-Aminoethyl)-N-(2-
ammonioethyl)amino]diazen-l-ium-l,2-diolate (DETA/NO), S-nitroso-N-valeryl-Dpenicillamine (SNVP) and S-nitrosoglutathione (GSNO), was carried out by a
combination of electrochemistry and electron paramagnetic resonance. Results
revealed the diazeniumdiolates, DEA/NO and DETA/NO, spontaneously liberate NO
radical in solution. However, GEA-3162 was found to release NO and O₂
concomitantly, and therefore, should be regarded as a ONOO generator. SNVP and
GSNO release only small amounts of free NO in solution, however, the mechanism
of action of these compounds is likely to also involve the transfer of NO⁺.
The effect of each of these compounds on cell viability was investigated in
human monocyte-derived macrophages (Mcp) and an aortic SMC line. In both Mcp
and SMC, only the ONOO" generator, GEA-3162, induced cell death. Analysis by
flow cytometry revealed GEA-3162 caused cell surface phosphatidylserine exposure
characteristic of apoptosis. Only a prolonged delivery of NO radical was able to
genuinely inhibit SMC proliferation. These results demonstrate, that contrary to
previous reports, NO per se is incapable of inducing cell death.
Finally, apoptotic cell death induced by GEA-3162 in human monocyte-derived
M(p was inhibited by pre-treatment of the cells with a low dose of the NO-releasing
compound, DETA/NO. This effect was enhanced by augmenting cGMP levels using
a direct guanylate cyclase stimulator, BAY 41-2272. Pre-treatment with DETA/NO
failed to protect SMC against GEA-3162-induced cell death, irrespective of inclusion
of BAY 41-2272. These results demonstrate that pre-conditioning of Mcp with
cGMP protects them against subsequent cell death.
Taken together, these studies demonstrate that the outcome of any NOinduced response depends on the concentration and precise NO-related species
generated in the microenvironment, as well as the nature of the target cell.
Exploiting these differences as a therapeutic strategy for the treatment of
atherosclerosis may be possible with the advent of cell-specific NO donor