Nitric oxide, superoxide, and peroxynitrite: Synthesis, interactions, and biological consequences.

The control of nitric oxide (NO) production and its interaction with other free radicals were examined in vitro and in vivo. First, the role of calmodulin in the regulation of inducible NO synthase (iNOS) was assessed. Second, the interaction between NO and superoxide (O2-) and the product of this reaction peroxynitrite (ONOO-) were investigated. Finally, the role of endothelial NO synthase (eNOS) and its regulation by tetrahydrobiopterin (BH4) and GTP cyclohydrolase-I (GTPCH-I) were examined in a mouse model of septic shock. Inducible NOS was expressed in a murine macrophage cell line, J774, and nitrite production measured as an index of NO production. This system was used to examine the effects of calmodulin antagonists on induction and activity of iNOS. Calmodulin antagonists inhibited induction and production of nitrite by J774 cells without significant inhibition of isolated iNOS. The rank order of inhibitory potency of calmodulin antagonists was similar to that on isolated calmodulin activity. Most antagonists exhibited significant cytotoxicity and over a similar concentration range inhibited nitrite production. However, ophiobolin A was identified as a calmodulin antagonist with minimal cytotoxicity that potently inhibited iNOS induction. Once NO is produced it reacts rapidly with target enzymes and other free radical species such as O2 Experiments were designed to determine whether rabbit aortic rings produced O2-. Under basal conditions, O2- measured by chemiluminescence was not detected. Inhibition of endogenous superoxide dismutase (SOD) resulted in detectable basal release, which was further increased, in a concentration-dependent manner, by NADH or NADPH. Incubation of aortic rings with S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP) resulted in concentration-dependent quenching of O2- chemiluminescence that was proportional to the amount of NO released. The interaction of NO and O2- was assessed by monitoring the formation of ONOO- by determining protein tyrosine nitration by Western blot analysis. Under basal conditions and in the presence of NADH, a single band immunoreactive to nitrotyrosine was detected. Incubation of rings with GSNO alone or GSNO and NADH resulted in the appearance of additional nitrotyrosine bands. The requirements for protein tyrosine nitration were further examined using an isolated system in which bovine serum albumin was exposed to various combinations of NO or O2-. Incubation with 3-morpholinosydnonimine (SIN-1; donor of NO and O2-) but not GSNO resulted in marked nitration of albumin, which was reduced in the presence of oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol (O2- generator), resulted in tyrosine nitration. It was concluded that nitrotyrosine formation observed in aortic rings exposed to NO donors is likely to have resulted from the interaction of exogenous NO and basal endogenous O2- resulting in the formation of ONOO-. In conditions such as septic shock induction of iNOS occurs, which results in significant formation of NO and nitrotyrosine. However, in human sepsis, it appears that an up-regulation of eNOS rather than induction of iNOS may occur. To assess the importance of eNOS, a conscious mouse model of sepsis was established using mice deficient in iNOS. Animals were injected with lipopolysaccharide (LPS) and the expression of GTPCH-I measured by Northern blotting. In addition plasma nitrite/nitrate and tissue biopterin levels (as an index of BH4 production) and GTPCH-I activity were measured. The expression of eNOS was not significantly altered by LPS treatment in wild-type and iNOS mutant mice. However, plasma nitrite/nitrate levels were increased by 14- and 2-fold in wild-type and iNOS mutant mice 12 h after LPS, respectively. This was associated with a significant 2.2- and 2.4-fold increase in heart GTPCH-I activity and a 3.8- and 2.8-fold increase in tissue biopterin levels of wild-type and iNOS mutant mice, respectively. Northern blotting showed the presence of two transcripts corresponding to 1.4 and 3.6 kb GTPCH-I mRNA in liver and heart tissue. In summary this thesis investigated the control of NO production and its subsequent interaction with O2 In conditions such as septic shock where enhanced NO production occurs through the inducible or constitutive pathways, therapies designed to inhibit the NO pathway could result in important therapeutic advances