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It has been demonstrated that nitric oxide (NO) has protective and harmful effects. This molecule is essential for numerous physiological activities, but it also has a role in various pathological situations, including neurological illness and cancer. Cytochrome oxidase (COX), the terminal enzyme of an electron transport chain (ETC), is a primary target of Nitric Oxide. NO reversibly binds to COX to control cellular respiration.
Although Nitric Oxide’s potential to change biological molecules is physiologically relevant, NO-dependent modifications, such as S-nitrosylation of proteins or even peroxynitrite generation, may lead to cell death. Prolonged NO exposure inhibits complex I of the ETC, most likely via S-nitrosylation. The inhibition of complex I by NO results in an increased discharge of oxidants from the ETC. In the presence of NO, an increase in oxidants promotes the creation of the highly reactive compound peroxynitrite, which can trigger p38 and c-Jun N-terminal kinase (JNK) to launch the intrinsic apoptotic pathway.
Nitric oxide can influence apoptotic signalling at numerous stages along the pathway:
1) Nitric Oxide can modulate the expression of death receptors in a cGMP-dependent way.
2) Nitric Oxide can change the expression of proteins such as acid sphingomyelinase, which aids in the regulation of early signalling events in death receptor signalling, such as ceramide formation. This decreases the creation of DISCs (Death Inducing Signalling Complexes) and is likewise mediated by the synthesis of cGMP.
3) Nitric Oxide can directly impact caspase action by nitrosylation of the active site, resulting in protein function suppression.
4) The Nitric Oxide caspase activity and effect upon the formation of DISC will degrade the cleavage of the bid, resulting in the deduction of amplification of apoptotic signalling via the mitochondria.
5) Nitric Oxide also has the potential to affect the expression of multiple Bcl-2 protein families and their members of the pro-and anti-apoptotic proteins. It can also affect the release of cytochrome C and other components from mitochondria and is also mediated via cGMP synthesis.
6) The activation of the anti-apoptotic protein cFLIP can potentially influence DISC formation. PKC is one of the proteins that can alter cFLIP recruitment to the DISC, and Nitric Oxide can modulate its activity via nitrosylation.
Cell death quantification
Cardiac myocyte mortality was measured using calcein-AM, a membrane-permeant dye that is not fluorescent until cellular esterases break the acetoxymethyl group in live cells (1, 39, 66). Cells were plated in 96-well plates at a density of 50,000 cells/well and left untreated (these cells served as control live cells) or treated with ethanol (40 percent; these cells served as control dead cells) or the appropriate quantities of S-nitroso-N-acetylpenicillamine (SNAP, Sigma, St. Louis, MO). After 4 hours of treatment (ethanol was introduced to wells 20 minutes before experiment termination), calcein AM (2 M final concentration; Molecular Probes, Eugene, OR) was applied to all wells.
In conclusion
To visualise myocytes directly, cells were plated on chamber slides with calcein AM and ethidium homodimer (4 M, Molecular Probes; this was always for nuclear identification); fluorescent images were acquired to use an Olympus System Microscope (model FLA-BX) with simultaneous excitation filters set at 485 and 530 nm to visualise ethidium-stained nuclear material and calcein-stained cytoplasm concurrently.
