Author: Aviral Vatsa
Nitric oxide (NO) is a short-lived, highly reactive, free radical which is ubiquitously present in the human body. Physiologically, it is widely used as a second messenger both an inter-cellular and intra-cellular signaling molecule. NO is produced when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS. Neuronal NOS and endothelial NOS are calcium-dependent enzymes, whereas inducible NOS is a calcium-independent inducible enzyme, that is activated and upregulated by cytokines during inflammatory processes. The tissue-specificity indicated in the names is not absolute as these subtypes have been discovered in wider locations in the body.
In bone, NO plays a vital role in mechanosensation and mechanotransduction. Osteocytes are widely accepted as the ‘professional’ mechanosensors in bone. They sense external mechanical loads on bone and produce chemical signals such as NO and prostaglandins. NO in turn has been shown to modulate the activity of both bone forming osteoblasts and bone resorbing osteoclasts. NO is essential for load-induced bone formation in vivo. Studies using single gene deletions have shown that NO is an important cog in the wheel for bone metabolism and bone remodelling. Although eNOS isotype is widely implicated in NO production in bone, but recent studies indicate that iNOS isotype might also be involved in NO production in bone in response to mechanical loading. Targeted deletion of eNOS shows mild osteoporotic phenotype in mice and iNOS pathway has been implicated in L-1-induced osteoclastic bone resorption. Hence both NOS isoforms have important role in bone remodelling.
Challenges to study NO: NO is a small, short-lived signalling molecule. It has a half life of less than 5 sec, which makes its online detection very difficult. Predominantly, the more stable metabolites of NO such as nitrites and nitrates are detected by using techniques such as Greiss reagent. They are however lited by the sensitivity levels and their inability to detect actual levels of NO. However, fluorescent dyes such as DAR 4M and DAF dyes are potent tools to detect online NO production at single cell level. These dyes are membrane-permeable, hence are taken up readily by the cells. Once inside the cell they are metabolised and rendered membrane-impermeable. When cell produces NO these dyes trap NO and get converted into fluorescent product, which can then be detected by using fluorescence microscopy. Moreover, by using these techniques, quantitative analyses of NO production (not only its metabolites) is feasible in live, single cells.
Molecular methods to investigate mRNA or protein levels of NOS enzymes are also used to corroborate with the changes in NO production levels.
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