Nitric oxide (NO) is a short lived signalling molecule involved in the regulation of physiological functions including vascular tension, neuroprotection, immune- and endocrine modulation.
The intracellular NO production relies on the activity of three isoforms of the nitric oxide synthase enzyme (NOS), the endothelial (eNOS), neuronal (nNOS) and inducile NOS (iNOS). The expression and activity of the isoforms is finely regulated, for instance by phosphorylation of target residues; as a consequence NOS function may result activated, due to the stabilisation of the dimeric structure, or even uncoupled, with loss of the physiological activity and by O2-. production instead of NO.
Recent evidence suggests that uncoupling of NOS represents a crucial turning point driving important changes in bioenergetics and cell redox homeostasis, with downstream effects on the induction of pathological conditions. This research is aimed at characterizing the NOS function in different cell lines, such as keratynocytes (HaCaT), endothelial (HUVEC) or hepatocytes (HepG2), following treatment with natural/synthetic compounds whose involvement in the NO signalling has already been envisaged. This is the case of lipoproteins, whose abundance and oxidation state have been shown to induce functional changes in endothelium, as well as of the alkylphenols (AP), recognised as environmental contaminants with proposed xenoestrogenic effects.
The project includes experiments in which, following cell incubation with the compounds described, the expression and activity level of the NOS enzymes will be detected by real time PCR and Western blot analysis, with particular attention in evaluating the level of eNOS uncoupling, by analysing the phosphorylation of specific target residues. The amount of the two different NOS catalytic products, i.e.: the phisyological one, NO, and the alternative one: O2-., will be quantified by nitrite/nitrate, ROS and peroxynitrite determination.
The intracellular role of NO has long been known and a number of scientific works may testify how an altered NO signaling represents a common condition found in different pathological states [1,2,6,10-12]. This notwithstanding, the molecular details concerning the biochemical activity of NO and its cell metabolism are still far from being clarified. Opposite effects may be envisaged when NO is acting as a physiological signal transducer, or when environmental conditions induce an alteration of the NO pathways, leading cells to a negative detrimental circle that results in a pathological state.
It is now evident that the intracellular concentration of NO represents a primary aspect in this context, but a number of important elements are still out of the reach of the investigators.
The activity of the different NOS isoforms, responsible for the endogenous NO production, specifically regulated at different levels, can be considered a central issue. eNOS and nNOS activity is normally modulated by substrates availability, dimer stabilisation and binding of cofactors, these last being influenced by aminoacidic modification of target residues, such as phosphorylation; in addition, the negative crosstalk among NOS isoforms may appear paradoxical, although it is an expression of the complexity of the regulatory apparatus. In this context the issue of NOS uncoupling represents a key element in the modulation of NO/ROS production. A scenario can be depicted where an incoming stress or insult rises the intracellular NO by iNOS activation, and joins the O2-. overproduction due to eNOS uncoupling: these are conditions ultimately determining peroxynitrite formation and irreversible cell damage [7-13,19,20].
Cell cultures constitute a suitable system for the study of NOS activity and regulation. Specific cell lines may undergo treatments allowing to mimic different pathological states. Settings may be easily controlled varying time and concentration of the 'stressor'. Following incubations, specific cell parameters can be evaluated in parallel, and compared to the control-untreated cells, in order to determine elements related to NOS expression and activity.
Based on the scientific bio-medical interest, two distinct conditions are meant to be approached by this research proposal.
The first is based on the treatment of endothelial cells with excess of native or oxidised VLDL. These natural aggregates are synthesised by the liver and constitutes the precursor of LDL, whose excess is known to be related to cardiovascular risk and atherosclerosis. Excess VLDL may thus represent a model of an early risk related to an altered lipid metabolism or overload of dietary lipidic component (obesity). At endothelial level, cell function is strictly linked to NO production, and in this context an altered NO signalling may represent a marker of cell dysfunction [12,14,15].
The second approach will pursue the evaluation of the toxic effects exerted by emerging environmental contaminants (the alkylphenols NP and OP) whose patho-physiological mechanisms are still poorly understood. The proposed xenoestrogenic effects of these by-products of the industrial manufacturing and treatment of plastic, grows the interest concerning their potential harmful effect on human health [16-22].
Advanced experimental designs will be exploited in both systems. The mRNA expression level of the different NOS isoforms will be analysed by real time PCR and the relative protein expression will be quantified by immune-detection (Western blot), using antibodies specific for the different NOS isoforms. A key investigation will be given by the characterisation of NOS activity, to be matched with quantification of specific phosphorylated residues. All these experiments will allow us to evaluate the level of NOS uncoupling vs the physiological NO signaling.
In the research here proposed, we focus the charaxterisation of an enzymatic factor (NOS) whose activity lay at branch point between physiological signals and pro-pathological cascade of effects. Simplified models (cell culture) will be adopted in order to clarify the roles carried out by the compound of interest (excess of VLDL native and oxidized, or alkylphenols OP/NP), in inducing cell stress and detrimental responses. Hopefully, the results obtained, will allow to take a step forward in the understanding the molecular mechanisms related to the onset and progression of common diseases like atherosclerosis, neurodegeneration and cancer.
[18] Perez-Albaladejo E, et al., Toxicol. In Vitro 2017vol. 38, 41-8
[19] Hishikawa K., et al., FEBS Lett, 1995 vol. 360, 291-3
[20] Wyckoff M.H. et al., J. Biol. Chem. 2001, vol. 276, 27071-76
[21] Liao TL et al., Ann NY. Acad. Sci, 2015 vol. 1350, 52-60
[22] Chen J et al. Carcinogenesis 1998. vol. 19, 2187-93