Ricerc@Sapienza

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.