Molecular mechanism of regulation of the production of nitric oxide (NO): physiopathological implications in different cell models.

Starting date
December 3, 2002
Duration (months)
24
Managers or local contacts
Suzuki Hisanori

Our recent hypothesis on the cross-talk between constitutive and inducible NO synthase (cNOS and iNOS, respectively) by means of NO pointed out the importance of the modulation of the production of NO by cNOS (neuronal and endothelial NOS (nNOS and eNOS, respectively) in physiopathological conditions. The induction of iNOS expression is finely regulated by both inducers and suppressors. LPS, IFN-gamma, TNF-alfa and IL1-beta, on the one hand, elicit the iNOS expression and, on the other hand, inhibit the cNOS activity. Suppressors of iNOS exptression such as dexamethasone, TGF-beta, estrogen and morphin up-regulte n/eNOS activity. NO itself suppresses the iNOS expression by inhibiting NF-kappaB activation.
The enzymatic activity of n/eNOS is finely modulated by a number of post-transcriptional modifications, including the phosphorylation of n/eNOS. Tyrosine-phosphorylation of both enzymes down-regulates their catalytic activity. Serine-phosphorylation of nNOS by calmodulin-dependent kinase inhibits the catalytic activity of the enzyme. Serine-phosphorylation of eNOS by Akt kinase leads to the activation of its enzymatic. Thus, NO normally produced by n/eNOS reflects the various phosphorylation state of these enzyme. Any condition shifting the equilibrium between native and variously phosphorylated state of n/eNOS may cause a rapid change in the intracellular amounts of NO. According to our hypothesis, this situation could strictly be correlated to the induction of iNOS expression.
In order to obtain a comprehensive view on the importance of the modulation of the production of NO we will study the following experimental models of diseases focusing on the evaluation of the cascade of NO:
1) Ischemia-reperfusion-induced cardiac injury (Research units: Bani and Suzuki)
An accurate study of the change in the amounts of NO, in the enzymatic activity of constitutive
NOS, in the activation of NF-kappaB and STAT1 and in the expression of iNOS mRNA at an early
phase following ischemia/reperfusion in cardiomyocytes will be performed to evaluate the
molecular mechanism of the putative cross-talk between constitutive and inducible NOS.
The effect of relaxin which induces iNOS expression in vascular smooth muscle cells and coronary endothelial cells on a widespread vasodilatation by increasing coronary flow and on counteracting ischemia-reperfusion-induced cardiac injury in in vivo and ex vivo models of myocardial infarction will also be examined.
2) Asthma (Research units: Bani and Suzuki)
As for many inflammatory diseases, iNOS induction plays a crucial role in the pathophysiology of asthma. It could be hypothesized that polyphenols and SOD-mimetic compounds may have a protective action against lung injury due to inflammatory infiltrate in asthma. It is well known that ROS and peroxynitrite may directly damage the DNA of cells in inflamed tissues, thereby inducing the activation of DNA repair enzymes such as poly(ADP-ribose)polymerase (PARP), which plays a crucial role in the inflammatory lung tissue damage in experimental models of acute lung inflammation in rats. Effect of polyphenols, SOD-mimetics and PARP inhibitors on experimental asthma will be examined.
3) Neurodegenerative disease-associated cell death (Research unit: Cantoni), including Alzheimer's disease (Research unit: Colasanti)
The cross-talk between phospholipase A and NOS will be studied in neurodegeneration associated with inflammatory processes.
The presence of neuritic plaques containing beta-amyloid (Abeta) constitutes the major pathohistological finding in Alzheimer's disease (AD). Abeta has been found to stimulate iNOS expression and NO production through a TNF-alfa) receptor-associated factor (TRAF6 and 2), and NF-kappaB-inducing kinase (NIK)-dependent signaling mechanism. The direct effect of intracellular Abeta or its fragments on NOS activity will be examined in the early stages of AD.

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Ministero dell'Istruzione dell'Università e della Ricerca
Funds: assigned and managed by the department

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