Mechanisms and Biological Significance of S-Nitrosation

Unlike most cellular messenger molecules, which act via specific interactions with ligand binding sites of target protein (enzymes, receptors, membrane transporters), nitric oxide (NO) exerts its effects through chemical reactions with cellular constituents. This renders the biological chemistry of NO an important topic that is essential for the understanding of NO physiology and pathophysiology. The biological activity of NO is determined by its reactions with molecular oxygen (O2 ), superoxide anion (O2 - ), and metal centres of enzymes. The reaction with O2 becomes relevant at pathophysiologically high NO concentrations, resulting in the formation of nitrosative intermediates that may contribute to NO-triggered mutagenesis and other toxic effects. The reaction with O 2 - is very rapid yielding peroxynitrite (ONOO- ) and peroxynitrous acid (ONOOH), highly reactive species leading to oxidation of various biomolecules and to nitration of tyrosine residues in proteins. The reactivity of ONOO - /ONOOH is thought to contribute to tissue injury associated with overproduction of NO and O 2 - in several cardiovascular, neuronal and other diseases. Finally, the prosthetic heme group of soluble guanylate cyclase (sGC) is a prominent example for protein metal centers that are affected by NO. NO binds at nearly diffusion-controlled rates as axial ligand to the heme iron, resulting in the activation of sGC and accumulation of the cellular messenger cyclic GMP, which regulates biological processes as diverse as vascular relaxation, platelet aggregation and synaptic plasticity of the brain. The current project is based on previous work showing that (i) the biosynthesis of NO is often accompanied by the production Of O2 - and that (ii) the generation of ONOO - from NO and O2 - is largely outcompeted by GSH, a thiol present at high concentrations in the cytoplasm of all mammalian cells. GSH is converted in a fairly efficient manner by NO/O 2 - to the corresponding S-nitroso compound (GSNO), which may function as relatively stable storage and transport form of NO but can also release NO through enzymatic and non-enzymatic mechanisms. In the course of this project three basic questions will be addressed that are related to the bio-chemistry and pharmacology of GSNO, and other biologically relevant S-nitrosothiols: 1. How does NO/O 2 - nitrosate thiols? This work should contribute to the understanding of the molecular mechanisms underlying (i) the biosynthesis of S-nitrosothiols in vivo and (ii) the vasorelaxant effects of sydnonimine-based drugs like molsidomine used clinically to treat coronary artery disease and myocardial infarction. 2. What is the metabolic fate of GSNO in cells? This part of the project is expected to identify (i) membrane transport systems involved in the cell-to-cell transfer of GSNO, (ii) biologically relevant pathways of NO release from GSNO, and (iii) novel drugs interfering with these processes in vivo. 3. Does the L-arginine/NO pathway trigger formation of GSNO in cells? In this part of the project a new, very sensitive analytical method will be used to study whether the physiological activation of NO synthase, leading to accumulation of tissue cGMP, does indeed involve nitrosation of cellular GSH.

Nitric oxide (NO) is a wide-spread biological signal molecule which is involved in biological processes as diverse as regulation of blood pressure, platelet aggregation, neurotransmission in the brain, penile erection, and immune defense against invading pathogens. Besides these important physiological functions, overproduction of NO may be deleterious and contribute to tissue injury in various inflammatory, infectious, and neurodegenerative diseases. Unlike other cellular messenger molecules, which act via specific interactions with ligand binding sites of target protein (enzymes, receptors, membrane transporters), nitric oxide (NO) exerts its effects through chemical reactions with cellular constituents, rendering the biological chemistry of NO important for the understanding of NO physiology and pathophysiology. In this project we studied the mechanism and biological significance of a particular chemical reaction of NO, called S-nitrosation, which, resembling the well known N-nitrosation reactions leading to the formation of carcinogenic nitrosamines, may confer potentially harmful modifications of cellular constituents containing essential sulfhydryl moieties (proteins or low-molecular mass thiols such as glutathione). In particular, we were interested in the biological conditions favoring S-nitrosation reactions and the interplay between S-nitrosation and the physiological actions of NO mediated by the most prominent target of NO, an enzyme called soluble guanylate cyclase, which converts GTP to the cyclic nucleotide cyclic GMP (cGMP) when activated by NO. The most important result we obtained refers to the potential mechanisms of cellular S-nitrosation. We identified two significant pathways of S-nitrosation, one triggered by NO autoxidation, the other triggered by co-generation of NO and superoxide anions. The autoxidation pathway takes place in cells in the presence of oxygen under conditions of sustained overproduction of NO, as occurring in inflamed tissues or upon induction of NO synthesis by bacterial infections. Using sophisticated biophysical techniques, we were able to demonstrate that nitrogen dioxide radical is the major active species which triggers S-nitrosation reactions in the course of NO autoxidation. The second pathway we have identified occurs at much lower NO concentrations but requires the additional presence of superoxide radicals, which are well known reactive species generated in inflamed tissues. According to our results, NO and superoxide combine to form the potent oxidant peroxynitrite, which oxidizes thiol anions to the corresponding thiyl radicals which rapidly react with NO to form S-nitrosothiols. So far, our studies with soluble guanylate cyclase did not provide evidence for an involvement of S-nitrosation in the physiological functions of NO, suggesting that drugs interfering with S-nitrosation could be therapeutically useful for the treatment of diseases associated with NO overproduction and nitrosative stress. The beneficial effects of several lead compounds, in particular polyphenolic scavengers of nitrogen dioxide radicals, are currently studied in our laboratory.