Nitric oxide (NO) and hydrogen sulfide (H2S) are two gasotransmitters with important physiological functions. NO exerts many different roles: it is a vasorelaxant, an inflammatory mediator and a neurotransmitter. Similarly, it has been shown that H2S relaxes blood vessels, is involved in inflammation and is a neurotransmitter too. The two gases are also involved in redox signalling reactions, being able to interact with reactive oxygen species (ROS). Interestingly, several pathological conditions, such as cardiovascular diseases and neurological disorders, are characterised by an imbalance in both the levels of . NO and H2S. All these observations contributed to develop the hypothesis of a possible interaction between the two molecules, especially in the cardiovascular system and during inflammation. While . NO biology is very well studied and despite the increasing interest in H2S biology, the mechanisms of action of H2S have not been fully elucidated yet. The aim of this work was to characterise some aspects of the cross-talk between . NO and H2S signalling pathways, with particular attention given to signalling pathways in the cardiovascular system (e.g. NO synthesis and cGMP production) and in inflammation (e.g. inflammasome complex activation). By using . NO derived metabolites of pharmacological interest for cardiovascular diseases (nitrite (N02-) and S-nitrosoalbumin (SNOA)) it was possible to study non-enzymatic H2S-dependent . NO synthesis. N02- and SNOA were reduced to ·NO by H2S donors (such as the sulfide salt NaSH and the slow releasing GYY 4137), as assessed by electron paramagnetic resonance spectroscopy (EPR) and gas-phase ozonebased chemiluminescence. The reactions were also chemically characterised and the effects of H2S-mediated . NO synthesis on smooth muscle and endothelial cells were studied. Endogenous H2S production was shown to significantly increase cGMP synthesis in N02--treated human aortic smooth muscle cells (compared to only N02- treatment). H2S-mediated . NO production from SNOA was also shown to increase nitrosothiol transport through cell membrane and subsequently to increase SNOA antioxidant properties in human microvascular endothelial cells. The study of the cross-talk between . NO and H2S was also carried forward by characterising the post-translational modification (S-nitrosation and sulfhydration) that they cause on key Cys residues of common target protein. S-nitrosation of albumin was performed using incubation with acidified N02- solutions and the presence of the modification on the protein was characterised by mass spectrometry. It was possible to show that SNOA had increased antioxidant capacity compared to unmodified albumin controls. Similarly it was done for H2Sinduced modification. It was possible to demonstrate that H2S was causing the decysteinylation of albumin samples, inducing an increase in albumin antioxidant properties (DMPD assay). Finally, the interaction between the two gases was also studied for key inflammatory processes such as the inflammasome complex activation and induction of IL-113 production. Unfortunately, with the assay used (IL-113 ELlSA) it was not possible to find a clear interaction between . NO and H2S, but it was possible to clarify some of the mechanisms behind H2S biological properties. It was indeed demonstrated the potential of H2S as antiinflammatory mediator by inhibition of inflammasome activation and IL- 113 production. In conclusion, with this work it was possible to demonstrate that H2S and . NO have "overlapping" (at least in part) signalling pathways and that consequently H2S is able to regulate non-enzymatic . NO production in the cardiovascular system. It was also possible to characterise novel post-translational modifications induced by H2S, potentially shedding new light on its biological mechanisms of action. The field of gasotransmitters biology is very exciting and more work is certainly needed to advance our understanding of the key cellular signalling processes that gases like· NO and H2S can modulate.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658029 |
| Date | January 2013 |
| Creators | Lo Faro, Maria Letizia |
| Publisher | Exeter and Plymouth Peninsula Medical School |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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