Date of Award

2001

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

The role of nitric oxide (NO) in the physiological system is complex. It functions as a signaling molecule in the neuronal, vascular and immunological systems. To date information regarding NO and its metabolites are plentiful offering various mechanisms of actions, regulation, transport and storage. One such important aspect is the discovery of S-nitrosothiols (RSNO). These thiol bound NO compounds act as storage and transport forms for NO while eliciting similar responses as NO. We developed a novel probe, N,N-didansylhomocystine, for the detection of RSNO influx into live cells. Utilizing the short range excited state energy transfer properties observed in N-dansyl-S-nitrosohomocysteine, we were able to monitor influx of S-nitroso-albumin (BSA-NO) and S-nitrosoglutathione in endothelial cells and fibroblasts. The KM values for RSNO uptake were 20--30muM. Furthermore, cell surface PDI (csPDI) was implicated in catalyzing denitrosation reactions at the cell surface and the subsequent transfer of NO into the cell. The reaction of NO and O2 was accelerated in the cell membrane producing N2O3 which was shown as the nitrosating agent of the intracellular thiols. Continued studies revealed the pathogenic effect of oxidized and reduced homocyst(e)ine (HCys, HCys2) in the vascular system was attributed to its effects on NO and RSNO metabolism. HCys and HCys2 inhibited L-Arg uptake and reduced the production of NO in endothelial cells. HCys/HCys 2 also inhibited uptake of RSNO in a csPDI dependent manner. In vitro studies revealed that the denitrosation activity was sensitive to HCys2 concentrations while isomerization activity was not affected. Therefore the pathophysiological role of HCys/HCys2 affects NO/RSNO metabolism. Decreasing endogenous levels of NO and decreasing denitrosation of extracellular RSNO, tend to promote cell-cell aggregation and cell-matrix aggregation. Leaving the isomerization activity intact promotes the csPDI-catalyzed binding of integrins to cell adhesion molecules. Its pro-aggregatory effects may explain the thrombotic and atherogenic behaviour observed in hyperhomocysteinaemia. An innovative study was designed to use Raman spectroscopy to probe biological samples. We were able to detect glutathione and S-nitrosoglutathione levels in solution with detection limits of 200mM. By derivatizing thiols with 5,5 '-dithio-bis-3-nitrobenzoate we were able to achieve sensitivities in the low mM range. Final attempts to observe thiols in live cells and model systems such as liposomes and red blood cells were not successful. This study has shown the possibilities and limitations of Raman spectroscopy with respect to its use in biological samples.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2001 .R35. Source: Dissertation Abstracts International, Volume: 63-04, Section: B, page: 1831. Thesis (Ph.D.)--University of Windsor (Canada), 2001.

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