Vitamin C (ascorbate or Asc) is an essential vitamin for humans. The transport of oxidized ascorbate occurs via Na^+-dependent vitamin C transporters (SVCTI/SVCT2). Ascorbate is a powerful antioxidant that may be beneficial in scavenging the reactive oxygen species associated with cardiovascular diseases. The objectives of this thesis were: to identify the SVCT isoform(s) expressed in pig coronary artery smooth muscle and endothelium, to determine if preloading cultured pig coronary artery smooth muscle cells with ascorbate protects them against oxidative stress, and to overexpress SVCT2 in these cells to see if an increase in ascorbate reserve helps protect the cells even more. Pig coronary artery smooth muscle tissue and cells cultured from the same tissue express SVCT2 and not SVCT1. Cultured pig coronary artery endothelial express SVCT2, however the limited amount of fresh endothelium isolated, restricted us from determining the isoform present in the fresh tissue. Ascorbate preloading (200 (mu)M overnight) did not decrease the damage caused by hydrogen peroxide as measured by oxidation of dichlorodihydrofluorescein diacetate or mitochondrial reductase activity. The mRNA and ^14C-Asc uptake was marginally greater in pig coronary artery smooth muscle cells stably transfected with a linear pcDNA3.1 SVCT2 plasmid than mock transfected controls. The ^14C-Asc uptake was 1.5 times greater than mock transfected cells after 60 min. A new SVCT2 plasmid, that contained SVCT2 coding region only, did not show greater ^14C-Asc accumulation compared to the plasmid that had the entire SVCT2 cDNA in transiently transfected HEK293T cells. This thesis is a beginning towards further study on the molecular and physiological role ascorbate plays in the coronary artery. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23258 |
| Date | 08 1900 |
| Creators | Best, Kelly |
| Contributors | Grover, A. K., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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