Date of Award

2011

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Lindon Young, PhD

Second Advisor

Qian Chen, PhD

Third Advisor

Brian Balin, PhD

Fourth Advisor

Marcus Bell, PhD

Abstract

Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte-endothelial interactions associated with vascular injury. However, when endothelial-derived NO is decreased, endothelial dysfunction results and promotes inflammation characterized by increased leukocyte-endothelial interactions. Under normal conditions, eNOS produces NO in the presence of an essential cofactor, tetrahydrobiopetrin (BH4) by facilitating the reduction of molecular oxygen to L-arginine oxidation and generation of L-citrulline. Whereas uncoupled eNOS refers to the electron transfer that becomes uncoupled to L-arginine oxidation and therefore superoxide (SO) is generated when the dihydrobiopetrin (BH2) to BH4 ratio is increased. SO is subsequently converted to hydrogen peroxide as a result. However, the role of uncoupled eNOS promoting endothelial dysfunction and leukocyte-endothelial interactions in not well characterized in vivo. This study examined the role of eNOS uncoupling by superfusing BH2 (100 or 200 μM) by itself and BH2 (100 μM) combined with BH4 (100 or 250 μM) in rat mesenteric venules on leukocyte rolling, adherence, and transmigration by using intravital microscopy. The effects of BH2 were compared to Krebs’ buffer, to NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 50 μM), and to the combination of BH2/BH4. We found that superfusion of BH2 (100 μM n=6, 200 μM n=6, both P