Effect of Previous Exposure to Nitric Oxide on Cyclooxygenase Signaling Pathway

This dissertation, "Effect of Previous Exposure to Nitric Oxide on Cyclooxygenase Signaling Pathway" by Hoi-ling, Angela, Chan, 陳海寧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract Endothelium can release endothelium-derived relaxing factor (EDRF), in which nitric oxide (NO) is the best characterized one. NO activates the soluble guanylyl cyclase (sGC) in the smooth muscle cells, increases the synthesis of cyclic guanosine-3', 5-monophosphate (cGMP) and leads to subsequent vascular relaxation. Besides, endothelium can release endothelium-derived contracting factor (EDCF). Prostanoids are among the major EDCF to cause contraction of underlying smooth muscle and they are products from the cyclooxygenase (COX) signaling pathway. The present study aims to examine the effect of previous exposure to NO on COX signaling pathway in endothelial cells. In addition, experiments were designed to determine whether or not oxidative stress, which is a common feature of different cardiovascular diseases, affects the interaction between NO and COX signaling. Human umbilical vein endothelial cells (HUVECs) were treated with L-NAME (inhibitor of NO synthases; 100 μM), sodium nitroprusside (SNP; nitric oxide donor; 1 μM), 8- Bromo-cGMP (analogue of cGMP; 100 μM), ODQ (sGC blocker; 10 μM) and/or hydrogen peroxide (H O; to induce oxidative stress; 1 μM) for 30 minutes or 24 2 2 2+ hours, followed by stimulation with or without A23187 (Ca ionophore; 1 μM). To determine the effects of different pharmacological treatments on the release of prostanoids, namely prostacyclin (PGI ), prostaglandin F (PGF ) and thromboxane 2 2α 2α iv A (TXA ), the concentration of their stable metabolites 6-keto prostaglandin F 2 2 1α (6-keto PGF ), PGF and thromboxane B (TXB ) in the medium incubating the cells 1α 2α 2 2 were measured with ELISA kits. The mRNA expressions of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and endothelial nitric oxide synthase (eNOS) were also measured by performing reverse transcriptase polymerase chain reaction assay. Results showed that pre-incubation of HUVECs with SNP or 8-bromo-cGMP for 24 2+ hours inhibited the production of PGI by the Ca ionophore A23187. ODQ pre-incubation for 24 hours inhibited A23187-induced release of PGI and had no effect on the inhibitory effect of SNP on PGI production H O pre-incubation for 24 2 . 2 2 2+ hours reduced the production of PGI by Ca ionophore, but increased the production 2+ of PGF of HUVECs that were not stimulated with Ca ionophore. In the presence of 2α H O, SNP and 8-bromo-cGMP no longer inhibited the production of PGI after 24 2 2 2 hours of incubation. Experiments to measure mRNA expression of COX-1 and COX-2 did not provide any clear pattern on the effects of different pharmacological treatments; whereas the mRNA bands for PGIS and eNOS were not detected. The present study confirms that prior long-term activation of NO signaling pathway (by 24 hours of incubation with SNP or 8-bromo-cGMP) can inhibit the release of COX product (PGI ). While H O activates the COX signaling pathway, it appears to 2 2 2 v suppress further activation of COX signaling pathway due to increase intracellular calcium concentration. Therefore, the present work provides the scientific information relating the detrimental effect of reactive oxygen species in the vascular function, and suggests that activation of NO signaling pathway with pharmacological agents, as an approach to inhibit endothelium-dependent contractions, is unlikely to be effective in conditions wi