No description available.

Tetrahydrobiopterin (BH4) is a limiting cofactor for nitric oxide synthase (NOS) catalysed conversion of L-arginine to nitric oxide and citrulline. Content of BH4 in mammalian cells is regulated at many levels, but most important is de novo biosynthesis from GTP. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for the de novo synthesis of BH4. While various immunostimulants, hormones and growth factors have been reported to increase GTPCH mRNA levels and intracellular biopterin (BH4 degradation product), it is not known whether these factors act at the level of GTPCH gene transcription. To test this I utilised 1, 3 and 6 kb 5'upstream GTPCH gene sequence in a secreted alkaline phosphatase reporter vector (SEAP). These constructs were stably transfected in PC-12 cells and rat aortic smooth muscle cells, and the cells were treated with various immunostimulants and growth factors in order to determine whether these factors could enhance GTPCH gene transcription. Intracellular biopterin levels were also measured to confirm that the upregulation of the SEAP-reporter correlated with a rise in biopterin. Our investigations conclude that transcriptional regulation of the GTPCH gene is indeed a major site for control of intracellular BH4 levels. In further experiments, we have characterised the binding of [3H]BH4 to endothelial NOS (eNOS) and examined influences of the substrate, arginine, on the BH4 binding. In addition we selected tetrahydropterins (that support NOS catalysis) and dihydropterins (that are catalytically incompetent) to determine the extent to which modifications of BH4 alter pterin binding affinity to eNOS. Dihydropterins are unable to support NOS catalysis. Studies showed for the first time that dihydropterins, but not tetrahydropterins, support superoxide generation by eNOS. We also have determined that eNOS may be able to produce NO in the absence of BH4 cofactor from the reaction intermediate hydroxyarginine. We have characterised this reaction and are able to provide a plausible mechanism for the NOx generation from eNOS in the absence of BH4 cofactor.

Electrocatalytic reduction and oxidation of nitric oxide (NO) using cobalt phthalocyanine complexes have been studied and compared to vitamin B₁₂ and other metallophthalocyanine (MPc) complexes. Modifying a glassy carbon electrode with these complexes resulted in improved sensitivity of the electrode allowing detection of NO to 10⁻⁹ mol dm⁻³. The mechanisms of catalysis were studied. Electrocatalysis of NO involves coordination of NO to the MPc complex. Hence catalytic activity is affected by the nature of the metal center. However coordination of NO to the MPc complex has to be reversible to eliminate poisoning of the electrode. Though FePc gave the best sensitivity and lowered the reduction potential more than CoPc, the strong Fe-NO bond resulted in the poisoning of the electrode hence, rendering the electrode unstable. Rate constants for NO coordination to the MPc complexes were studied. These rates were smaller than the studied NO porphyrin coordination rates. Electrocatalytic reduction of NO using MPc complexes involves a transfer of an electron from the metal center to the NO ligand. Hence, substitution of electron-donating grohps on the cobalt pthalocyanine complex resulted in improved sensitivity and catalytic activity. A CoPc modified microelectrode (11μm) was used to monitor NO in human blood components and to detect NO in a rat brain. Detections of NO were also done in aqueous solutions in the presence of interfering species such as dopamine and serotonin. An interaction between NO and serotonin was observed.

[No abstract available] / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

No description available.

No / Elevated plasma concentrations of the sulphur-containing amino acid homocysteine (Hcy) is associated with increased risk of atherosclerosis and arterial thrombosis. The mechanism by which Hcy exerts these effects has yet to be fully elucidated, although a variety of possible mechanisms have been proposed, including endothelial dysfunction or haemostatic abnormalities. However, the influence of Hcy on platelets, cells central to the atherothrombotic process, has never been addressed directly in patient studies. Here, the influence of mild hyperhomocysteinaemia (hHcy) on platelet function was explored in patients with peripheral occlusive arterial disease as evidence by intermittent claudication. Claudicants (n=39) were assigned to one of two subgroups depending on their plasma Hcy concentrations. hHcy claudicants had plasma Hcy concentrations of 18.9±1.0 ¿M (n=24), compared to 11.3±0.5 ¿M for normohomocysteinemic (nHcy) claudicants (n=15) and 12.6±0.7 ¿M for age-matched controls (n=15). Platelet function was evaluated ex vivo in both groups and compared to age-matched controls. Platelet activation and sensitivity to nitric oxide-mediated inhibition was assessed by platelet fibrinogen binding and P-selectin expression. At low concentrations of adenosine diphosphate (ADP; 0.1 ¿M) and thrombin (0.02 U/ml), platelets from hHcy claudicants were more reactive than those from age-matched controls, but not nHcy claudicants. Agonist-induced P-selectin expression was significantly raised in hHcy claudicants compared to all other groups. Interestingly no differences were observed between nHcy claudicants and age-matched controls, indicating that claudication per se did not affect platelet function. Since platelet activity in vivo is determined by the exposure to both agonists and antagonists, we subsequently tested the sensitivity of platelets to inhibition by nitric oxide (NO), using the same platelet markers. Platelets from hHcy claudicants were significantly less sensitive to GSNO (1¿100 ¿M)-mediated inhibition than all other groups. GSNO (1 ¿M) induced 42.6±10 and 39±11.5% inhibition of ADP-induced fibrinogen binding for the nHcy claudicants and age-matched controls, respectively. However, in hHcy claudicants only 16.4±9.7% inhibition was observed, significantly less than the other groups (P<0.01). Again no differences between nHCy claudicants and controls were observed. These results suggest the presence of claudication alone does not influence platelet function but if complicated with mild hyperhomocysteinemia, the sensitivity to agonists is increased, and more importantly, their sensitivity to inhibition is greatly reduced. The overall effect would be an increased propensity for platelet activation. The presence of even mildly elevated plasma Hcy could dramatically increase thrombotic risk.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Nitroglycerin and isoamyl nitrate have been used as nitrovasodilators since the nineteeth century. However, not until recently has it been known that these compounds were useful since they promoted the release of NO in the body. More recently, a new class of drugs, NO donors, has been developed. These include S-nitrosothiols (RSNO), sydnonimines, and nucleophilic NO adducts.

一氧化氮（NO）和內皮源性超極化因子（EDHFs）是內皮衍生的血管舒張因子兩大類。 EETs是構成EDHFs的主要類型，這是由花生四烯酸通過細胞色素P450 （CYP）表氧化酶的催化活性得到。雖然這兩個EET和NO誘導血管舒張，從而降低血壓，許多報告表明，NO對EET引起的血管舒張起抑製作用。然而，不管它的重要性，有關一氧化氮對EETs的抑制作用的機理尚未完全了解。 / 在本研究中，我調查了一氧化氮對EET的負調控。通過膜電位和動脈張力測量，我們發現， 11,12-EET可引起內皮剝脫豬冠狀動脈平滑肌細胞膜超極化和血管舒張。該反應被S-亞硝基-N-乙酰青黴胺（SNAP）和8-Br-cGMP，一個NO的供體和cGMP的膜穿透物類似物，分別抑制。 SNAP和8-Br-cGMP對11,12-EET引起的細胞膜超極化和血管舒張的抑製作用被羥鈷胺，一氧化氮清除劑; ODQ ，鳥苷酸環化酶抑製劑;和KT5823 ，蛋白激酶G（PKG）抑製劑逆轉。 SNAP和8-Br-cGMP對EET反應的抑製作用也被過度供應外源性激酶底物， TAT-TRPC1S¹⁷²和TAT -TRPC1T³¹³廢除。羥鈷胺，ODQ， KT5823， TAT -TRPC1，和TAT -scrambled獨自使用不影響11,12-EET引起的細胞膜超極化和血管舒張作用。然而，獨自使用14，15-EEZE（EET的拮抗劑）抑制了11,12-EET的作用。 此外，磷酸化試驗表明， PKG可以直接在Ser172和Thr313位點磷酸化TRPC1 。此外，TRPV4 ， TRPC1 ，或KCa1.1被選擇性地抑制時，11,12-EET未能引起細胞膜超極化和血管舒張。免疫共沉澱研究表明， TRPV4 ， TRPC1和KCa1.1物理上彼此相關聯。 / 以上結果表明，NO-cGMP-PKG通路可通過TRPC1的磷酸化來抑制11,12- EETs在冠狀動脈血管平滑肌細胞上的作用。此外，TRPV4，TRPC1和KCa1.1參與11,12-EET誘導平滑肌超極化和血管舒張，他們可能互相關聯。從本研究的結果表明，NO和cGMP可通過PKG-介導的TRPC1的磷酸化，抑製EET誘導的平滑肌超極化和血管舒張。 / Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHFs) are two main classes of endothelium-derived vascular relaxant factors. EETs constitute a major type of EDHFs, which are derived from arachidonic acids via the catalytic activity of cytochrome P450 (CYP) epoxygenases. Although both EET and NO induce vascular relaxation, thus reduce blood pressure, numerous reports demonstrated that NO exerts an inhibitory action on EET-induced vascular relaxation. However, despite of its importance, the mechanisms related to the inhibitory effects of NO on EETs are incompletely understood. / In the present study, I investigated the scheme for negative regulation of NO on EET action. Through measurements of membrane potential and arterial tension, we showed that 11,12-EET could induce membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. The responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, a NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, a NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-Br-cGMP on EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with excessive supply of exogenous PKG substrates, TAT-TRPC1S¹⁷² and TAT-TRPC1T³¹³. Hydroxocobalamin, ODQ, KT5823, TAT-TRPC1 and TAT-scrambled alone has no effect on 11,12-EET-induced membrane hyperpolarization and vascular relaxation. However, 14,15-EEZE (a selective EET antagonist) alone inhibits the action of 11,12-EET. Furthermore, phosphorylation assay was performed and it demonstrated that PKG could directly phosphorylate TRPC1 at Ser¹⁷² and Thr³¹³. In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1 and KCa1.1 physically associated with each other in smooth muscle cells. / Taking together, our findings demonstrated that the NO-cGMP-PKG pathway may act through the phosphorylation of TRPC1 to inhibit the action of 11,12-EETs in coronary arterial smooth muscle cells. Furthermore, TRPV4, TRPC1 and KCa1.1 are critically involved in the 11,12-EET-induced smooth muscle hyperpolarization and relaxation and that they may physically associate with each other. The results from this study demonstrated that NO and cGMP could lead to PKG-mediated phosphorylation of TRPC1, resulting in an inhibition of EET-induced smooth muscle hyperpolarization and vascular relaxation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Peng. / "Ca" on title page is subscript. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 115-133). / Abstracts also in Chinese.