OBJECTIVE - Exercise training has been shown to restore vasodilation to nitric oxide synthase (NOS) activation in arterioles distal to coronary artery occlusion. Because reactive oxygen species are generated during NOS uncoupling and the production of vasodilator H2O2 is increased during exercise in patients with coronary disease, we proposed that H2O2 may contribute to the restoration of vasodilation in porcine coronary occlusion model. METHODS AND RESULTS - Left circumflex (LCX) coronary artery of miniature swine was progressively occluded for 8 weeks followed by exercise training (EX; 5 days/wk treadmill) or sedentary (SED) protocols for 12 weeks. Arterioles were isolated from distal LCX and nonoccluded left anterior descending (LAD) artery for in vitro study. Vasodilation to NOS activators adenosine and ionomycin was impaired in SED LCX, but not LAD, arterioles. This impairment was restored by L-arginine. NO production induced by adenosine was also reduced in SED LCX arterioles. EX had no effect on LAD arterioles but improved NO production and restored dilation of LCX arterioles. NOS blockade (L-NAME) inhibited vasodilation to NOS activators in LAD (SED & EX) arterioles but was ineffective in SED LCX arterioles. In EX LCX arterioles, vasodilation to NOS activators was slightly inhibited by L-NAME but abolished by catalase. H2O2 production was markedly increased by adenosine in EX LCX arterioles. CONCLUSIONS - This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H2O2, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion.

C-reactive protein (CRP) is made in liver and its serum concentration increases in inflammation. Measurement of serum CRP is recommended for use as an indicator of inflammation and predictor of atherosclerosis. Cholesterol-lowering drugs statins also lower CRP. To evaluate statin-mediated CRP reduction and to reassess clinical usefulness of CRP, we investigated regulation of CRP gene expression. Here, we show that pravastatin and simvastatin prevent the induction of CRP expression in human hepatoma Hep3B cells exposed to proinflammatory cytokines IL-6 and IL-1β. The nitric oxide (NO) donor, sodium nitroprusside, also prevented the induction of CRP expression while the CRP inducers IL-6 and IL-1β were present with the cells. The effect of NO on CRP expression was at the level of transcription. These findings suggest that the decrease in CRP level in vivo after statin-treatment does not necessarily reflect absence of inflammation, and that NO-releasing drugs have the potential to reduce serum CRP levels. Thus, the measurement of serum CRP levels alone in individuals on statin/NO-therapy is not as useful as was imagined.

Objective: Determine if aspirin inhibits the IL-1β-stimulated expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and prostaglandin E2 (PGE2) in rat ovarian dispersates cultured in vitro. Design: Prospective, controlled in vitro study. Setting: Academic research laboratory. Animals: Ovaries collected from immature rats. Intervention(s): Ovaries were collected from immature rats and enzymatically dispersed. Ovarian dispersates were placed into plates containing media alone or media supplemented with IL-1β (100 U/mL) and varying concentrations of aspirin (0, 1, 3, 5 and 10 mM). Ovarian dispersates were cultured in a humidified environment of 5% CO2 in air at 37°C for 24 or 48 hours. Main Outcome Measure(s): Twenty-four- and 48-hour iNOS, nitrite (a stable metabolite of NO), and PGE2 levels were determined from ovarian dispersates cultured in vitro. Result(s): Administration of IL-1β increased nitrite and PGE2 levels over that observed in the control group after culture of ovarian dispersates for 24 and 48 hours. Aspirin dose dependently reduced the IL-1β-stimulated increase in nitrite production from ovarian dispersates after culture for 24 and 48 hours. Aspirin completely (24 hours) or dose dependently (48 hours) prevented the IL-β-stimulated increase in PGE2. Coadministration of IL-1β and aspirin (10 mM) attenuates IL-1β-stimulated iNOS expression after culture for 24 and 48 hours. Conclusion(s): Aspirin significantly inhibits the IL-1β-stimulated expression of iNOS, NO, and PGE2 in ovarian dispersates cultured in vitro.

Nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two electronreduction of NO to N2O (2NO + 2H+ + 2e- → N2O + H2O) as part of the process ofdenitrification, the step-wise reduction of nitrate to dinitogen. The NOR-catalyzedreaction is central in the nitrogen cycle, since in this step the N=N double bond isformed. NOR is a deviant heme copper oxidase, located in the cytoplasmic membrane,containing four redox active cofactors. Like cytochrome c oxidase (CcO), NOR canreduce oxygen to water as a side reaction, but in contrast to CcO it does not contributeto the proton motive force that drives the conversion of ADP to ATP by ATP synthase.The active site in the catalytic subunit NorB consists of a non-heme iron FeB and a hemeb3 that are anti-ferromagnetically coupled. Additionally a low-spin heme b in NorB isinvolved in accepting electrons from heme c of NorC, a membrane anchored cytochromec, which is the second subunit of the purified NorBC heterodimer.We have studied the terminal region of the proton entry channel and possible ligands tothe binuclear active site of NOR using the flow-flash technique and could demonstratethat the putative proton channel residues Glu(E)198 and E267 in NorB are essential forproton uptake. We propose that they define the terminal proton channel region close tothe binuclear site. An alanine variant of the fully conserved amino acid residue E202 ofNOR that, according to the model of NOR (47), is located in the vicinity to the active site,is neither essential for catalytic activity nor integrity of the active site.Furthermore, we were able to demonstrate the [NO] dependency of NOR in the reactionbetween fully reduced protein and NO using the flow-flash technique (21, 24). Thebinding of NO to the fully reduced enzyme is clearly concentration dependent,inconsistent with a previously proposed obligatory binding of NO first to FeB before itligates to heme b3, where it, in the first turnover, is reduced by the electrons from theactive site. Further oxidation involves electron transfer from the low-spin hemes, which isaccelerated at lower [NO]. This acceleration at lower substrate concentration is evenlarger at decreased pH. We could demonstrate that substrate inhibition, observed insteady-state measurements, occurs already on oxidizing the fully reduced enzyme,indicating that NO binds to its inhibitory site before electrons can redistribute to theactive site from the low-spin hemes.

A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection).

Indiana University-Purdue University Indianapolis (IUPUI) / Stimulation of N-­methyl-­D-­aspartic acid receptors (NMDARs) and the resulting activation of neuronal nitric oxide synthase (nNOS) are critical for fear memory formation. A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear memory, but they also affect many other CNS functions. Following NMDAR stimulation, efficient activation of nNOS requires linking nNOS to a scaffolding protein, the postsynaptic density protein 95 (PSD95). We hypothesized that PSD95-­nNOS interaction in critical limbic regions (such as amygdala and hippocampus) during fear conditioning is important in regulating fear memory formation, and disruption of this protein-­protein binding may cause impairments in conditioned fear memory. Utilizing co-­immunoprecipitation, electrophysiology and behavioral paradigms, we first showed that fear conditioning results in significant increases in PSD95-­nNOS binding within the basolateral amygdala (BLA) and the ventral hippocampus (vHP) in a time-­dependent manner, but not in the medial prefrontal cortex (mPFC). Secondly, by using ZL006, a small molecule disruptor of PSD95-­ nNOS interaction, it was found that systemic and intra-­BLA disruption of PSD95-­ nNOS interaction by ZL006 impaired the consolidation of cue-­induced fear. In contrast, disruption of PSD95-­nNOS interaction within the vHP did not affect the consolidation of cue-­induced fear, but significantly impaired the consolidation of context-­induced fear. At the cellular level, disruption of PSD95-­nNOS interaction with ZL006 was found to impair long-­term potentiation (LTP) in the BLA neurons. Finally, unlike NMDAR antagonist MK-­801, ZL006 is devoid of adverse effects on many other CNS functions, such as motor function, social activity, cognitive functions in tasks of object recognition memory and spatial memory. These findings collectively demonstrated that PSD95-­nNOS interaction within the conditioned fear network appears to be a key molecular step in regulating synaptic plasticity and the consolidation of conditioned fear. Disruption of PSD95-­nNOS interaction holds promise as a novel treatment strategy for fear-­ motivated disorders, such as post-­traumatic stress disorder and phobias.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18233号 / 工博第3825号 / 新制||工||1586(附属図書館) / 31091 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18160号 / 医博第3880号 / 新制||医||1003(附属図書館) / 31018 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 生田 宏一, 教授 宮地 良樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20968号 / 医博第4314号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 上杉 志成, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21799号 / 工博第4616号 / 新制||工||1719(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 跡見 晴幸, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM