Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome.

Cocaine is interrelated with the opioid system on many levels, especially via the mu opioid receptor (MOR). Also, cocaine has been involved in modulating nitric oxide (NO) actions within the cell. The effect of cocaine was first assessed on the MOR, and then on transcription by the use of 1 µg/ mL actinomycin D inhibitor. Several signaling pathways that cocaine may exert its action in modulating the MOR up-regulation in protein expression were also explored. Two dosage regimens were used in cocaine treatment, single continuous treatment (SCT), and repeated intermittent treatment (RIT). Different pathway inhibitors were used on PC12 cells, as follows: the PLC-PKC inhibitors 5 µM U-73122 and 10 µM BIS-1 used to investigate the involvement of the PKC signaling pathways in MOR expression levels, the evaluation of MAPK pathway by the use of 50 µM U0126 inhibitor, and the 10 µM LY94002 inhibitor was used to investigate the PI3K/Akt pathway. Moreover, the effect of NO on these signaling pathways was investigated by the use of 20 mM nonselective L-NAME inhibitor and qualitatively by DAF-2 florescence. Western blot analysis indicated that cocaine up-regulated MOR protein expression. Also, RIT cocaine treatment increased MOR protein levels via transcription. All three signaling pathways, MAPK, Akt and PKC modulated cocaine-induced increase of MOR following SCT cocaine treatment (post-transcriptional). Both MAPK and Akt have been found to modulate the cocaine-induced transcription of MOR via the two dosage regimens of cocaine, SCT and RIT. Also, inhibition of both PLC and PKC did not prevent cocaine-induced increase in MOR transcription, according to RIT of cocaine. Furthermore, Akt and PKC appeared to modulate cocaine-induced NO production while MAPK did not. NO seemed to be involved with the PKC and Akt pathways in up-regulating MOR in RIT of cocaine directly by the Akt pathway, and indirectly by the PKC pathway. On the other hand, NO and MAPK modulated the MOR up-regulation expression simultaneously, but in an individual/parallel manner. Furthermore, signaling pathway activation levels were tested using L-NAME which concluded that NO modulated cocaine-induced increase in total Akt protein levels, but did not appear to have an effect on phosphorylated MAPK activation levels. In conclusion, different treatment regimens of cocaine activate different pathways; SCT of cocaine activated all three signaling pathways, however, RIT of cocaine activated only the MAPK and Akt pathways. / Saudi Bureau in Canada

Nitric oxide (NO) plays a role in regulating the rate of orthodontic tooth movement (OTM) in rat models; however, in humans this role remains less clear. In this study, samples of gingival crevicular fluid (GCF) were collected from each maxillary central incisor and first and second molar immediately before (T0), 1 hour after (T1), and 3-4 days after (T2) application of light orthodontic forces in thirteen male participants (ages 11-18 years) undergoing orthodontic therapy. NO levels were measured in each GCF sample, and significantly higher NO levels (p<0.05) were found at T1 at the buccal surfaces of the central incisors when compared to the posterior teeth. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the PDL of human teeth are affected by the magnitude of an applied force.

The electronegativity of thiolate ligation in the hemeprotein nitric oxide synthase (NOS) proteins has been identified as an influence on autoinhibition in this enzyme. The mutation of a conserved tryptophan residue, which hydrogen bonds to the coordinating thiolate ligand and therefore influences its electronegativity, to either phenylalanine or tyrosine has had various effects including heme loss and dimer disruption in the inducible isoforms, while hyperactivity occurs in the neuronal isoforms. I have performed the analogous mutations in W180 of eNOSoxy, the endothelial isoform. UV/visible and resonance Raman spectroscopy have demonstrated that the mutants experienced increased basicity of the thiolate due to loss of the hydrogen bond between the mutated residue in the absence of the cofactor (6R)5,6,7,8-tetrahydrobiopterin (H4B). The mutants also displayed relative rates of NO2- production that were comparable to the nNOSoxy mutants, which is consistent with the nNOSoxy results. The presence of H4B alters porphyrin planarity, which enabled hydrogen bonding to occur in W180Y, thus restoring thiolate basicity to that of wild-type eNOSoxy. Reduced overall activities by the proteins suggest that H4B stabilizes the heme. The analogous W56 mutants of saNOS, a NOS oxygenase domain-like protein from Staphylococcus aureus (saNOS), have been previously characterized using resonance Raman spectroscopy. These mutants also exhibit increased thiolate electronegativity over wild-type. As the homodimers had already been investigated, saNOS was an ideal system in which to explore heterodimers. Heterodimers were generated through the co-expression of one wild-type and one mutated subunit, enabling the examination of each subunit individually through resonance Raman spectroscopy. The subunits of the resulting proteins were shown to have heme environments that resembled those of their corresponding homodimers. The activity of saNOS did not vary significantly for the various W56 mutants, suggesting that saNOS catalysis may be unaffected by thiolate electronegativity. / Thesis (Master, Chemistry) -- Queen's University, 2008-09-04 11:37:38.688

Blockade of the renin-angiotensin system lowers the rate of cardiovascular events in patients at risk for vascular disease and also improves endothelial function but the mechanism remains unclear. HUVECs were stimulated with Ang II (100 nM). Ang II produced a 2-fold increase in O2- production, which was measured by lucigenin-enhanced chemiluminescence. This increase was blocked by NAD(P)H oxidase inhibitor DPI, but not by eNOS inhibitor L-NAME. Ang II increased monocyte adhesion to ECs by 4.5-fold, and this increase was blocked by candesartan (AT1 receptor antagonist), DPI, L-NAME, wortmannin (PI3K inhibitor), dominant negative-AKT, and p22phox siRNA. Dominant active-AKT increased adhesion by 1.5-fold. Our findings indicate that the simultaneous activation by Ang II of eNOS and NAD(P)H oxidase leads to endothelial activation. This process can partially explain the therapeutic benefits of reducing the action of Ang II.

The use of mouthwash is a common complement to oral care. However, the physiological implication of this use, besides of effects on oral hygiene, is poorly known. The research of the gut micro flora and its implications on the host is a very active area of research today. Many important connections between the gut micro flora and obesity and diabetes have been found. These billions of bacteria are part of the immune system, they produce essential vitamins and they make inaccessible polysaccharides more digestible to the host, just to mention a few of their symbiotic roles for the host. A less explored area is the micro flora in the oral cavity. On the back of the tongue, anaerobic bacteria can reduce dietary nitrate to nitrite which then further can be reduced to nitric oxide, NO. NO is important in several important biological functions, e.g. as a signal substance, vasoregulation, mucus production and antibacterial effects. Vegetables as beetroot and spinach are dietary sources with a high nitrate content. Also drinking water and processed meats can be of relevance. Nitrite is added to processed meat for the prevention of botulism but also adds taste and color.   Experiments on humans indicate that mitochondrial efficiency increases after nitrate load, manifested as a decreased oxygen demand during physical exercise. This can also be relevant under conditions where the mitochondrial function is impaired, such as in diabetes and cardiovascular diseases. First a pilot study was made to evaluate the nitrate reducing effect from the antibacterial mouthwash. The mouthwash proved very effective. The concentrations of nitrate and nitrite in saliva was analyzed by HPLC and saliva from the antibacterial treatment showed greatly reduced concentrations of nitrite and high concentrations of nitrate. Saliva from placebo mouthwash showed high concentrations of nitrite and low concentrations of nitrate as expected. To study the importance of oral bacteria on metabolism, we performed a randomized, cross-over double-blinded study with 19 healthy males between 22-43 years. During two separate three-day periods they used an antibacterial and placebo mouthwash, respectively. On the fourth day their basal metabolic rate (BMR) was measured with an indirect calorimetric system. Moreover, samples from saliva, urine and blood were collected but these results are not included in this thesis. An earlier, unpublished study has demonstrated that nitrate administration reduces the basal metabolic rate. Accordingly, our aim was to study potential effects on the basal metabolic rate following reduction of the number of oral bacteria by aid of antibacterial mouthwash. Our hypothesis was that the reduced availability of nitrite would decrease the availability of NO in the body and manifest as an increased basal metabolic rate. The results from indirect calorimetry measurements showed no significant difference between placebo and antibacterial mouthwash, but there may be confounding factors. Further study is needed to assess the potential effects on host metabolism by these bacteria.

Nitric oxide (NO) plays a role in regulating the rate of orthodontic tooth movement (OTM) in rat models; however, in humans this role remains less clear. In this study, samples of gingival crevicular fluid (GCF) were collected from each maxillary central incisor and first and second molar immediately before (T0), 1 hour after (T1), and 3-4 days after (T2) application of light orthodontic forces in thirteen male participants (ages 11-18 years) undergoing orthodontic therapy. NO levels were measured in each GCF sample, and significantly higher NO levels (p<0.05) were found at T1 at the buccal surfaces of the central incisors when compared to the posterior teeth. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the PDL of human teeth are affected by the magnitude of an applied force.

The significant role that nitric oxide plays in human physiology is linked to the ability of NO to bind to iron forming mono-nitrosyl iron complexes. Protein-bound and low-molecular-weight dinitrosyl iron complexes (DNICs) are known to form in excess NO. Studies of such biological DNICs have relied on their paramagnetism and characteristic EPR signal of g value of 2.03. It has been suggested that DNICs act in vivo as NO storage (when protein-bound) and transfer agents (when released by, for example, free cysteine). Biological DNICs, mainly resulting from iron-sulfur cluster degradation, are difficult to extract and isolate, thereby preventing their full characterization. Thus, development of synthetic DNICs is a promising approach to model and better understand the formation and function of biological DNICs, the scope of donor ligands that might coexist with Fe(NO)2 units, the redox levels of bio-DNICs, and establish other spectroscopic techniques appropriate for characterization. A series of N-heterocyclic carbene (NHC) and imidazole (Imid) complexes has been characterized as mimics of histidine-containing DNICs. The pseudo-tetrahedral L2Fe(NO)2 complexes have NO stretching frequencies and redox potentials that suggest the NHCs are slightly better donors than Imids, however the two types of ligands have similar steric properties. Both the EPR-active, {Fe(NO)2}9 and the EPR-silent, {Fe(NO)2}10 states can be accessed and stabilized by the NHC. Nitric oxide transfer studies have shown that only the {Fe(NO)2}9 complexes are capable of transferring NO to a suitable NO trapping agent. Deprotonation of the distal nitrogen functionality in the imidazolate ligands of [(Imidazole)2Fe(NO)2]- leads to aggregation forming molecular squares of {Fe(NO)2}9 units bridged by the imidazolates. These interesting tetrameric complexes are examined by X-ray diffraction, EPR, and Mössbauer studies. The paramagnetic tetrameric complexes have multiple redox events observed by cyclic voltammetry. Mössbauer spectral data of the tetrameric complexes are compared with Mössbauer data obtained for a series of NHC-containing DNICs. Iron and cobalt-containing mononitrosyl N2S2 model complexes of the nitrile hydratase enzyme active site demonstrate sulfur-based reactivity resulting in the formation of polymetallic complexes. In all cases, shifts in the nitrosyl stretching frequencies demonstrate substantial transfer of electron density from the (NO)M(N2S2) moiety to the metal-acceptor site.

Previous research has shown that nitric oxide signalling in the lateral nucleus of the amygdala is required for the consolidation of Pavlovian conditioned fear. Given the evidence that nitric oxide can act as a retrograde signalling molecule in in vitro models of memory consolidation the question arises whether this is also occurring within behavioural memory models? Using auditory fear conditioning this research shows that nitric oxide does indeed act as retrograde signalling molecule in the fear system. Its synthesis in the lateral nucleus of the amygdala regulates conditioning induced expression of the immediate early gene early growth response gene 1 (EGR-1) in cells of the auditory thalamus that project to the lateral nucleus of the amygdala. The regulation of EGR-1 expression by the lateral nucleus of the amygdala was proven to be dependent on amygdala-based cellular excitation, nitric oxide synthesis and NR2B-NMDA receptor activation but not ERK/MAPK activity. Using an EGR-1 antisense oligonucleotide to prevent training induced EGR-1 expressions in the auditory thalamus it was shown that this gene upregulation is necessary for the consolidation of conditioned fear. Finally, inhibition of EGR-1 upregulation in the auditory thalamus was proven to impair conditioning induced increases in the presynaptic proteins synaptophysin, and synapsin II and II back in the lateral nucleus of the amygdala. Overall, the results of this dissertation have shown that nitric oxide acts as a retrograde messenger in a mammalian memory system by modulating gene expression in presynaptic cells. This modulation of gene expression serves to increase levels of presynaptic proteins back at the origin of nitric oxide synthesis. This supports the long standing doctrine that nitric oxide acts as a retrograde signalling molecule to coordinate presynaptic changes associated with memory formation.

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo. Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) mainly through oxidative stress and altered metabolism. Mitochondria are isolated from post-mortem brain samples from selective regions of AD and control patients and, utilizing the cryopreservation strategy, analyzed for respiration and oxidative damage. While we did not observe increases in free radicals, we did observe decreased respiration and increases in oxidative damage markers in AD patients, suggesting a role for oxidative stress in mitochondrial dysfunction. While in the mitochondria, calcium (Ca2+) increases free radical generation by processes not completely understood. A new isoform of nitric oxide synthase (mtNOS) has been isolated and localized to mitochondria; though its existence and physiological role is debated. Nitric oxide synthase (NOS), when activated by Ca2+, produces nitric oxide (NO•) that can interact with ROS producing various reactive nitrogen species (RNS). These highly reactive radical species can damage DNA, proteins and lipids, ultimately resulting in cell death via apoptosis or necrosis. The current research is aimed at understanding the role of Ca2+ and NOS in oxidative stress leading to mitochondrial dysfunction. We observed a significant reduction in mitochondrial respiration with increasing doses of calcium. We also observed NOS enzyme activity and detected NOS protein in the purified mitochondrial fraction. Lastly, we were also able to show that Ca2+ increased the levels of free radicals and changes in oxidative damage markers. These results suggest the presence of NOS in mitochondria that could play a role in Ca2+ induced mitochondrial dysfunction and potentially leading to cell death as relevant to aging and neurodegenerative diseases.