Master of Science / Department of Kinesiology / Carl Ade / Background: Cancer survivors treated with anthracycline-based chemotherapy have a high risk of developing anthracycline-induced cardiotoxicities, including cardiac abnormalities, endothelial dysfunction, and dilated cardiomyopathy. Notably, the imbalance of decreased nitric oxide (NO) production and increased reactive oxygen species has been shown to cause significant damage to cardiac tissue and mitochondria. Therefore, the aim of the current investigation was to determine if an inorganic dietary nitrate (NO3-) supplementation period could restore normal cardiac function in cancer survivors with a history of anthracycline chemotherapy. Methods: Ten cancer survivors, 9 with breast cancer and 1 with lymphoma, completed the experiment. Standard and Tissue Doppler echocardiography were used to assess LV and carotid artery function during systole and diastole at rest. Results: There were no differences in ventricular-arterial coupling (p=0.10), arterial stiffness (p=0.38) or strain of the LV (p=0.49). However, NO₃- supplementation improved strain rate in early filling, early mitral septal wall annular velocity, and mitral A-wave velocity or late diastolic filling. Conclusion: Following NO₃- supplementation, cancer survivors with a history of anthracycline chemotherapy showed significant improvements in diastolic function compared to placebo treatments. These findings add support to the literature of the therapeutic benefits of inorganic dietary NO₃- supplementation on cardiovascular function in clinical populations.

Three biologically-active aminothiols cysteamine (CA), DL-cysteine (CYSH) and DL-homocysteine, were studied in this thesis. These aminothiols react with nitrous acid (HNO2), prepared in situ, to produce S-nitrosothiols (RSNOs): S-nitrosocyteamine (CANO), S-nitrosocysteine (CYSNO) and S-nitrosohomocysteine (HCYSNO). They also react with S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) through a transnitrosation reaction to produce their corresponding RSNOs. A detailed kinetics and mechanistic study on the formation of these RSNOs and their subsequent decomposition to release nitric oxide (NO) were studied. For all three aminothiols the stoichiometry of their reaction with nitrous acid is strictly 1:1 with the formation of one mole of RSNO from one mole of HNO2. In all cases, the nitrosation reaction is first order in nitrous acid, thus implicating it as a nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating another nitrosating agent, the nitrosonium cation, NO+ ( which is produced from the protonation of nitrous acid) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Nitric oxide itself was not detected as a nitrosant. Bimolecular rate constants for the nitrosation of CA, CYSH and HCYSH were deduced to be 17.9, 6.4, 0.09 M-1 s-1 for the nitrosation by nitrous acid and 8.25 x 1010, 2.89 x 1010 and 6.57 x 1010 M-1 s-1 for the nitrosation by nitrosonium cation respectively. A linear correlation was obtained between the rate constants and the pKa of the sulfur center of the aminothiols for nitrosation by NO+. The stabilities of the three RSNOs were found to be affected by metal ions. They were unstable in the presence of metal ions, with half-lives of few seconds. However, in the presence of metal ion chelators, they were found to be relatively stable with half-lives of 10, 30 and 198 hours for CYSNO, CANO and HCYSNO respectively. The relative stability of HCYSNO may be an advantage in the prevention of its metabolic conversion to homocysteine thiolactone, the major culprit in HCYSH pathogenesis. This dissertation has thus revealed new potential therapeutic way for the modulation of HCYSH related cardiovascular diseases.

Many types of chemotherapeutic agents have been developed to target specific mechanisms within the body that control the progression of cancer, though few have been able to circumvent the existing problems associated with the treatments. The current remedies entail grueling drug regimens and toxic side effects that may undermine the effectiveness of the drugs. Cisplatin, a common nitroplatinum(II) drug widely used to treat a variety of cancers, is administered intravenously and circulates systemically, affecting healthy regions of the body as well. Resistance to cisplatin is increasing and the need for new, less toxic medication must be met for future success in cancer therapy. Our lab has synthesized novel nitroplatinum(IV) cisplatin complexes (PH1-14) that may evade these problems. We examined the effects of these compounds on cell viability, as well as effects on cancer-specific mechanisms such as nitric oxide (NO) production, angiogenesis, and the STAT signaling pathways. In vitro studies demonstrated that PH1-11 and PH14 demonstrated greater efficacy at inhibiting cell proliferation with lower IC50 values that ranged from 41-58 uM (as compared with cisplatin IC50 = 66 uM). Data from NO assays were inconclusive, though there was elevated expression of inducible nitric oxide synthase in cells treated with PH3 and PH11. We also found that PH9 was able to inhibit STAT dimerization at concentrations as low as 0.3 uM. PH9 also decreased VEGF and HIF-1α expression, thereby inhibiting angiogenesis. The activity of the PH complexes was also studied in C57BL/6 mice inoculated with murine bladder MB49 tumors. The experimental group showed significantly slower tumorigenesis and smaller tumors as compared with the control group. Toxicological analyses of the blood via metabolic assays showed that no nephrotoxicity was observed in dosages of less than 7 mg drug/kg. We conclude from these results the potential for the use of novel mechanisms in the treatment of cancers. This work will guide future investigations of these drugs in further preclinical trials and also introduce an alternative to the traditional chemotherapeutic agents.

Introduction: The enzyme, inducible nitric oxide synthase (iNOS), is upregulated in the airway epithelium in patients with allergic asthma resulting increased nitric oxide production. The concentration of nitric oxide in exhaled air (FENO) correlates with the degree of eosinophilic airway inflammation and has been proposed as a new breath test to assist in the management of asthma. Key Aims: To determine the predictive accuracy of FENO compared with conventional testing in the diagnosis of asthma. To assess the performance characteristics of FENO to predict steroid responsiveness compared with the conventional approach in patients presenting with non-specific respiratory symptoms. To assess the role of FENO compared with conventional guidelines-based approach as a guide to adjusting inhaled corticosteroid therapy in patients with chronic persistent asthma. Methods: Consecutive patients referred with chronic undiagnosed respiratory symptoms were enrolled. Comparisons were made between FENO, induced sputum analysis and other conventional tests (including peak flow variation, spirometry, response to oral steroid challenge) for predicting the presence of asthma in the first study, and for predicting response to four weeks inhaled fluticasone treatment in the second study. In the third study, 110 subjects with chronic persistent asthma were enrolled into a single-blind placebo-controlled study during which subjects were randomly allocated to have their corticosteroid (fluticasone) dose adjusted on the basis of either FENO measurements or an algorithm based on conventional guidelines. The main outcomes were the frequency of asthma exacerbations and the mean daily dose of inhaled corticosteroid. Results: In the first study, 17 of 47 consecutive patients were diagnosed with asthma. Sensitivities for the conventional tests (0-47%) were lower than for FENO (88%) and sputum eosinophils (86%), with overall significantly greater diagnostic accuracy for FENO and sputum eosinophils. Fifty-two consecutive subjects completed the second study. When compared to the conventional tests, the predictive powers for FENO were consistently greater than for almost all other baseline measurements used to predict steroid responsiveness, with an optimum cut point of 47 ppb. In the third study, the exacerbation rates were 0.49 (95% C.I. 0.20, 0.78) per patient per year in the FENO group and 0.90 (95% C.I. 0.31, 1.49) in the control group, representing a nonsignificant reduction of 45.6 percent (95% C.I. -78.5, 54.5%) in the FENO group. The final mean daily doses of fluticasone were significantly lower in for the FENO group compared with the conventional group (370 [mu]g/day (95% C.I. 263, 477) vs 641 [mu]g/day (95% C.I. 526, 756) respectively; p=0.003). There were no significant differences in other markers of asthma control, use of oral prednisone, pulmonary function, or levels of airway inflammation (sputum eosinophils). Conclusions: FENO is comparable to induced sputum analysis and superior to the conventional methods currently used including peak flows and spirometry as a dignositic for asthma and as a predictor of steroid responsiveness in patients with chronic respiratory symptoms. FENO measurements as a guide to adjusting inhaled corticosteroid therapy compared to the conventional guidelines based approach results in significantly lower maintenance doses being achieved without compromising asthma control. The results of this thesis provide evidence to support the use of FENO measurements in routine clinical practice as a tool to improve the overall management and diagnosis of asthma.

Essential hypertension is defined as a chronic elevation of blood pressure of unknown cause. Though a definitive trigger for this change in blood pressure has not been established, there is a strong association with an upregulation of sympathetic output from the central nervous system. There are a number of central autonomic nuclei involved in the maintenance of blood pressure, including the brainstem regions of the nucleus tractus solitarii (NTS), caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM), the sympathetic preganglionic neurons (SPNs) within the intermediolateral cell column (IML) of the spinal cord, as well as forebrain regions such as the paraventricular nucleus (PVN) of the hypothalamus. Within these centers, a vast number of neurotransmitters have been identified that contribute to the control of blood pressure, including glutamate, angiotensin II, serotonin, neurotensin, neuropeptide Y, opioids and catecholamines. Recognition of the role of nitric oxide (NO) and its multiple influences over the neural control of blood pressure is gaining increasing significance. Nitric oxide is a unique modulatory molecule that acts as a non-conventional neurotransmitter. As NO is a gas with a short half-life of 4 – 6 seconds, its’ synthesising enzyme, nitric oxide synthase (NOS) is often used as a marker of location of production. Once activated, the best-known “receptor” for NO is soluble guanylate cyclase (sGC), which drives the production of cyclic guanosine monophosphate (cGMP). Identifying the presence of cGMP can therefore be used to determine sites receptive to NO. Previous studies examining the role of NO in the central autonomic control of blood pressure have focused predominantly upon application of either excitatory or inhibitory drugs into the key central autonomic regions and assessing pressor or depressor effects. This thesis aims instead to study the neuroanatomical relationship and functional significance of NO and cGMP expression in the brain and spinal cord of a hypertensive and normotensive rat model. In the first experimental chapter (Chapter 3), a comparative neuroanatomical analysis of neuronal NOS expression and its relationship with cGMP in the SPN of mature Spontaneously Hypertensive Rats (SHR) and their controls, Wistar Kyoto (WKY) was undertaken. Fluorescence immunohistochemistry confirmed the expression of nNOS in the majority of SPN located within the IML region of both strains. However, a strain specific anatomical arrangement of SPN cell clusters was evident and while there was no significant difference between the total number of SPN in each strain, there were significantly fewer nNOS positive SPN in the SHR animals. All nNOS positive SPN were found to express cGMP, and a novel subpopulation of nNOS negative, cGMP-positive SPN was identified. These cells were located in the medial edge of the IML SPN cell group. These results suggest that cGMP is a key signalling molecule in SPN, and that a reduced number of nNOS positive SPN in the SHR may be associated with the increase in sympathetic tone seen in essential hypertension. The second experimental chapter (Chapter 4) aimed to determine if reduced numbers of nNOS containing SPN translated into reduced detectable cGMP. The functional significance of cGMP signalling in the two strains was then examined. Based on previous work by our group, it was predicted that reduced nNOS in the SHR would translate into reduced cGMP and that intrathecal administration of exogenous cGMP in the spinal cord would drive a differential pressor response in the two animal strains. Immunohistochemical techniques confirmed that within each SPN, the relative level of cGMP expression was significantly reduced in the SHR when compared to the WKY. Intrathecal application of 8-bromo-cGMP, a drug analogous to cGMP, increased blood pressure in both strains and had a differential and dose dependent effect, causing only a small increase in blood pressure in anaesthetised WKY animals, while driving a significant pressor response in the SHR. This finding raised the novel hypothesis that in the SHR, reduced nNOS expression is not a driver of hypertension, but is instead a protective mechanism limiting the potent pressor effects of cGMP within SPN. The third experimental chapter (Chapter 5) examines the expression of neuronal and inducible isoforms of NOS (nNOS, iNOS) within the RVLM of SHR and WKY rats. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyse the level of mRNA expression and immunohistochemistry was then used to further analyse protein levels of nNOS. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR. Quantitative real-time PCR indicated that relative to WKY, mRNA levels for nNOS was significantly higher in RVLM of the SHR. This was confirmed immunohistochemically. When compared to iNOS, nNOS was expressed at significantly higher levels overall, however there was no difference in iNOS mRNA expression between the two strains. This demonstration of differential expression levels of nNOS and iNOS in the RVLM raises the possibilities that (i) NO production is up-regulated in the RVLM in SHR in response to increased sympathetic activity in order to re-establish homeostatic balance or alternatively that (ii) an alteration in the balance between nNOS and iNOS activity may underlie the genesis of augmented sympathetic vasomotor tone during hypertension. The fourth experimental chapter (Chapter 6) extends the observations in Chapter 5 through examination of the expression of cGMP and sGC within the RVLM. There is strong functional evidence to suggest that NO signalling in the RVLM relies on cGMP as an intracellular signalling molecule and that this pathway is impaired in hypertension. Immunohistochemistry was used to assess cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, again comparing SHR and WKY animals. Fluorescence immunohistochemistry on sections of the RVLM, double labelled for cGMP and either nNOS or phenylethylamine methyl-transferase (PNMT) failed to reveal cGMP positive neurons in the RVLM from aged animals of either strain, despite consistent detection of cGMP immunoreactivity neurons in the nucleus ambiguus from the same or adjacent sections. This was demonstrated both in the presence and absence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and in young vs. aged animals. In-vitro incubation of RVLM slices in the NO donor DETA-NO or NMDA did not reveal any additional cGMP neuronal staining within the RVLM. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase immunoreactivity was found throughout the RVLM, although it did not co-localise with the PNMT or nNOS neuronal populations. Overall, results suggest that within the RVLM, cGMP is not detectable in the resting state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application. A short time course of cGMP signalling or degradation not inhibited by the phosphodiesterase inhibitor utilised (IBMX) in the RVLM cannot be excluded. The final experimental chapter (Chapter 7) examines cGMP expression in magnocellular and preautonomic parvocellular neurons of the PVN. Retrograde tracing techniques and immunohistochemistry were used to visualise cGMP immunoreactivity within functionally, neurochemically and topographically defined PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. Cyclic GMP immunoreactivity was significantly higher in magnocellular compared to preautonomic neuronal populations. In preautonomic neurons, the level of cGMP expression was independent on their subnuclei location, innervated target or neurochemical phenotype. The data presented in this chapter indicates a highly heterogeneous distribution of basal cGMP levels within the PVN, and supports work by others indicating that constitutive NO inhibitory actions on preautonomic PVN neurons are likely mediated indirectly through activation of interneurons. Summary Together, these studies comprise a detailed analysis of the neuroanatomical expression of NO and its signalling molecule cGMP in key central autonomic regions involved in the regulation of blood pressure. Under resting or basal conditions, the studies demonstrate notable differences in the expression of NO synthesising enzymes between normotensive and hypertensive animals, and correlating changes in the downstream signalling molecule cGMP. In the spinal cord, novel functional differences in cGMP activity were also demonstrated. In the RVLM, although differences in nNOS were demonstrated, cGMP expression could not be readily detected in either the WKY or SHR, while in contrast within the PVN, cGMP was detected in both magnocellular and parvocellular neuronal populations. Conclusion This thesis gives insight into the physiological role of NO and cGMP as mediators of central blood pressure control. The results presented indicate that the NO-cGMP dependent signalling pathway may not be the dominant driver responsible for maintaining high blood pressure in the SHR model of essential hypertension, and that there is no globally consistent pattern of expression, and indeed the role of NO as a mediator of pressor and depressor function may vary between the autonomic regions examined. Further, it is possible that this pathway is only recruited during activation of reflex homeostatic pathways or during times of marked physiological stress, and that the differences we see in basal expression between the normotensive and SHR animals are instead a result of compensatory mechanisms.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--Ohio University, March, 2006. / Includes bibliographical references (leaves 152-160)

Thesis (Ph. D.)--University of Akron, Dept. of Chemistry, 2006. / "May, 2006." Title from electronic dissertation title page (viewed 10/11/2006). Advisor, Daniel J. Smith; Committee members, Michael J. Taschner, Wiley J. Youngs, Kim C. Calvo, Darrell H. Reneker; Department Chair, Michael J. Taschner; Dean of the College, Ronald F. Levant; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.