Asthma is a chronic respiratory disease with a high prevalence in Western countries, including Canada, and increased exacerbations have been associated with ambient air pollution. The maintenance of airways tone is critically dependent on the endogenous bronchodilator, nitric oxide (NO). The nitric oxide synthase (NOS) isoenzymes produce NO from the amino acid, L-arginine, and competition for substrate with the arginase isoenzymes can limit NO production. Imbalances between these pathways have been implicated in the airways hyperresponsiveness (AHR) of asthma. The overall objective of this work was to determine whether arginase and downstream polyamine metabolites are functionally involved in airways responsiveness in animal models of asthma and the adverse responses of allergic animals to air pollution. To this purpose, the expression profiles of proteins involved in L-arginine metabolism were determined in lung tissues from human asthmatics and murine models of ovalbumin (OVA)-induced airways inflammation. Expression of arginase 1 was increased in human asthma and animal models. Competitive inhibition of arginase attenuated AHR in vivo. The roles of the downstream metabolites of arginase, the polyamines (putrescine, spermidine and spermine) were examined by administering them via inhalation to anaesthetized mice. It was demonstrated that spermine increases methacholine responsiveness in normal and allergic mice. Additionally, inhibition of polyamine synthesis improved AHR in a murine model. Thus, arginase and downstream polyamine metabolites contribute to AHR in asthma. Finally, the potential role of arginase in the exacerbation of asthma by air pollution was investigated. For this purpose, murine sub-acute and chronic murine models of allergic airways inflammation were employed, which exhibit inflammatory cell influx and remodeling/AHR, respectively, to determine the role of arginase in the response to concentrated ambient fine particles plus ozone. Allergic mice that were exposed to air pollution exhibited increased arginase activity and expression, compared to filtered air-exposed controls. Furthermore, inhibition of arginase attenuated the air pollution-induced AHR. Thus, the studies of the arginase pathway and downstream metabolites described in this thesis indicate that arginase inhibition may be a therapeutic target in asthma and may also protect susceptible populations against the adverse health effects of air pollution.

Nitric oxide (NO) is an important regulator of pulmonary blood flow and attenuates hypoxic pulmonary vasoconstriction (HPV). Nitric oxide is synthesized enzymatically in a number of tissues, including the lungs, and can also be generated from reduction of nitrite during hypoxia and acidosis. Inhaled nitric oxide (INO) is a selective pulmonary vasodilator, with no effects on systemic arterial blood pressure due to inactivation by hemoglobin in the blood. INO has distant effects both within the lungs and in other organs, since NO can be transported to remote tissues bound to proteins, or as more stable molecules of nitrite and nitrate. In healthy pigs, INO causes vasoconstriction and down regulation of endogenous NO production in lung regions not reached by INO, and predominantly so in hypoxic lung regions, i.e. augmentation of HPV. In this thesis, distant effects of INO in pigs with endotoxemic- and lavage-induced lung injuries were studied. INO increased the NO production in lung regions not reached by INO in endotoxemic pigs, whereas endogenous NO production was unaffected in pigs with lavage-induced injury. Metabolic and/or hypercapnic acidosis frequently occurs in critically ill patients, but whether acidosis affects the endogenous pulmonary NO production is unclear. The regional NO production and blood flow in hyperoxic and hypoxic lung regions, were studied during metabolic and hypercapnic acidosis. Neither metabolic, nor hypercapnic acidosis changed the endogenous NO production in hyperoxic or hypoxic lung regions. Metabolic acidosis potentiated HPV, whereas hypercapnic acidosis transiently attenuated HPV. In conclusion, the present thesis has demonstrated that INO in experimental sepsis increases the endogenous NO production in lung regions not reached by INO, which may cause increased shunt and poor response to INO. This distant effect is not seen in lavage injuried lungs, an experimental model with less inflammation. Acidosis does not affect the endogenous pulmonary NO production in hyperoxic or hypoxic lung regions. Whereas metabolic acidosis potentiates HPV, hypercapnic acidosis transiently attenuates HPV, due to a combination of hypercapnia-induced increase in cardiac output and a probable vasodilating effect of the CO2-molecule.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xxiii, 251 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 191-209).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Nitric oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzymatic conversion of arginine to citrulline, NO, has known roles in circulatory, immune and nervous tissues. In the nervous system nitric oxide has been implicated in long-term potentiation, neurotransmitter release, channel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitric oxide in cells, namely, neuronal differentiation. During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To deternine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53 and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In further studies of this pathway, we have examined the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB 203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we determine that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0l26 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Having shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arrest and neuronal differentiation. Unlike NOS inhibition, the inhibition of soluble guanylate cylcase does not block the induction of neuronal markers. Moreover, treatment of NGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence, cGMP is not a component of this pathway and we had to consider other mechanisms of NO action. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm2 regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies in order to disrupt Hdm2-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.