published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Orthostatic intolerance following exposure to simulated or actual microgravity is observed following spaceflight and extended periods of bed rest, and is not always associated with simultaneous hypotension. Differential adaptation of cephalic and caudal arterial vasculatures (as a result of removal of the normal hydrostatic gradient) is proposed as a potential mechanism underlying this phenomenon. A potential role for changes to the L-arginine/nitric oxide pathway in such adaptations has been suggested, predominantly from previous in vitro studies; using an established model of simulated microgravity (head-down tilt; HDT). This thesis investigates whether findings in isolated vessels are reflected by in vivo measurements of cephalic and caudal vascular function. Using carotid or iliac artery flow normalized to mean arterial pressure as an index of cerebral or hind limb vascular conductance, autoregulatory cerebral vasodilatation in response to lower body negative pressure was found to be impaired following HDT. In addition, α¬1-adrenoceptor agonist-mediated vasoconstriction was decreased in the cerebral vasculature and increased in the peripheral and hind limb vasculature. Administration of acetylcholine or the non-selective nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) demonstrated a decreased contribution of NOS to cerebrovascular tone, but an increased contribution of NOS to peripheral vascular resistance and tone of the hind limb vasculature. Together with a lack of difference in the response to the selective inducible NOS (iNOS) inhibitor 1400W, these results suggest that differential adaptation of eNOS may account for the observed differences between control and HDT animals. Further investigation of the changes to the L-arginine/nitric oxide pathway suggest that these changes are not associated with changes in eNOS expression, but may be related to altered activity of eNOS. Furthermore, the bioavailability (as measured by pharmacokinetic half life) or the vascular effector mechanisms (as measured by the haemodynamic response to exogenously administered nitric oxide) responsible for the effects of nitric oxide were also shown to be unaffected by HDT. These findings suggest that differential adaptation of the L-arginine/nitric oxide pathway may contribute to the inability to raise total peripheral resistance and impaired cerebral autoregulation following HDT, thereby representing a mechanism of orthostatic intolerance following exposure to microgravity.

No description available.

Nitric oxide (NO) is produced in different mammalian tissues by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All NOS isoforms contain two domains, an oxygenase domain and a reductase domain. NO is an important transmitter of information between cells in many physiological processes; however, overproduction of this molecule may lead to health problems. Therefore, selective inhibition of NOS isoforms has useful therapeutic potential for treatment of certain diseases that can appear because of the pathological overproduction of nitric oxide. Producing useful isoform selective-inhibitors that bind to the active site in the oxygenase domain has proven to be difficult when based solely on the structure of these enzymes. Biophysical studies in combination with structural properties should provide better insights into isoform-specific inhibitor development. The first step of this study was to produce and purify truncated versions of NOS isozymes consisting of the oxygenase domain as they contain the active site of the enzyme. As a result of differences between humans and other mammals in the amino acids found in the second and third shells/layers surrounding the active site, all the experiments were performed with genes coding for human proteins. The major result of this project was the development of an Escherichia coli (E. coli) expression system to produce large amounts of pure protein. This system will allow for the testing of inhibitors that bind to the active site of NOS enzymes.

Orthostatic intolerance following exposure to simulated or actual microgravity is observed following spaceflight and extended periods of bed rest, and is not always associated with simultaneous hypotension. Differential adaptation of cephalic and caudal arterial vasculatures (as a result of removal of the normal hydrostatic gradient) is proposed as a potential mechanism underlying this phenomenon. A potential role for changes to the L-arginine/nitric oxide pathway in such adaptations has been suggested, predominantly from previous in vitro studies; using an established model of simulated microgravity (head-down tilt; HDT). This thesis investigates whether findings in isolated vessels are reflected by in vivo measurements of cephalic and caudal vascular function. Using carotid or iliac artery flow normalized to mean arterial pressure as an index of cerebral or hind limb vascular conductance, autoregulatory cerebral vasodilatation in response to lower body negative pressure was found to be impaired following HDT. In addition, α¬1-adrenoceptor agonist-mediated vasoconstriction was decreased in the cerebral vasculature and increased in the peripheral and hind limb vasculature. Administration of acetylcholine or the non-selective nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) demonstrated a decreased contribution of NOS to cerebrovascular tone, but an increased contribution of NOS to peripheral vascular resistance and tone of the hind limb vasculature. Together with a lack of difference in the response to the selective inducible NOS (iNOS) inhibitor 1400W, these results suggest that differential adaptation of eNOS may account for the observed differences between control and HDT animals. Further investigation of the changes to the L-arginine/nitric oxide pathway suggest that these changes are not associated with changes in eNOS expression, but may be related to altered activity of eNOS. Furthermore, the bioavailability (as measured by pharmacokinetic half life) or the vascular effector mechanisms (as measured by the haemodynamic response to exogenously administered nitric oxide) responsible for the effects of nitric oxide were also shown to be unaffected by HDT. These findings suggest that differential adaptation of the L-arginine/nitric oxide pathway may contribute to the inability to raise total peripheral resistance and impaired cerebral autoregulation following HDT, thereby representing a mechanism of orthostatic intolerance following exposure to microgravity.

Thesis (Ph. D.)--Georgia State University, 2007. / Vincent Rehder, committee chair; Sarah Pallas, Walter William Walthall, committee members. Electronic text (248 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 28, 2008; title from file title page. Includes bibliographical references (p. 218-248).

Thesis (M.D.) - University of Glasgow, 2008. / M.D. thesis submitted to the Faculty of Medicine, Division of Cardiovascular and Medical Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.