Reactor Controlado por Tiristores (TCR)

The findings of this thesis contribute significantly towards elucidating the abundance of CNP in the CNS, by relating changes in CSF concentrations of CNP peptides to their distribution, form and regulation in related tissues. In vivo studies such as those described in this thesis are necessary as they are an essential part of establishing fundamental aspects of physiology — particularly as CNP may be an invaluable therapeutic and/or diagnostic tool in certain settings of central

pathophysiological disorders (Mahinrad et al. 2016). While numerous studies have mapped out sites of NPPC and NPRB expression in the CNS (Minamino et al. 1991, Yeung et al. 1996b,

Moriyama et al. 2006), relatively little has been reported on sites and abundance of CNP peptides themselves. Advancing our knowledge about the regulation of CNP synthesis in vivo and

identifying central sources of CNP in the brain is part of the crucial groundwork necessary to allow for the development of practical applications in future.

The demonstration that markedly elevated concentrations of CNP and NTproCNP in the

circulation — as occurs during ruminant pregnancy — do not result in increased levels in the CSF, is supportive of the hypothesis that central sources of CNP peptides exist (Chapter 4). This

observation is in agreement with the single report by Schouten et al. (2011) describing concurrent levels of CNP peptides in human CSF and plasma, where concentrations were independent in the respective fluids. Prior to this study, there were no known physiological states or compounds capable of acutely altering concentrations of CNP peptides in CSF or nervous tissue. A method for cannulation of the cisterna magna was developed to facilitate repeated collection of CSF samples from conscious sheep (Chapter 3), following which a series of pilot studies were conducted in order to identify a compound or physiological state capable of altering CSF concentrations of CNP peptides (5.1.2). The consequences of the finding that CSF concentrations of CNP peptides concentration were not altered during or after anaesthesia were two-fold; firstly, it added to the growing body of evidence that CNP peptide concentrations in CSF are remarkably stable and presumably necessary for some aspect of brain homeostasis. Secondly, it established that anaesthesia was unlikely to be a confounding factor in studies where anaesthesia was necessary for obtaining single samples of CSF.

Upon finding that a single intravenous dose of dexamethasone resulted in elevated

120 in response to different doses of dexamethasone was characterised in the respective fluids (Chapter 6) which indicated a dose-response relationship with dexamethasone and a differential response between CSF and plasma. Hypothesising that the increase in CSF concentration of CNP peptides was a result of increased peptide synthesis and release into extracellular fluid/CSF from one or more regions of brain tissue, CNP and NTproCNP concentration was measured in a wide selection of tissues sampled from brains of dexamethasone- and saline-treated sheep (Chapter 6). This revealed a widespread response to dexamethasone in central tissues. The demonstration that gene expression levels of NPPC increased in response to stimulation with dexamethasone suggests that increased peptide concentration is a result of increased synthesis of CNP peptides. Of the natriuretic peptide family, this effect was shown to be specific to CNP, as no response was shown for ANP or BNP in plasma, CSF and brain tissue. The measurement of the gene expression levels of NPRB and NPRC, which encode for the CNP receptor and clearance receptor,

respectively, provided an insight into CNP signalling and clearance pathways in response to dexamethasone. Characterisation of different molecular size forms of CNP through size-exclusion HPLC revealed different profiles and proportions of the respective forms in CSF, plasma, brain tissue and anterior and posterior pituitary glands (Chapter 6).

These studies establish multiple sources for CNP in the CNS. The identification of dexamethasone as a secretagogue for CNP in the CNS implicates CNP as a potentially important mediator of glucocorticoid-mediated pathways. Together, these findings highlight a difference in the processing of proCNP among brain and anterior and posterior pituitary glands and portray the pituitary gland as a major peripheral source. The novel contributions of this thesis are outlined in Table 8.1.

121 Table 8.1 Summary of novel findings presented in this thesis.

The demonstration that both CSF and plasma concentrations of CNP and NTproCNP remained stable throughout anaesthesia

The finding that markedly elevated plasma concentrations of CNP and NTproCNP throughout gestation in sheep do not lead to increased concentrations in CSF Demonstration of independent regulation of peripheral and central levels of CNP and NTproCNP in pregnant sheep and red deer stags

First report of a stimulus (dexamethasone) capable of acutely increasing CSF concentrations of CNP and NTproCNP

The finding that concentrations of CNP and NTproCNP are increased in multiple brain tissues following dexamethasone administration

The demonstration that the stimulating effect of dexamethasone on peptide synthesis is specific to CNP, as ANP and BNP concentrations were unchanged in plasma, CSF and brain tissue

Demonstration of increased NPPC expression in brain tissues following dexamethasone, suggesting that increased peptide concentrations reflect increased synthesis

The finding that the ratio of NTproCNP: CNP in the anterior and posterior pituitary gland (1:1) is markedly different from the ratio in brain tissues (5:1 to 10:1)

Evidence from differing HPLC profiles suggesting that CNP processing differs between the anterior and posterior pituitary gland

Identification of large irCNP fragments in plasma, consistent with proCNP (1-103)

The finding that NPRC gene expression levels are similar between brain and both lobes of the pituitary gland— despite large differences in the NTproCNP:CNP ratio between the brain and pituitary gland