A study of C-type natriuretic peptide in cerebrospinal fluid and related tissues of sheep, and its regulation by dexamethasone

C-type natriuretic peptide (CNP) has high abundance in cerebrospinal fluid (CSF) and central tissues, and has been implicated in the regulation of important processes in the central nervous system (CNS) including the stimulation of neuronal branching, regulation of blood-brain barrier permeability and neuroprotection. Despite the many important putative roles that have been attributed to CNP during development and in adult life through in vitro studies, little is known about the regulation of CNP in vivo — and its usefulness in clinical settings of pathophysiological CNS disorders remains untapped. The aim of this PhD project was to explore the regulation of CSF concentrations of CNP and the amino-terminal fragment of proCNP (NTproCNP) in sheep, and relate this to central sources. To determine whether CSF concentrations of CNP peptides are affected by altered levels in plasma, concurrent CSF and plasma samples were collected from 15 pregnant sheep throughout the gestation period (days 4, 87 and 116) — a time when plasma concentrations become markedly increased. Compared with non pregnant sheep (n = 15), plasma concentrations of CNP peptides were elevated by 30-fold at days 87 and 116 in pregnant sheep, yet CSF concentrations of CNP and NTproCNP did not differ between the groups — which indicated that central levels of CNP peptides were independently regulated from those in plasma in sheep. As there were no known compounds or physiological setting capable of acutely affecting CSF concentrations of CNP peptides, a series of pilot studies was carried out to screen a number of candidate drugs in sheep, and to examine the effect of changes in live weight and appetite in red deer stags. Despite the dramatic changes in appetite and live weight that occurred throughout the breeding season in red deer stags (> 30 kg live weight gain), CSF concentrations of CNP peptides remained stable. Similarly, CNP and NTproCNP concentration in CSF of sheep remained unchanged following administration with all compounds (including anaesthetics, morphine, a pyrogen and l-deprenyl) except for one; a single iii i.v. dose of dexamethasone (0.25 mg/live weight) was shown to reliably induce a marked increase in CNP and NTproCNP concentration in CSF within 8 h. Following the successful identification of a secretagogue for CNP, subsequent studies were carried out to examine the increases in CNP peptide concentration in CSF and plasma following different doses of dexamethasone (0, 0.025, 0.063, 0.125, 0.25 mg/kg live weight), and to associate dexamethasone-stimulated increases in peptide levels in CSF with changes in peptide concentration and gene expression of NPPC, NPRB and NPRC in tissues sampled from the brain and anterior and posterior pituitary glands. Whereas plasma concentrations of CNP peptides were increased following all doses of dexamethasone, concentrations in CSF were elevated only at the two highest doses. Compared with saline-treated sheep, CNP and NTproCNP content in nervous tissue of dexamethasone-treated sheep (0.25 mg/kg, i.v.) was significantly higher throughout the brain in 6 and 11 of the 14 tissues considered, respectively. Gene expression of NPPC was upregulated in several tissues (anterior pituitary gland, posterior pituitary gland, hypothalamus, hippocampus and pons), which is supportive of increased synthesis of CNP following dexamethasone. The pituitary gland (anterior and posterior), which is known for its enriched levels of CNP content — had a similar concentration of NTproCNP when compared with most tissues sampled from the brain. Furthermore, the NTproCNP:CNP concentration ratio in pituitary gland (1:1) differed from brain (5:1 to 10:1), suggesting that little CNP degradation occurs in the pituitary gland — however the expression of NPRC did not differ between pituitary and brain tissue. Gene expression of NPPC was upregulated in both the anterior and posterior pituitary gland following dexamethasone, despite no significant increase in CNP or NTproCNP concentration in these tissues. Analysis of the molecular forms present in various fluids and tissues using size-exclusion high performance liquid chromatography revealed similar profiles for anterior pituitary gland and plasma extracts, whereby proCNP was present, CNP-53 was the prevalent form and CNP-22 was virtually absent. Together, these findings suggest that CNP processing differs between brain, anterior and posterior pituitary glands and supports the possibility that CNP is secreted directly from the anterior pituitary gland into the circulation. The widespread increase of CNP secretion in multiple tissues across the brain in response to dexamethasone implicates CNP in glucocorticoid actions — possibly related to inflammation or local fluid dynamics mediated by glial cells. These findings pave the way for future studies designed to establish the role of CNP in the CNS in normal iv health in vivo, and to establish a clinical application for this peptide in central settings of pathophysiological disorders in the CNS.