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Title: Regulation of murine corticotroph cell excitability
Author: Duncan, Peter James
ISNI:       0000 0004 5990 9606
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.
Supervisor: Shipston, Michael ; Ludwig, Mike Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: corticotroph ; HPA axis. ; stress ; glucocorticoids ; BK channel