|dc.description.abstract||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
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
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.||en