| dc.description.abstract | Anterior pituitary corticotrophs are integral components of the hypothalamus-pituitary-adrenal (HPA) axis controlling the neuroendocrine stress response. A variety
of ion channels regulate intrinsic electrical excitability of corticotrophs and are also
targets for control of excitability by the hypothalamic neuropeptides CRH/AVP as well
as negative feedback by glucocorticoids. The context under which ion channels are
regulated allows corticotrophs to transition between excitability patterns including
from single spikes to ‘pseudoplateau bursting’. Hence, ion channels are regulators of
feed forward mechanisms of ACTH secretion and feedback mechanisms of
glucocorticoids (GCs) regulating the HPA axis function at the pituitary level.
However, a comprehensive analysis of the expression of various ion channels has not
yet been determined in corticotrophs. Furthermore, the functional role of different ion
channels is poorly understood and whether ion channel expression is changed under
different conditions, including during chronic stress or is different between sexes is
unknown.
In Chapter 3, analysis of ion channel mRNA expression by RNA-seq from
corticotrophs FACS purified from POMC-GFP mice was undertaken to determine the
landscape of ion channel expression and to determine whether this was different
between males and females or was modified in response to chronic stress or long term
glucocorticoid exposure. Corticotrophs express a wide range of ion channels however
differential expression between sexes points to the possibility of changes in
corticotroph excitability between sexes. In males, exposure to 2 weeks of daily
restraint stress resulted in modest changes in ion channel gene expression however,
following 4 weeks after recovery a number of ion channel mRNAs were significantly
downregulated. In males treated for 4 weeks with dexamethasone in the drinking water
relatively small changes in ion channel expression were observed after treatment or up
to 4 weeks after dexamethasone withdrawal. Indeed, few ion channels were
differentially regulated by both chronic stress and dexamethasone exposure although
members of the Kv 12.x, Trp and Ttyh ion channel families were differentially
expressed in both datasets.
In Chapter 4, the functional role of Kcnh2 (Kv 11.1) one of the most highly expressed
ion channel mRNAs in our FACS-purified male and female corticotrophs was
determined by patch clamp electrophysiology. In several pituitary cell types, members
of ether-a-go-go related (ERG, Kv 11.x) family of voltage dependent potassium
channels are reported to control both spontaneous and hormone-induced regulation of
excitability; however, their role in corticotrophs is not known. Erg-like currents were
biophysically and pharmacologically isolated in corticotrophs from both sexes using
E4031 (5 µM), a selective inhibitor of ERG family. Both male and female
corticotrophs displayed variable Erg-like current densities with a V50 that would
suggest they are active during spontaneous activity in both sexes. E4031 did not alter
spontaneous electrical activity in either sex, however, CRH/AVP evoked excitability
was inhibited by E4031. In particular, CRH-induced bursting in males and CRH/AVP
evoked event frequency in both males and females was reduced by E4031 as well as
by another Erg family inhibitor, dofetilide (DOF). However, CRH/AVP did not
directly regulate erg-like currents in males or females in voltage clamp studies. This
indicates ERG family plays a role in evoked corticotroph excitability, however Erg-ike currents are not a direct target for CRH/AVP.
In Chapter 5, characterisation of the NMDG+
sensitive background sodium
conductance previously identified in female corticotrophs was examined. The
background current was characterised at the potassium reversal potential (EK) and
NMDG+
decreased the background currents while external low calcium increased the
background currents. While CRH/AVP failed to depolarise corticotrophs in the
presence of NMDG+
, CRH/AVP had no significant effect on the background sodium
current at EK suggesting it is not a direct target for regulation. NALCN, previously
reported to encode background sodium currents in a number of cell types was one of
the highly expressed ion channel mRNAs from our RNA seq analysis. While the
background sodium current in corticotrophs displayed characteristics of NALCN more
studies need to be undertaken to identity if they are responsible.
In conclusion, murine corticotrophs exhibit differential expression of ion channel
mRNAs between sexes, which may explain the differences in their intrinsic
excitability. Gene expression of ion channel changes in response to chronic stress and
long-term dexamethasone treatment seems evident, however the functional
significance remains to be examined. ERG family plays an indirect role in evoked
corticotroph excitability, however their role in CRH-induced bursting activity needs
to be further explored. The background NMDG+
sensitive sodium conductances are
important for electrical excitability and are sensitive to low extracellular calcium but
do not appear to be direct targets for CRH/AVP. However, whether NALCN channels
directly contribute to these needs are to be further explored. Thus, this thesis has
revealed that challenge to HPA axis can change the expression of ion channels in
corticotrophs and that ERG family potassium channels are an important determinant
of corticotroph excitability. | en |
