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Title: Regulation of calcium signalling in murine corticotrophs
Author: Deng, Mengdie
ISNI:       0000 0004 8509 2281
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Anterior pituitary corticotrophs, the central components of the hypothalamic-pituitaryadrenal (HPA) axis, are important for controlling the neuroendocrine response to stress. In response to a stressor, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are released from the hypothalamic neuroendocrine neurones and act synergistically on corticotrophs to stimulate the release of adrenocorticotrophin hormone (ACTH). In turn, ACTH stimulates the secretion of glucocorticoids from adrenal glands that exerts negative feedback on the HPA axis. CRH and AVP have been shown to stimulate different patterns of electrical excitability in corticotrophs, with CRH inducing electrical bursting behaviour that is dependent upon large conductance voltage- and calcium-activated potassium (BK) channels whereas AVP-induced spiking is BK channel independent. Although CRH and AVP are known to control intracellular free calcium ([Ca2+]i) by different signalling pathways, several important questions remain. Firstly, most previous studies have examined CRH and/or AVP-evoked changes in calcium signalling in response to a single supraphysiological concentration of secretagogue whereas in vivo corticotrophs must respond to repeated changes in CRH and AVP. Secondly, although CRH and AVP synergise at the level of ACTH secretion, whether synergy is also observed at the level of intracellular free calcium is essentially unknown. Thirdly, although CRH and AVP regulate different patterns of electrical excitability, whether CRH-, or AVP-induced calcium signalling is modified by BK channels is not known. Fourthly, BK channels are subject to regulation by post-translational modifications, such as S-acylation by zDHHC23, however, whether S-acylation controls CRH-induced calcium signalling is not known. To address these questions and to test the overarching hypothesis that CRH-, but not AVP-induced [Ca2+]i signalling is dependent upon functional BK channels, a new lentiviral-mediated POMC-GCaMP6s reporter was developed and tested to allow specific labelling of live murine corticotrophs in vitro for calcium imaging recordings. [Ca2+]i signalling was characterised in wild-type corticotrophs under basal conditions and following repeated exposure to pulses of physiological levels of CRH (0.2 nM) and/or AVP (2 nM). Murine wild-type corticotrophs showed highly variable spontaneous [Ca2+]i signalling. Stimulation with CRH induced a significant sustained and repeatable increase of [Ca2+]i that lasted longer than the exposure to CRH. In contrast, AVP evoked two phenotypes of [Ca2+]i responses, oscillations and sustained increases, that were highly reproducible. No significant differences were observed between corticotrophs from male or female mice. Synergistic [Ca2+]i responses between CRH and AVP were observed in ~ 30% of female wild-type corticotrophs, although this was not significantly different at the population level. However, this was never observed in male corticotrophs. To test the role of BK channels in [Ca2+]i responses, we took both genetic and pharmacological approaches. Genetic deletion of BK channels displayed sex differences in regulating corticotroph spontaneous [Ca2+]i signalling. Repeated CRH stimulation induced significantly reduced [Ca2+]i responses in male, but not female, BK-KO corticotrophs compared to wild-type controls. However, CRH-evoked [Ca2+]i signalling was unaffected by acute pharmacological inhibition of BK channels with paxilline suggesting a compensatory mechanism may underlie the change in CRH-evoked [Ca2+]i responses in BK-KO corticotrophs. Genetic deletion of BK channels had no impact on [Ca2+]i responses to repeated AVP stimulation in either male or female corticotrophs. Genetic deletion of the S-acyl transferase zDHHC23 had no significant effect on spontaneous [Ca2+]i signalling. Both male and female zDHHC23-KO corticotrophs showed a progressive attenuation in [Ca2+]i responses to repeated CRH stimulation. However, compared to wild-type corticotrophs, [Ca2+]i responses evoked by repeated CRH or AVP stimulation were unaffected by knockout of zDHHC23 in either male or female corticotrophs. In conclusion, development of the lentiviral POMC-GCaMP6 calcium reporter allowed analysis of spontaneous and secretagogue-evoked [Ca2+]i responses specifically in corticotrophs in vitro. CRH evoked sustained elevations of [Ca2+]i whereas AVP evoked two patterns of [Ca2+]i response: sustained elevation and oscillation. No significant differences were observed between wild-type male and female corticotrophs although synergy between CRH and AVP was only observed in female corticotrophs. Genetic deletion of BK channels reduced CRH- but not AVP-induced [Ca2+]i signalling in male but not female corticotrophs. However, this is likely a compensatory mechanism as acute pharmacological inhibition of BK channels in wild-type corticotrophs did not show the same effect. zDHHC23 plays a minor role in regulating [Ca2+]i signalling in corticotrophs. Further studies are warranted to investigate the link between changes in electrical excitability, calcium signalling and the control of ACTH release in corticotrophs. The approach developed here should allow us to probe the mechanism of [Ca2+]i signalling and its regulation that is central to understand the physiological role of corticotrophs and the control of the stress axis.
Supervisor: Shipston, Michael ; Le Tissier, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: BK channel ; calcium signalling ; corticotroph ; zDHHC23