Corticotroph isolation from Pomc-eGFP mice reveals sustained transcriptional dysregulation characterising a mouse model of glucocorticoid-induced suppression of the hypothalamus-pituitary-adrenal axis.

Glucocorticoids (GC) are prescribed for periods > 3 months to 1%-3% of the UK population; 10%-50% of these patients develop hypothalamus-pituitary-adrenal (HPA) axis suppression, which may last over 6 months and is associated with morbidity and mortality. Recovery of the pituitary and hypothalamus is necessary for recovery of adrenal function. We developed a mouse model of dexamethasone (DEX)-induced HPA axis dysfunction aiming to further explore recovery in the pituitary. Adult male wild-type C57BL6/J or Pomc-eGFP transgenic mice were randomly assigned to receive DEX (approximately 0.4 mg kg-1 bodyweight day-1 ) or vehicle via drinking water for 4 weeks following which treatment was withdrawn and tissues were harvested after another 0, 1, and 4 weeks. Corticotrophs were isolated from Pomc-eGFP pituitaries using fluorescence-activated cell sorting, and RNA extracted for RNA-sequencing. DEX treatment suppressed corticosterone production, which remained partially suppressed at least 1 week following DEX withdrawal. In the adrenal, Hsd3b2, Cyp11a1, and Mc2r mRNA levels were significantly reduced at time 0, with Mc2r and Cyp11a1 remaining reduced 1 week following DEX withdrawal. The corticotroph transcriptome was modified by DEX treatment, with some differences between groups persisting 4 weeks following withdrawal. No genes supressed by DEX exhibited ongoing attenuation 1 and 4 weeks following withdrawal, whereas only two genes were upregulated and remained so following withdrawal. A pattern of rebound at 1 and 4 weeks was observed in 14 genes that increased following suppression, and in six genes that were reduced by DEX and then increased. Chronic GC treatment may induce persistent changes in the pituitary that may influence future response to GC treatment or stress.

Heterogeneity of Calcium Responses to Secretagogues in Corticotrophs From Male Rats.

Heterogeneity in homotypic cellular responses is an important feature of many biological systems, and it has been shown to be prominent in most anterior pituitary hormonal cell types. In this study, we analyze heterogeneity in the responses to hypothalamic secretagogues in the corticotroph cell population of adult male rats. Using the genetically encoded calcium indicator GCaMP6s, we determined the intracellular calcium responses of these cells to corticotropin-releasing hormone and arginine-vasopressin. Our experiments revealed marked population heterogeneity in the response to these peptides, in terms of amplitude and dynamics of the responses, as well as the sensitivity to different concentrations and duration of stimuli. However, repeated stimuli to the same cell produced remarkably consistent responses, indicating that these are deterministic on a cell-by-cell level. We also describe similar heterogeneity in the sensitivity of cells to inhibition by corticosterone. In summary, our results highlight a large degree of heterogeneity in the cellular mechanisms that govern corticotroph responses to their physiological stimuli; this could provide a mechanism to extend the dynamic range of the responses at the population level to allow adaptation to different physiological challenges.

Reduced hypothalamic vasopressin secretion underlies attenuated adrenocorticotropin stress responses in pregnant rats.

We sought to explain decreased ACTH secretory responses to stress in pregnant rats by investigating hypothalamic CRH and vasopressin secretion and actions on anterior pituitary corticotrophs. In late pregnancy median eminence, CRH content was reduced (by 12%). Anterior pituitary proopiomelanocortin mRNA expression, measured by in situ hybridization but not radioimmunoassayed ACTH content, was also reduced (by 45% on d 21); CRH receptor (CRHR)1 mRNA expression was unaltered in pregnancy, but V1b receptor mRNA expression was reduced (by 19%). ACTH secretory responses, measured in jugular blood, to CRH (200 ng/kg iv) or vasopressin (1.7 microg/kg, iv) were reduced on d 21 vs. virgins (49% and 44%), but the response to combined CRH and vasopressin injection was intact. Either antalarmin (CRHR1 antagonist; 20 mg/kg ip) or dP(Tyr(Me)2),Arg-NH2(9))AVP (V1a/b antagonist; 10 microg/kg, iv) pretreatment reduced the ACTH secretory response to forced swimming (90 sec) in virgin rats (by 57% and 40%), but only antalarmin was effective in pregnant rats (53% decrease). In vitro, measuring ACTH secretion from acutely dispersed anterior pituitary cells showed increased corticotroph sensitivity in pregnancy to CRH and to CRH augmentation by vasopressin, attributable to increased intracellular cAMP action. Hence, in late pregnancy, reduced anterior pituitary CRHR1 or V1b receptor expression did not impair corticotroph responses to CRH or vasopressin. Rather, diminished secretagogue secretion in vivo accounts for reduced action of stress levels of exogenous CRH or vasopressin alone; the late pregnancy attenuated ACTH secretory response to swim stress is deduced to be due to reduced vasopressin release by parvocellular paraventricular nuclei neurones.

Efficacy of Semliki Forest virus transduction of bovine adrenal chromaffin cells: an analysis of heterologous protein targeting and distribution.

In using chromaffin cells as a model for studying the mechanism of regulated exocytosis, there is a requirement for an efficient, safe, and robust system for the transduction and expression of heterologous cDNA in these cells. We have used Semliki Forest virus to transduce cDNAs encoding various proteins fused to enhanced green fluorescent protein (EGFP) into cultured bovine adrenal cells. Transduction is highly efficient but has no significant effect on the steady state levels of several endogenous proteins or of catecholamines in the transfected cells. Furthermore, the transfected cells show depolarization-induced calcium currents and nicotine-induced catecholamine release. We present data to show that virally transduced proteins are targeted to their intracellular locations correctly in chromaffin cells. The fusion protein pro-ANF-EGFP is specifically targeted to large dense-core vesicles as shown by its colocalization with acidophilic dyes and chromogranin A, making this a useful system for the study of secretory vesicle dynamics.