Negotiating with a pea-sized hormone factory - the mediatory role of pituitary formyl peptide receptor (FPR) ligands in times of stress

Vance Naughton1, Bradley Spencer-Dene2, Chris John1, Julia Buckingham1. 1Imperial College London, Centre for Integrative Mammalian Physiology and Pharmacology, W12 0NN, London, United Kingdom, 2London Research Institute, Experimental Pathology, WC2A 3LY, London, United Kingdom.

Background and purpose: Vertebrates typically respond to stress by secreting glucocorticoids (GCs) to regain homeostasis. In turn, GCs inhibit secretion of hypothalamic corticotrophic hormone (CRH) and pituitary adreno-corticotrophic hormone (ACTH), ending the stress response by negative feedback. The pituitary folliculo-stellate cells express annexin A1 (ANXA1)(1), a mediator protein shown to be necessary for the negative feedback actions of GCs(2). Previous work has demonstrated that ANXA1 acts via formyl peptide receptors (FPR in man, Fpr in rodents)(3). Anterior pituitary tissue does not express the Fpr1 subtype, whilst functional data has implied ligands selective for Fpr2 or Fpr3 mediated feedback-like inhibition of ACTH(4). In the present work, we hypothesised that Fpr2-selective ligands would inhibit secretagogue-induced ACTH release from pituitary tissue in vitro.

Experimental approach: Firstly, we cultured anterior pituitary cells from male Sprague-Dawley rats (n = 12, male, 200-220g) in CRH-stimulated [100nmol.L-1] or basal conditions, along with peptide agonists for Fpr2 (WKYMVm [10-10, 10-9, 10-8(5), 10-7, and 10-6 mol.L-1] and F2L [50, 100, 200(6), 400, and 800 nmol.L-1]) or Fpr1 (fMLF [10-9, 10-8, 10-7, 10-6, and 10-5 mol.L-1]). ACTH was measured by radioimmunoassay (RIA) after 4 hours. Data (mean ± SEM) were analysed by two-way ANOVA with Bonferonni and Dunnet’s post hoc tests. Secondly, to determine the spatial distribution of Fpr2/3 in the anterior pituitary, we used fluorescently-labelled antibodies against markers of pituitary cell subtypes and green fluorescent protein (GFP) signal from formalin-fixed paraffin-embedded pituitary sections of Fpr2/3-null/GFP-positive mice (n = 4, male).

Key results: Concordant with previous data (4), low dose fMLF did not affect ACTH release, confirming that Fpr1-selective ligands are not instrumental in ACTH regulation. Surprisingly, WKYMVm and F2L both stimulated ACTH release. In separate experiments, two-way ANOVA revealed a significant main effect of WKYMVm and F2L on ACTH release (F(7,176) = 71.38 and 49.17 respectively; P<0.0001) and post hoc tests revealed ACTH release from CRH-stimulated, WKYMVm-treated [10-9 mol.L-1] and CRH-stimulated, F2L-treated cells [800 nmol.L-1] versus CRH control was 20% (1680 pg.ml-1 ± 111 vs 1320 pg.ml-1 ± 75) and 60% (2953 pg.ml-1 ± 188 vs 1817 pg.ml-1 ± 227) higher, respectively (P<0.05). The signal for GFP as a proxy for Fpr2/3, was evident in corticotrophs (anti-ACTH), somatotrophs (anti-growth hormone) and lactotrophs (anti-prolactin), but, importantly, not in folliculo-stellates (anti-S100b).

Conclusions and implications: In conclusion, our work shows rodent Fpr2-selective ligands stimulate ACTH release and suggests Fpr2 might not be instrumental in mediating the feedback-like regulation of pituitary ACTH release. Ongoing work will determine whether inhibition of ACTH is mediated by Fpr3 ligands, namely, 15-epi lipoxin A4.

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