Impact of Dopamine D2 and Bradykinin B2 receptors interaction. Preliminary study in human pituitary adenomas

ABELEDO MACHADO A; ARGERICH BERGADÀ J; PEÑA ZANONI M; BORNANCINI D; VIDAL N; GARCIA C; GIRONACCI MM; CIRUELA ALFÉREZ F; DIAZ-TORGA G

Congreso; International Congress of Neuroendocrinology; 2022

Pan American Neuroendocrine Society

Introduction/Aim: Most prolactinomas are effectively treated with dopamine D2 receptors (D2R) agonists. Nevertheless, a subset (~20%) became resistant to the treatment and require extirpation. The molecular mechanisms underlying the escape from dopamine inhibition include alterations in D2R signalling. In double transfected HEK293T cells it was demonstrated that bradykinin B2 receptor (B2R) can heteromerize with D2R, abolishing Gi signalling of D2R. On the other hand, it was reported that B2R is highly expressed in human prolactinomas. In the present study, we proposed to assess: 1- the impact of B2R on prolactin secretion. 2-the physical and 3- the functional interaction of D2R-B2R and 4- to determine whether those receptors interact in human pituitary adenomas. Methods/Results: 1-Pituitary explants of C57 adult mice were incubated ex vivo with B2R ligands for 30min. B2R agonist (10-7M to 10-9M) induced a dose-response PRL release in male pituitaries whereas it had a clear dose-response inhibition on PRL secretion in females. Those effects were abolished when pituitary explants were pre-incubated with the specific B2R antagonist (HOE 140, 10-6M). 2-By using the NanoBiT protein-protein interaction assay (NB-assay): 2a- we validated the formation of BR2-D2R measuring NanoLuciferase (NL) activity in HEK293T cell line transiently transfected with human D2R and B2R fused to inactive fragments of the split NL. 2b- we studied the coupling of B2R to G proteins in cells transiently transfected with B2R and Gi, Gs, G12/13 or Gq, fused to inactive fragments of the split NL. We found that B2R recruits Gq and Gi after stimulation with B2R specific agonist.3-We measured the Ca2+ mobilization by Fluo4-NW Assay in HEK293T transiently transfected with B2R alone or B2R-D2R. B2R agonist (100nM) increased [Ca2+]i in cells expressing B2R alone or B2R-D2R. D2R agonist nor antagonist did not alter B2R signal, suggesting that the coupling of B2R to Gq protein is not altered in the B2R-D2R heteromer. 4-B2R-D2R interaction was measured in human pituitary adenomas by AlphaLisa assay. We found B2R-D2R complexes in prolactinomas and non-secreting tumours, but not in mixed tumours (GH+PRL). Conclusions: A- The opposite effect of B2R activation observed among genders on PRL secretion suggests a gender-specific G protein coupling of B2R (and or B2R-D2R complex), and a gender specific function of pituitary kinins. B- We hypothesized that D2R-B2R dimerization is increased in prolactinomas and non-secreting pituitary adenomas disturbing D2R signalling by prevailing the D2R-B2R coupling to Gq, promoting resistance to D2R agonists.