CGRP adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

摘要

a Expression of CALCR, CALCRL, RAMP1, RAMP2, and RAMP3 genes in HUVECs. Data normalised to GAPDH expression. n = 3 independent experiments. b–f Dose–response curves were constructed for HUVECs stimulated with CGRP, AM or AM2 and the cAMP levels quantified relative to forskolin (100 μM) (n = 7) (b), mobilisation of Ca2+i relative to ionomycin (10 μM) (n = 6) (c), intracellular ERK1/2 phosphorylation relative to PMA (10 μM) (n = 4) (d), total nitric oxide production relative to acetylcholine (10 μM) (n = 3) (e), and extent of cell proliferation (after 72 h) relative to vector treated control and VEGF (n = 3–6) (f). Data are analysed using a three-parameter non-linear regression curve or the operational model of receptor agonism27. g–h Signalling bias plots were calculated as ∆Log(τ/KA) (g) or ∆∆Log(τ/KA) (h) for each agonist and for each signalling pathway. Determination of values requires normalisation to a reference agonist (AM) alone in (g), while for (h) values were normalised to both a reference agonist (AM) and a reference pathway (cAMP). i Heatmaps representing the signalling properties between HUVEC and HUAEC cells for potency, effector maximum and the transducer coefficient. All data represent mean ± SEM for n repeats. j Representation of the signalling outcomes as a result of AM-mediated receptor activation in a HUVEC. Solid arrows indicate known pathways. Dashed arrows represent possible pathways.