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Determination of the relative efficacies of dopamine D2 receptor agonists by global fitting of the operational model in the absence and presence of phenoxybenzamine. The Black and Leff operational model (1) can estimate agonist efficacy from concentration response curves (CRCs) alone through receptor depletion by irreversible antagonists. We investigate the feasibility of this approach in dopamine D2 receptor (D2R) reporter gene and internalisation assays, by comparing agonist CRCs with or without phenoxybenzamine (PBz), a reported D2R irreversible antagonist (2). Experiments used CHO cells stably expressing SNAP-tagged human D2R (long isoform) and the CRE-SPAP reporter gene cDNAs. For receptor internalisation (3), cells were labelled with SNAPsurface BG-AF488 (30 min, 0.1μM) before 1h agonist treatment and PFA fixation. Plates were imaged (MDC IX Ultra plate reader) and D2R internalisation quantified with a granularity algorithm. For SPAP (4), serum starved cells were agonist treated for 1h prior to a 5h, 3μM forskolin incubation. SPAP activity was determined as previously described. Where appropriate, cells were preincubated for 30min with 1µM or 3µM PBz before agonist stimulation. Agonist pKAs and τ estimates were determined from global fitting of the PBz treated SPAP agonist CRCs from individual experiments using the operational model in GraphPad Prism v6.0. In the SPAP reporter gene assay, dopamine (DA) and bromocriptine (BC) were full agonists and aripiprazole (AP) a partial agonist relative to the quinpirole (QP) inhibitory response, with an order of potency BC>AP>QP=DA (Table 1). QP was 19-fold less potent in the internalisation compared to SPAP assay (p< 0.01, Student’s t test), while BC became a partial agonist relative to QP and AP had no effect (Table 1). PBz pretreatment abolished QP internalisation responses (at 1 µM, data not shown) and progressively decreased the potency and Emax of agonist responses in the SPAP assay. Global fitting of the operational model to the SPAP CRCs (Table 1) generated a rank order of log τ values in the absence of PBz of QP=DA>BC>AP and an order of pKA affinity estimates AP=BC>DA=QP. Effects of PBz persisted after removal by wash out prior to agonist treatment (data not shown).
Table 1: Maximal responses, potencies and τ values of ligands. Data represent mean ± s.e.m. of 3-4 experiments performed in triplicate with Emax normalised to 10µM QP. Operational model fits gave global pKA estimates and log τ values for vehicle, 1µM and 3µM PBz (log τ absolute values also dependent on receptor number). NE: No effect; * P<0.05 compared to 10µM QP (1 way ANOVA, Dunnett’s post test). We conclude that the increased receptor reserve from the SPAP assay over receptor internalisation sufficiently explains the differences in relative agonist potency and Emax between the assays. Operational model fitting of SPAP agonist CRCs in the absence or presence of PBz provides estimates of agonist affinity and relative efficacy consistent with the literature (5), with τ values as estimates of efficacy. (1)Black et al, Br J Pharmacol 84: 561, 1985 (2)Hamblin & Creese, Mol Pharm 21:44, 1981 (3)Kilpatrick et al, Biochim. Biophys. Acta 1823: 1068, 2012 (4)Baker JG, PLoS One 5: e15487, 2010 (5)Gardner & Strange, Br J Pharmacol 124: 978, 1998 Supported by a CASE studentship funded by the BBSRC and GSK
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