The effects of different cellular environments on opioid receptor binding

Background: Imaging endogenous opioid peptide (EOP) release using positron emission tomography (PET) would increase our understanding of the opioid systems' role in health and disease. [11C]Carfentanil and [11C]diprenorphine binding is suggested to be sensitive to fluctuations in EOP levels.1,2 Receptor internalisation may contribute to signal changes observed during endogenous release studies. 3 We assessed the binding affinities of five EOPs to rodent opioid receptors (ORs) using [11C]carfentanil and [3H]diprenorphine. Additionally we examined the in vitro binding parameters of both these radioligands in cellular environments representative of those experienced by a receptor following agonistinduced internalisation and assessed the extent each cellular compartment may contribute to overall basal signal observed with [3H]diprenorphine. Methods: Rat whole brain binding assays were performed using [3H]diprenorphine and [11C]carfentanil. Saturation studies: a range of concentrations (0.003-10nM) of both ligands were performed in the presence of three buffers representative of different cellular compartments: Extracellular-50 mMTris-HCl, 140 mMNaCl, 5 mMKCl, 1.5 mMMgCl2, 1.5 mMCaCl2, pH 7.4, 371C Intracellular-50mMTris-HCl, 10mMNaCl, 140 mMKCl, 0.5 mMMgCl2, pH 7.0, 371C Endosomal- 20 mMMES, 10 mMNaCl, 140 mMKCl, 0.5 mMMgCl2, 0.003 mMCaCl2, pH 6.0, 371C. To determine EOP affinity: unlabelled peptides were used in the presence of [3H]diprenorphine and [11C]carfentanil (both 0.3nM) in extracellular buffer at a range of concentrations: b-endorophin (10 pM-10 mM), endomorphin- 1 (3 pM-100 mM), endomorphin-2 (3pM- 100 mM), met-enkephalin (3 pM-100 mM), leu-enkephalin (3 pM-100 mM). To achieve plasma-membrane, microsomal and cytosolic cell compartments, subcellular fractionation assays were performed according to Laduron.4 For each fraction, radioligand binding assays were performed using [3H]diprenorphine (5 nM) and Western blot analysis (30 mg/well) using polyclonal antibodies for m/d/k. Results: A significant reduction in OR density (Bmax) was observed in the endosomal versus the extracellular condition for both radioligands (Table 1; p < 0.001/p < 0.05 for [3H]diprenorphine/[11C]carfentanil). A trend for a reduced affinity (KD) for [11C] carfentanil but not [3H]diprenorphine was observed in the endosomal environment versus extracellular and intracellular (Table 1 p