The effects of different cellular environments on opioid receptor binding

Darren Quelch, Christine Parker, David Nutt, Robin Tyacke

Research output: Contribution to journalConference articlepeer-review


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
Original languageEnglish
Article numberP016
Pages (from-to)53-54
Number of pages2
JournalJournal of Cerebral Blood Flow and Metabolism
Issue numberIssue_1_Suppl
Publication statusPublished - 1 Aug 2012
Externally publishedYes
Event9th International Symposium on Functional Neuroreceptor
Mapping of the Living Brain
- Baltimore, United States
Duration: 9 Aug 201212 Aug 2012
Conference number: 9th


  • opiate receptor
  • diprenorphine
  • carbon 11
  • carfentanil
  • leucine enkephalin
  • ligand
  • endomorphin 1
  • peptide
  • endorphin
  • metenkephalin
  • endomorphin 2
  • receptor
  • opiate peptide
  • cell protein
  • polyclonal antibody
  • radioligand
  • receptor binding
  • brain
  • environment
  • intracellular space
  • pH
  • tissues
  • binding assay
  • binding site
  • in vitro study
  • cell membrane
  • internalization (cell)
  • dopaminergic system
  • cytosolic fraction
  • cytosol
  • fractionation
  • competition
  • agonist
  • density
  • Western blotting
  • rat
  • assay
  • cell fractionation
  • parameters
  • rodent
  • immunoreactivity
  • binding affinity
  • health
  • positron emission tomography
  • imaging


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