Abstract
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 language | English |
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Article number | P016 |
Pages (from-to) | 53-54 |
Number of pages | 2 |
Journal | Journal of Cerebral Blood Flow and Metabolism |
Volume | 32 |
Issue number | Issue_1_Suppl |
DOIs | |
Publication status | Published - 1 Aug 2012 |
Externally published | Yes |
Event | 9th International Symposium on Functional Neuroreceptor Mapping of the Living Brain - Baltimore, United States Duration: 9 Aug 2012 → 12 Aug 2012 Conference number: 9th |
Keywords
- 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