Down-regulation of 3H-lofentanil binding to opiate receptors in different cultured neuronal cells

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Abstract
Summary There was stereospecific binding of 3H-lofentanil (K D value = 1.53 nM) to membranes of neuroblastoma-glioma NG 108-15 cells which are known to bear high affinity binding sites for enkephalin derivatives (δ-opiate receptor subtype). There was no high affinity specific binding of the μ-opiate specific ligand 3H-sufentanil. The specific binding of 3H-lofentanil to δ-opiate receptor subtype was down-regulated (decrease in B max value without change in the K D value) after prolonged incubation of the cells in the presence of leu- and met- enkephalin (0.1 μM). There was no down-regulation of the opiate receptors (3H-lofentanil and 3H-d-ala-d-leu-enkephalin specific binding) after incubation of NG 108-15 cells with drugs from the fentanyl series (alfentanil or sufentanil). In cultured neurones from rat forebrain (15 day old embryos), the 3H-lofentanil binding was specific with high affinity (K D: 0.048 nM) and a slow dissociation rate similar to that in adult rat cortex. Drugs of the fentanyl series (4-anilino-piperidines) were potent displacers whereas agonists of the δ- (enkephalin derivatives), σ (phencyclidine, haloperidol, 3-hydroxyphenyl-propylpiperidine) or K- (U 50488) opiate sites had a low affinity (K i > 0.5 μM) for 3H-lofentanil specific binding sites. Since there was also specific binding of 3H-sufentanil, the opiate receptors in cultured neurones seem to be mainly of the μ-subtype and this is consistent with the ontogeny of opiate receptors subtypes. These receptors were down-regulated after incubation in the presence of etorphine, sufentanil and alfentanil but not enkephalin derivatives. These results strongly suggest specific binding of 3H-sufentanil and 3H-lofentanil mainly to the so-called μ-opiate receptors in cultured neurones and a specific binding of 3H-lofentanil to lower affinity δ-opiate receptors in neuroblastoma-glioma cells. The down-regulation of the μ-opiate binding sites in cultured neurones and that of the δ-site in neuroblastoma × glioma hybrid cells were dose-and temperature-dependent, induced by the corresponding high affinity agonists and prevented by naloxone. Morphine did not induce down-regulation of μ or δ receptor sites, possibly because of a partial antagonist effect on both receptor subtypes.