Neutral antagonist activity of naltrexone and 6β-naltrexol in naïve and opioid-dependent C6 cells expressing a μ-opioid receptor

Abstract

Background and purpose: Adenylyl cyclase sensitization occurs on chronic agonist activation of μ-opioid receptors and is manifested by an increase in cAMP levels (overshoot) on challenge with antagonist. It has been proposed that a long lasting constitutively active receptor is formed on chronic μ-opioid exposure and that antagonists with inverse agonist activity rapidly return the receptor to a basal state causing a cAMP overshoot and a more severe withdrawal response in vivo. This hypothesis depends on an accurate characterization of neutral and inverse agonist properties of opioid antagonists. Experimental approach: C6 glioma and HEK293 cells expressing μ-opioid receptors were used. Opioid antagonists were examined for their ability to induce a cAMP overshoot following chronic treatment with the agonist DAMGO ([D-Ala 2,N-Me- Phe 4,Glyol 5]-enkephalin). The compounds were also characterized as agonists, inverse agonists or neutral antagonists by using assays for competitive binding, [ 35S]GTPγS (guanosine-5′- O-(3-[ 35S]thio)triphosphate) binding and changes in cell surface receptor expression. Key results: Naltrexone, 6β-naltrexol and naloxone were indistinguishable to the μ-opioid receptor in the opioid-naïve or dependent state and acted as neutral antagonists. The δ-opioid receptor inverse agonist RTI-5989-25 [(+)-N-[trans-4′-(2-methylphenyl)-2′- butenyl]-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine], a 3,4-dimethyl-4-(3- hydroxyphenyl)-piperidine, was an inverse agonist at the μ-opioid receptor, and the peptide antagonist CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2) showed variable, assay-dependent properties. All the antagonists precipitated the same degree of cAMP overshoot in opioid-dependent cells. Conclusions and implications: Antagonists at the μ-opioid receptor may be neutral or show inverse agonist activity. Formation of a constitutively active μ-opioid receptor is not a requirement for the development or expression of adenylyl cyclase sensitization. © 2009 The British Pharmacological Society.