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

Abstract

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, [ 35 S]GTPγS (guanosine-5′-O-(3-[ 35 S]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]-(3 R ,4 R )-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.