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

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‐Ala2,N‐Me‐Phe4,Glyol5]‐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‐NH2) 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.