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Keywords:

  • µ-opioid receptor;
  • constitutive activity;
  • inverse agonism;
  • protean agonism;
  • adenylyl cyclase;
  • cAMP overshoot;
  • naltrexone;
  • 6β-naltrexol;
  • RTI-5989-25;
  • CTAP

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.