enkephalin--ala(2)-mephe(4)-gly(5)- and 6-beta-hydroxynaltrexone

Adenylyl cyclase sensitization occurs on chronic agonist activation of mu-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 mu-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.. C6 glioma and HEK293 cells expressing mu-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]GTPgammaS (guanosine-5'-O-(3-[(35)S]thio)triphosphate) binding and changes in cell surface receptor expression.. Naltrexone, 6beta-naltrexol and naloxone were indistinguishable to the mu-opioid receptor in the opioid-naïve or dependent state and acted as neutral antagonists. The delta-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 mu-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.. Antagonists at the mu-opioid receptor may be neutral or show inverse agonist activity. Formation of a constitutively active mu-opioid receptor is not a requirement for the development or expression of adenylyl cyclase sensitization.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Line, Tumor; Cyclic AMP; Drug Inverse Agonism; Drug Partial Agonism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Morphine; Naloxone; Naltrexone; Peptide Fragments; Piperidines; Radioligand Assay; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Somatostatin

Constitutively active mu-opioid receptors (mu* receptors) are reported to be formed following prolonged agonist treatment of cells or whole animals. mu* receptors signal in the absence of activating ligand and a blockade of mu* activation of G-proteins by naloxone and naltrexone has been suggested to underlie the profound withdrawal syndrome precipitated by these antagonists in vivo. In this issue of the Journal, Divin et al. examined whether treatment of C6 glioma cells with mu-opioid receptor agonists produced constitutively active mu-opioid receptors or other commonly reported adaptations to prolonged agonist treatment. Adenylyl cyclase superactivation was readily apparent following agonist treatment but there was no evidence of the formation of constitutively active mu-opioid receptors. This result challenges the notion that prolonged agonist exposure inevitably produces mu* receptors, and is consistent with many studies of adaptations in neurons produced by chronic agonist treatment. The investigators provide no explanation of their failure to see mu* receptors in C6 cells, but this is perhaps understandable because the molecular nature of mu* receptors remains elusive, and the precise mechanisms that lead to their formation are unknown. Without knowing exactly what mu* receptors are, how they are formed and how they signal, understanding their role in cellular adaptations to prolonged opioid treatment will remain impossible. Studies such as this should refocus attention on establishing the molecular mechanisms that underlie that phenomenon of mu* receptors.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Line, Tumor; Drug Inverse Agonism; Drug Partial Agonism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Morphine; Naltrexone; Radioligand Assay; Rats; Receptors, Opioid, mu

Alpha- and beta-naltrexol derived esters 9 and 10 and ethers 11 and 12, each containing the alpha, beta-unsaturated ester functionality, were prepared as conformationally more flexible analogues of spiro-alpha-methylene-gamma-lactones 5 and 6. All were active in the opioid radioreceptor binding assay against [3H]bremazocine and more active against [3H]DAGO, indicating mu-subtype selectivity, but only ether 12 showed significant irreversible activity. We conclude that small structural changes, made in very closely related electrophilic opioids, lead to changes in receptor binding. All four compounds were long-acting antagonists to morphine in mice, with ester 10 being approximately equipotent with naltrexone.

Topics: Animals; Benzomorphans; Binding, Competitive; Cattle; Caudate Nucleus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Esters; Ethers; In Vitro Techniques; Ligands; Methacrylates; Naltrexone; Receptors, Opioid; Structure-Activity Relationship