Neutral antagonist activity of naltrexone and 6beta-naltrexol in naïve and opioid-dependent C6 cells expressing a mu-opioid receptor.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Divin
M F
MF
Department of Pharmacology, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-5632, USA.
Bradbury
F A
FA
Carroll
F I
FI
Traynor
J R
JR
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D054314:Drug Inverse Agonism; D054333:Drug Partial Agonism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D006801:Humans; D009020:Morphine / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D010880:Piperidines / Q000737:chemistry; D011869:Radioligand Assay; D051381:Rats; D017465:Receptors, Opioid, delta / Q000037:antagonists & inhibitors; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013004:Somatostatin / Q000494:pharmacology
C529311:CTAP octapeptide; D010446:Peptide Fragments; D010880:Piperidines; C541709:RTI 5989-25; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; C010410:6 beta-hydroxynaltrexone; D013004:Somatostatin; D009271:Naltrexone; D009020:Morphine; D000242:Cyclic AMP; D000262:Adenylyl Cyclases