[35S]GTPγS binding and opioid tolerance and efficacy in mouse spinal cord

The present study examined efficacy of a series of opioid agonists and then using chronic in vivo treatment protocols, determined tolerance to opioid agonist stimulated [S-35]GTP gamma S (guanosine 5'-O-(3-[S-35] thio)triphosphate) binding in mouse spinal cord membranes and compared it directly to spinal analgesic tolerance. The [S-35]GTP gamma S binding assay was used to estimate efficacy (E-max and tau; Operational Model of Agonism) of a series of opioid agonists for G-protein activation in mouse spinal cord. The rank order of opioid agonist efficacy determined in the [S-35]GTP gamma S assay using the Operational Model and E-max was similar. These efficacy estimates correlated with historical analgesic efficacy estimates. For tolerance studies, mice were continuously treated s.c. for 7 days with morphine, oxycodone, hydromorphone, etorphine or fentanyl and [S-35]GTP gamma S studies were conducted in spinal cord membranes. Other mice were tested in i.t. analgesia dose response studies (tailflick). Tolerance to DAMGO ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin) or morphine stimulated [S-35]GTP gamma S binding (decrease in E-max) was observed following etorphine and fentanyl treatment only. These treatment protocols downregulate mu-opioid receptor density whereas morphine, oxycodone and hydromorphone do not. Spinal analgesic tolerance was observed following all treatment protocols examined (morphine, oxycodone and etorphine). Opioid antagonist treatment that specifically upregulates (chronic naltrexone) or downregulates (clocinnamox) mu-opioid receptor density produced a corresponding change in opioid agonist stimulated [S-35]GTP gamma S binding. Although receptor downregulation and G-protein uncoupling are among potential mechanisms of opioid tolerance, the present results suggest that uncoupling in mouse spinal cord plays a minor role and that the [S-35]GTP gamma S assay is particularly responsive to changes in mu-opioid receptor density. (C) 2011 Elsevier Inc. All rights reserved.