title: [(35)S]GTPγS binding and opioid tolerance and efficacy in mouse spinal cord.
abstract: 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 [(35)S]GTPγS (guanosine 5'-O-(3-[(35)S] thio)triphosphate) binding in mouse spinal cord membranes and compared it directly to spinal analgesic tolerance. The [(35)S]GTPγS binding assay was used to estimate efficacy (E(max) and τ; 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 [(35)S]GTPγS 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 7days with morphine, oxycodone, hydromorphone, etorphine or fentanyl and [(35)S]GTPγS 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 [(35)S]GTPγS binding (decrease in E(max)) was observed following etorphine and fentanyl treatment only. These treatment protocols downregulate μ-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) μ-opioid receptor density produced a corresponding change in opioid agonist stimulated [(35)S]GTPγS 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 [(35)S]GTPγS assay is particularly responsive to changes in μ-opioid receptor density.
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, 8000 Utopia Parkway, Queens, NY 11439, United States.
authors: Priyanka A
authors: PA
authors: Madia
authors: Dipesh M
authors: DM
authors: Navani
authors: Byron C
authors: BC
authors: Yoburn
mesh_terms: D000465:Algorithms; D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004361:Drug Tolerance; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D007278:Injections, Spinal; D008297:Male; D008566:Membranes / Q000187:drug effects; D051379:Mice; D009020:Morphine / Q000494:pharmacology; D010147:Pain Measurement / Q000187:drug effects; D011869:Radioligand Assay; D011930:Reaction Time / Q000187:drug effects; D017450:Receptors, Opioid, mu / Q000187:drug effects; D013116:Spinal Cord / Q000187:drug effects*; D013462:Sulfur Radioisotopes
chemical_list: D000701:Analgesics, Opioid; D017450:Receptors, Opioid, mu; D013462:Sulfur Radioisotopes; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009020:Morphine; D019204:GTP-Binding Proteins