Tags

Type your tag names separated by a space and hit enter

Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells.
Mol Pharmacol. 1995 Apr; 47(4):848-54.MP

Abstract

The ability of mu-opioid agonists to activate G proteins has been demonstrated by studying the binding of the GTP analogue guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) to membranes from the human neuroblastoma SH-SY5Y cell line. The potent opioid agonist fentanyl caused an approximate doubling of basal [35S]GTP gamma S binding in a naloxone-sensitive manner, confirming this to be an opioid receptor-mediated process. The presence of GDP was necessary to observe this effect. Pretreatment of the cells with pertussis toxin (100 ng/ml, for 24 hr) completely prevented the fentanyl-stimulated increase in [35S]GTP gamma S binding and lowered the basal binding of [35S]GTP gamma S. These latter data suggest an involvement of Gi and/or Go proteins and their activation by added membrane-bound receptors even in the absence of agonist. The order of potency of a series of opioid agonists in stimulating the binding of [35S]GTP gamma S was buprenorphine > cyclazocine = levallorphan > nalorphine > [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) > fentanyl > morphine > pentazocine. DAMGO, fentanyl, and morphine were full agonists but the remaining compounds showed decreasing levels of intrinsic activity in the order buprenorphine > pentazocine > cyclazocine = nalorphine > levallorphan. The opioid antagonist naloxone was without effect. Under the conditions of the [35S]GTP gamma S assay, binding of agonists was to a high affinity site, indicating that a high agonist affinity state of the mu-opioid receptor is responsible for the observed stimulation of [35S]GTP gamma S binding. The level of [35S]GTP gamma S binding (597 fmol/mg of protein) stimulated by DAMGO was 2-fold greater than the maximal number of mu-opioid agonist binding sites (Bmax) determined using [3H]DAMGO (254 fmol/mg of protein). The opioid agonist-mediated stimulation of [35S]GTP gamma S binding in SH-SY5Y cell membranes thus provides a "functional" measure of agonist occupation of mu-opioid receptors and offers a simple method for the determination of efficacy and intrinsic activity of mu-opioid agonists.

Authors+Show Affiliations

Department of Cell Physiology and Pharmacology, University of Leicester, UK.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7723747

Citation

Traynor, J R., and S R. Nahorski. "Modulation By Mu-opioid Agonists of guanosine-5'-O-(3-[35S]thio)triphosphate Binding to Membranes From Human Neuroblastoma SH-SY5Y Cells." Molecular Pharmacology, vol. 47, no. 4, 1995, pp. 848-54.
Traynor JR, Nahorski SR. Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol Pharmacol. 1995;47(4):848-54.
Traynor, J. R., & Nahorski, S. R. (1995). Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Molecular Pharmacology, 47(4), 848-54.
Traynor JR, Nahorski SR. Modulation By Mu-opioid Agonists of guanosine-5'-O-(3-[35S]thio)triphosphate Binding to Membranes From Human Neuroblastoma SH-SY5Y Cells. Mol Pharmacol. 1995;47(4):848-54. PubMed PMID: 7723747.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. AU - Traynor,J R, AU - Nahorski,S R, PY - 1995/4/1/pubmed PY - 1995/4/1/medline PY - 1995/4/1/entrez SP - 848 EP - 54 JF - Molecular pharmacology JO - Mol Pharmacol VL - 47 IS - 4 N2 - The ability of mu-opioid agonists to activate G proteins has been demonstrated by studying the binding of the GTP analogue guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) to membranes from the human neuroblastoma SH-SY5Y cell line. The potent opioid agonist fentanyl caused an approximate doubling of basal [35S]GTP gamma S binding in a naloxone-sensitive manner, confirming this to be an opioid receptor-mediated process. The presence of GDP was necessary to observe this effect. Pretreatment of the cells with pertussis toxin (100 ng/ml, for 24 hr) completely prevented the fentanyl-stimulated increase in [35S]GTP gamma S binding and lowered the basal binding of [35S]GTP gamma S. These latter data suggest an involvement of Gi and/or Go proteins and their activation by added membrane-bound receptors even in the absence of agonist. The order of potency of a series of opioid agonists in stimulating the binding of [35S]GTP gamma S was buprenorphine > cyclazocine = levallorphan > nalorphine > [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) > fentanyl > morphine > pentazocine. DAMGO, fentanyl, and morphine were full agonists but the remaining compounds showed decreasing levels of intrinsic activity in the order buprenorphine > pentazocine > cyclazocine = nalorphine > levallorphan. The opioid antagonist naloxone was without effect. Under the conditions of the [35S]GTP gamma S assay, binding of agonists was to a high affinity site, indicating that a high agonist affinity state of the mu-opioid receptor is responsible for the observed stimulation of [35S]GTP gamma S binding. The level of [35S]GTP gamma S binding (597 fmol/mg of protein) stimulated by DAMGO was 2-fold greater than the maximal number of mu-opioid agonist binding sites (Bmax) determined using [3H]DAMGO (254 fmol/mg of protein). The opioid agonist-mediated stimulation of [35S]GTP gamma S binding in SH-SY5Y cell membranes thus provides a "functional" measure of agonist occupation of mu-opioid receptors and offers a simple method for the determination of efficacy and intrinsic activity of mu-opioid agonists. SN - 0026-895X UR - https://www.unboundmedicine.com/medline/citation/7723747/Modulation_by_mu_opioid_agonists_of_guanosine_5'_O__3_[35S]thio_triphosphate_binding_to_membranes_from_human_neuroblastoma_SH_SY5Y_cells_ L2 - http://molpharm.aspetjournals.org/cgi/pmidlookup?view=long&pmid=7723747 DB - PRIME DP - Unbound Medicine ER -