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Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes.
Mol Pharmacol. 1988 Apr; 33(4):423-31.MP

Abstract

Multiple affinity states of opioid receptors of the mu and delta types have been identified in membranes prepared from cells which bear only one type of opioid receptor (mu receptors in 7315c cells, delta receptors in NG 108-15 cells), and in guinea pig cortical membranes where both types of receptors were present in the membrane preparations. States of mu and delta receptors which have agonist affinities too low to be identified by radiolabeled agonist have been measured indirectly by agonist competition for sites labeled by radioactive antagonist. Using analogues of guanyl nucleotides, we have examined the competition of the mu and delta agonists DAGO and DSLET against [3H]DIP or [3H]NAL binding to opioid receptors and identified several agonist affinity states. In the absence of added nucleotide, competition of DSLET for [3H]DIP binding to delta opioid receptors revealed the presence of two binding sites with differing apparent agonist affinities. Addition of GDP beta S produced a steep monophasic curve which was best fit by a one-site model. In contrast, in the presence of added GTP or GTP gamma S, two affinity states were again apparent for DSLET competition at the delta receptor. The competition curve with GTP was shifted to the right relative to that produced in the absence of added guanyl nucleotide, indicating the presence of a lower apparent affinity state than any observed under other treatment conditions. DAGO competed against [3H]DIP or [3H]NAL binding to mu receptors over a wide concentration range in the absence of added guanyl nucleotide, consistent with the occupation by this ligand of more than one agonist affinity state of the mu receptor. However, when GDP beta S was added to the incubation mixture, only a single binding site was identified. Two mu receptor affinity states were again observed in the presence of added GTP or GTP gamma S. One of these had significantly lower apparent affinity than those states detected in the absence of added nucleotide or with GDP beta S. Pertussis toxin treatment resulted in a monophasic agonist competition curve which was best fitted by a single-site model in both 7315c and NG108-15 cell membranes. Addition of 100 microM GTP did not affect the agonist Kapp or Bmax after pertussis toxin treatment, suggesting that sites labeled under these conditions were not functionally associated with a G protein. In general, the effects of guanyl nucleotides were qualitatively similar at mu and delta receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Authors+Show Affiliations

Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2833686

Citation

Werling, L L., et al. "Multiple Agonist-affinity States of Opioid Receptors: Regulation of Binding By Guanyl Nucleotides in Guinea Pig Cortical, NG108-15, and 7315c Cell Membranes." Molecular Pharmacology, vol. 33, no. 4, 1988, pp. 423-31.
Werling LL, Puttfarcken PS, Cox BM. Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. Mol Pharmacol. 1988;33(4):423-31.
Werling, L. L., Puttfarcken, P. S., & Cox, B. M. (1988). Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. Molecular Pharmacology, 33(4), 423-31.
Werling LL, Puttfarcken PS, Cox BM. Multiple Agonist-affinity States of Opioid Receptors: Regulation of Binding By Guanyl Nucleotides in Guinea Pig Cortical, NG108-15, and 7315c Cell Membranes. Mol Pharmacol. 1988;33(4):423-31. PubMed PMID: 2833686.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. AU - Werling,L L, AU - Puttfarcken,P S, AU - Cox,B M, PY - 1988/4/1/pubmed PY - 1988/4/1/medline PY - 1988/4/1/entrez SP - 423 EP - 31 JF - Molecular pharmacology JO - Mol Pharmacol VL - 33 IS - 4 N2 - Multiple affinity states of opioid receptors of the mu and delta types have been identified in membranes prepared from cells which bear only one type of opioid receptor (mu receptors in 7315c cells, delta receptors in NG 108-15 cells), and in guinea pig cortical membranes where both types of receptors were present in the membrane preparations. States of mu and delta receptors which have agonist affinities too low to be identified by radiolabeled agonist have been measured indirectly by agonist competition for sites labeled by radioactive antagonist. Using analogues of guanyl nucleotides, we have examined the competition of the mu and delta agonists DAGO and DSLET against [3H]DIP or [3H]NAL binding to opioid receptors and identified several agonist affinity states. In the absence of added nucleotide, competition of DSLET for [3H]DIP binding to delta opioid receptors revealed the presence of two binding sites with differing apparent agonist affinities. Addition of GDP beta S produced a steep monophasic curve which was best fit by a one-site model. In contrast, in the presence of added GTP or GTP gamma S, two affinity states were again apparent for DSLET competition at the delta receptor. The competition curve with GTP was shifted to the right relative to that produced in the absence of added guanyl nucleotide, indicating the presence of a lower apparent affinity state than any observed under other treatment conditions. DAGO competed against [3H]DIP or [3H]NAL binding to mu receptors over a wide concentration range in the absence of added guanyl nucleotide, consistent with the occupation by this ligand of more than one agonist affinity state of the mu receptor. However, when GDP beta S was added to the incubation mixture, only a single binding site was identified. Two mu receptor affinity states were again observed in the presence of added GTP or GTP gamma S. One of these had significantly lower apparent affinity than those states detected in the absence of added nucleotide or with GDP beta S. Pertussis toxin treatment resulted in a monophasic agonist competition curve which was best fitted by a single-site model in both 7315c and NG108-15 cell membranes. Addition of 100 microM GTP did not affect the agonist Kapp or Bmax after pertussis toxin treatment, suggesting that sites labeled under these conditions were not functionally associated with a G protein. In general, the effects of guanyl nucleotides were qualitatively similar at mu and delta receptors.(ABSTRACT TRUNCATED AT 400 WORDS) SN - 0026-895X UR - https://www.unboundmedicine.com/medline/citation/2833686/Multiple_agonist_affinity_states_of_opioid_receptors:_regulation_of_binding_by_guanyl_nucleotides_in_guinea_pig_cortical_NG108_15_and_7315c_cell_membranes_ L2 - http://molpharm.aspetjournals.org/cgi/pmidlookup?view=long&pmid=2833686 DB - PRIME DP - Unbound Medicine ER -