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Tolerance to μ‐opioid agonists in human neuroblastoma SH‐SY5Y cells as determined by changes in guanosine‐5′‐O‐(3‐[ 35 S]‐thio)triphosphate binding
Author(s) -
Elliott Jackie,
Guo Li,
Traynor John R
Publication year - 1997
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0701253
Subject(s) - sh sy5y , damgo , agonist , chemistry , gtp' , diprenorphine , (+) naloxone , endocrinology , medicine , pentazocine , g protein , morphine , partial agonist , pharmacology , opioid receptor , receptor , opioid , biology , biochemistry , cell culture , neuroblastoma , genetics , enzyme
The agonist action of morphine on membranes prepared from human neuroblastoma SH‐SY5Y cells was measured by an increase in the binding of the GTP analogue [ 35 S]‐GTPγS. Morphine increased the binding of [ 35 S]‐GTPγS to SH‐SY5Y cell membranes by 30 fmol mg −1 protein with an EC 50 value of 76±10 n M . Incubation of SH‐SY5Y cells with 10 μ M morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC 50 value with a 31±6% decrease in the maximum stimulation of [ 35 S]‐GTPγS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. Chronic treatment of the cells with the more efficacious μ‐ligand [ D ‐Ala 2 , MePhe 4 , Gly‐ol 5 ]enkephalin (DAMGO, 10 μ M ) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9±6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8±4%. There was a complete loss of agonist activity for pentazocine. The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative β‐adrenoceptor kinase (β‐ARK) inhibitor suramin. The observed tolerance effect was accompanied by a down‐regulation of μ‐opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [ 3 H]‐diprenorphine of 66±4%, but with no change in binding affinity. Binding of the agonist [ 3 H]‐DAMGO was similarly reduced. The modulation of [ 35 S]‐GTPγS binding in SH‐SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.