Article date: July 1997
By: Jackie Elliott, Li Guo, John R Traynor, in Volume 121, Issue 7, pages 1422-1428
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 [35S]‐GTPγS. Morphine increased the binding of [35S]‐GTPγS to SH‐SY5Y cell membranes by 30 fmol mg−1 protein with an EC50 value of 76±10 nM.
Incubation of SH‐SY5Y cells with 10 μM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 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‐Ala2, MePhe4, Gly‐ol5]enkephalin (DAMGO, 10 μM) 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 [3H]‐diprenorphine of 66±4%, but with no change in binding affinity. Binding of the agonist [3H]‐DAMGO was similarly reduced.
The modulation of [35S]‐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.
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 [35S]‐GTPγS. Morphine increased the binding of [35S]‐GTPγS to SH‐SY5Y cell membranes by 30 fmol mg−1 protein with an EC50 value of 76±10 nM.
Incubation of SH‐SY5Y cells with 10 μM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 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‐Ala2, MePhe4, Gly‐ol5]enkephalin (DAMGO, 10 μM) 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 [3H]‐diprenorphine of 66±4%, but with no change in binding affinity. Binding of the agonist [3H]‐DAMGO was similarly reduced.
The modulation of [35S]‐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.
DOI: 10.1038/sj.bjp.0701253
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