Tolerance to mu-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5'-O-(3-[35S]-thio)triphosphate binding.
1. 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]-GTPgammaS. Morphine increased the binding of [35S]-GTPgammaS to SH-SY5Y cell membranes by 30 fmol mg(-1) protein with an EC50 value of 76 +/- 10 nM. 2. Incubation of SH-SY5Y cells with 10 microM 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 [35S]-GTPgammaS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. 3. Chronic treatment of the cells with the more efficacious mu-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO, 10 microM) 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. 4. 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 beta-adrenoceptor kinase (beta-ARK) inhibitor suramin. 5. The observed tolerance effect was accompanied by a down-regulation of mu-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. 6. The modulation of [35S]-GTPgammaS 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.
Elliott
J
J
Department of Chemistry, Loughborough University, Leics.
Guo
L
L
Traynor
J R
JR
D004361:Drug Tolerance; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D009020:Morphine / Q000494:pharmacology*; D009447:Neuroblastoma / Q000473:pathology; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000502:physiology; D014407:Tumor Cells, Cultured
D004745:Enkephalins; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009020:Morphine