Tolerance to μ-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5′-O-(3-[³⁵S]-thio)triphosphate binding

Abstract

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]-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.

2. 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.

3. 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.

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 β-adrenoceptor kinase (β-ARK) inhibitor suramin.

5. 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.

6. 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.

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