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.