REVERSIBLE MODULATION OF OPIOID RECEPTOR-BINDING IN INTACT NEURAL CELLS BY ENDOGENOUS GUANOSINE TRIPHOSPHATE

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an in hibitor of inosine monophosphate dehydrogenase (the key enzyme in puri ne nucleotide biosynthesis), reduced the cellular content of GTP by 94 % relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cel ls the receptor binding parameters (K-d and B-max) of the opioid antag onist [H-3]naltrexone were unchanged from those in untreated cells, th e binding affinity of the mu-selective opioid agonist [H-3]Tyr-D-Ala-G ly-(Me)-Phe-Gly-ol ([H-3]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotid e-depleted cells, the dissociation rate constant for [H-3]DAMGO was re duced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin red uced high-affinity ligand binding by 95% and abolished the effect of M PA treatment. Renewed incubation of the GTP-depleted cells with guanos ine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the B-max of [H-3]DAMGO binding. Forsk olin- and prostaglandin E(1)-stimulated adenylyl cyclase activities we re decreased significantly in GTP-depleted cells. DAMGO and [D-Pen(2), D-Pen(5)]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenyl yl cyclase activity by both of the agonists was reduced, whereas MPA c aused a 2-fold reduction in potency for DAMGO. The results show that r eduction in endogenous GTP levels leads to noticeable changes in agoni st, receptor, and G protein interactions, as measured by agonist bindi ng, and to subsequent diminution of the signal transduction, as reflec ted by the cAMP levels.