title: Reversible modulation of opioid receptor binding in intact neural cells by endogenous guanosine triphosphate.
abstract: Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine 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 cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin reduced high-affinity ligand binding by 95% and abolished the effect of MPA treatment. Renewed incubation of the GTP-depleted cells with guanosine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the Bmax of [3H]DAMGO binding. Forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were decreased significantly in GTP-depleted cells. DAMGO and [D-Pen2,D-Pen5]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenylyl cyclase activity by both of the agonists was reduced, whereas MPA caused a 2-fold reduction in potency for DAMGO. The results show that reduction in endogenous GTP levels leads to noticeable changes in agonist, receptor, and G protein interactions, as measured by agonist binding, and to subsequent diminution of the signal transduction, as reflected by the cAMP levels.
mesh_terms: D000262:Adenylyl Cyclases / Q000032:analysis; D000595:Amino Acid Sequence; D000700:Analgesics / Q000378:metabolism / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009173:Mycophenolic Acid / Q000494:pharmacology; D009271:Naltrexone / Q000378:metabolism; D009292:Narcotic Antagonists / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D000700:Analgesics; D004745:Enkephalins; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D014316:Tritium; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009271:Naltrexone; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D009173:Mycophenolic Acid