title: The intact human neuroblastoma cell (SH-SY5Y) exhibits high-affinity [3H]pirenzepine binding associated with hydrolysis of phosphatidylinositols.
abstract: The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.
authors: Department of Pharmacology, College of Medicine, University of Arizona, Tucson 85724.
authors: M
authors: M
authors: Serra
authors: L
authors: L
authors: Mei
authors: W R
authors: WR
authors: Roeske
authors: G K
authors: GK
authors: Lui
authors: M
authors: M
authors: Watson
authors: H I
authors: HI
authors: Yamamura
mesh_terms: D001285:Atropine / Q000378:metabolism; D001667:Binding, Competitive; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009447:Neuroblastoma / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000031:analogs & derivatives / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; C054940:clidinium; C048593:otenzepad