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 × 10⁶ M⁻¹ min⁻¹ and 1.1 ± 0.06 × 10⁻¹ min⁻¹, respectively. Saturation experiments revealed a single class of binding sites (n_H= 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 vas atropine sulfate > pirenzepine > AF‐DX116, with K_0.5 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.