Solubilization and characterization of binding sites for [H-3]NE-100, a novel and potent sigma1 ligand, from guinea pig brain

The binding sites for [H-3]NE-100, a newly defined sigmal ligand, was solubilized from guinea pig brain, using zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), and the properties of the solubilized binding sites were compared to those for [H-3](+)-pentazocine, a selective sigmal ligand. The pharmacological selectivity of solubilized sites for both [H-3]NE-100 and [H-3](+)-pentazocine was identical to that obtained from membrane preparations. Stereoselectivity of benzomorphan such as pentazocine and SKF10,047 was preserved in displacing [H-3]NE-100 binding in solubilized preparations as observed in membrane preparations. The inhibitory potencies of several sigma ligands on [H-3]NE-100 binding were similar to those on [H-3](+)-pentazocine binding, indicating that the pharmacological characteristics of the binding sites for [H-3]NE-100 are retained after solubilization. Phenytoin augmented the binding of [H-3](+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine hydrochloride (3-PPP) to solubilized sigma binding sites while it had no effect on the binding of [H-3]NE-100. Furthermore, the inhibitory effect of putative sigma receptor agonists such as (+)-3-PPP and dextromethorphan were enhanced by phenytoin; the effects of haloperidol, a putative sigma receptor antagonist, were unaltered. Molecular weight of [H-3]NE-100 binding protein was estimated to be 440KDa by Sepharose CL-6B gel filtration chromatography, and the value was identical to that of [H-3](+)-pentazocine binding protein, a putative sigmal binding protein. These findings indicate that [H-3]NE-100 binding sites are putative sigmal binding sites, and that NE-100 may act as an antagonist at sigmal binding sites.