Human beta-endorphin: specific binding in neuroblastoma N18TG2 cells.

Abstract

The mouse neuroblastoma N18TG2 has specific, saturable binding sites for human -endorphin (h-EP). The affinity and number of sites are 1.1 nM and 280,000 per cell, respectively. (lh-EP binding is not inhibited by [Leu]enkephalin or morphine at concentrations up to 0.1 mM; Ph-EP-(6-31)is a potent inhibitor ofbinding, and camel PEP is much less potent. The data suggest the importance of the nonenkephalin segment of the Phl-EP molecule' for interaction with the binding site in NL8TG2 cells.We have previously reported the binding of f3endorphin ((3-EP) in NG108-15 cells to be homogeneous by the criterion of Scatchard analysis but clearly heterogeneous by displacement with enkephalins (1). In broadening the scope of our studies to include the two parent cell lines (N18TG2 and C6) that gave rise to NG108-15 cells (2), we have found that the N18TG2 cell (2) Ea-gle's medium (H21 medium) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, penicillin G at 10 units/ml, and streptomycin at 10 ,g/ml in a 90% air/10% CO2 atmosphere. NG108-15 cells also received 0.1 mM hypoxanthine, 1 AM aminopterin, and 16 ,M thymidine, N18TG2 cells received 80 pM 6-thiogaunine. For suspension binding assays, cells were harvested, washed with Ca2O-, Mg2+-free phosphate-buffered saline, and transferred to the assay buffer (290 mM sucrose/25 mM Tris HCl, pH 7.5/0.1% bovine serum albumin/0. 1% bacitracin). Cells (1-5 x 105/ml) and labeled peptides were incubated together for 30 min at 24°C in the presence or absence of unlabeled ligand -in plastic tubes. Assays were terminated by filtering and washing on glass fiber filters presoaked in 0.1% myelin basic protein in assay buffer. The filters were assayed after incubation overnight in scintillation fluid.

An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs

“…Our binding data do not exclude the possibility that the cannabinoid and nantradol drugs are acting at the p-endorphin binding sites (E) detected in N18TG2 cells by Li and co-workers (Hammonds and Li, 1981;Westphal and Li, 1984). However, 10 K M naloxone, an antagonist with an adequate affinity for the E opioid sites (Hazum et al, 1979;Law et al, 1979h), failed to affect the cannabinoid-induced inhibition of adenylate cyclase.…”