Evidence for [D‐Ala 2 ,D‐Leu 5 ]Enkephalin‐Induced Supersensitivity to 5‐Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB‐20)

A neuroblastoma × Chinese hamster embryonic brain explant hybrid cell line (NCB‐20) expressed 5‐hydroxytryptamine (5‐HT 1 ) receptors, linked to adenylate cyclase, which closely resembled 5‐HT 1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5‐HT was only 150 n M compared to 5 n M in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB‐20 cells produced by 5‐HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E 1 (PGE 1 ) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5‐HT. The elevation of cyclic AMP levels by either 5‐HT or PGE 1 was reversed by [D‐Ala 2 ,D‐Leu 5 ]enkephalin (DADLE), morphine, clonidine, and 3,4‐dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5‐HT or PGE 1 alone. When the DADLE exposure time was increased to 48 h, 5‐HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE 1 unless naloxone was added 1 h prior to treatment with PGE 1 . Scatchard analysis showed that the increased potency of 5‐HT resulted from an increase in receptor affinity for 5‐HT (from a K D of 150 ± 20 n M to one of 20 ± 7 n M ), with a reduction in the number of apparent binding sites. The 5‐HT supersensitivity observed in NCB‐20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.