DIFFERENTIAL REGULATION OF MU-OPIATE AND DELTA-OPIATE RECEPTORS BY MORPHINE, SELECTIVE AGONISTS AND ANTAGONISTS AND DIFFERENTIATING AGENTS IN SH-SY5Y HUMAN NEUROBLASTOMA-CELLS

Mu and delta opiate receptor regulation by opiate agonists and antagon ists was studied in the human neuroblastoma cell line SH-SY5Y. Morphin e down-regulated both mu and delta receptors, but its effects on each subtype could be dissociated by use of specific antagonists. The selec tive mu antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) blocked the down-regulation of mu, but not delta receptors. Conversely, the de lta antagonist eu-OH([N,N-diallyl-Tyr(1),Aib(2,3)]Leuenkephalin)} ICI 174,864 blocked morphine-induced down-regulation of delta but not mu r eceptors. These selective antagonists also were studied alone for thei r effects on both receptors. CTAP alone at doses of 0.1 mu M and highe r up-regulated mu receptors. CTAP did not affect delta receptors at 0. 3 mu M or less, but it down-regulated them at doses of 1 mu M or more, apparently due to its delta agonist activity at higher doses, which w as reversed by ICI 174,864. ICI 174,864 alone also showed complex effe cts on the two subtypes, up-regulating both mu and delta sites. Its ef fects were most selective at a low dose (0.1 mu M), which upregulated delta sites with minimal effects on mu sites. The nonselective antagon ist naloxone provided a more robust upregulation (>40%) of both mu and delta receptors than either selective antagonist alone or in combinat ion. The mu-to-delta ratio (1.4 to 1) was not altered by differentiati on of the cells with retinoic acid, which up-regulated both mu and del ta receptors. Differentiation with the phorbol agent 12-O-tetradecanoy l-phorbol-13-acetate, however, up-regulated mu, but not delta receptor s. The selective mu agonist Tyr-Pro-MePhe-D-Pro-NH2 (PL017) down-regul ated mu receptors with a half-maximal effect at 180 nM, but was withou t effect on delta receptors at concentrations up to 10 mu M. Conversel y, the selective delta agonist Tyr-D-Pen-Gly-Phe-D-Pen([D-Pen(2,5)]-en kephalin) (DPDPE) potently down-regulated delta receptors, producing h alf-maximal decreases at 0.5 nM. At doses above those that reduced the maximum binding of [H-3]pCl-DPDPE binding to the delta site, DPDPE al so induced an apparent loss of affinity (increased K-d) at the delta s ite. It was without effect on mu receptors, however, at doses up to 10 mu M. Thus, down-regulation of mu and delta receptors was homologous, because selective agonists downregulated their respective receptors w ithout effect on the heterologous opiate receptor. These studies show that the use of SH-SY5Y cells in combination with selective pharmacolo gical agents permits the study of selective regulation of mu and delta opiate receptors, as well as the effects of compounds such as morphin e and naloxone, that can affect both receptors in the same cell line.