Novel opioid binding sites associated with nuclei of NG108-15 neurohybrid cells

Nuclear opioid binding sites have been discovered in NG108-15 neurohybrid cells. Marker enzyme analyses as well as electron and fluorescence microscopy studies attested to the high degree of purity of the nuclear preparations. Immunohistochemical studies on cryostat sections of NG108-15 cells with an antibody to the opioid receptor corroborated a nuclear localization. 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE), 3H-[D-Ala2,D-Leu5]enkephalin (3H-DADLE), and 3H-diprenorphine binding parameters, Kd and Bmax values, and heterologous competition binding and stereospecificity data satisfied criteria for the presence of delta-opioid sites in purified nuclear preparations. Neither mu-([D-Ala2,mephe4,gly-ol5] enkephalin), dihydromorphine, nor kappa-(U69593) specific binding was detectable in purified nuclear preparations. Rates of association and dissociation of 3H-[D-Ser2,L-Leu5]enkephalyl-Thr were comparable to values obtained previously for opioid receptors. Opioid binding was also shown in subnuclear preparations from NG108-15 cell cultures. Agonists, 3H-DADLE and 3H-DPDPE, bind with high affinity to nuclear membranes and with lower affinity to chromatin. In contrast, partial agonist 3H-diprenorphine high-affinity binding sites were predominant in chromatin, while low-affinity binding was found in the nuclear membrane. Accordingly, 5'-guanylylimidodiphosphate sensitivity of 3H-DADLE binding was detected in nuclear membranes but not in chromatin. Both agonist and partial agonist opioid binding to nuclear membrane and chromatin were abolished upon cycloheximide treatment of NG108-15 cells. Taken together, the results suggest that NG108-15 cells contain newly synthesized GTP binding regulatory protein (G-protein)-coupled delta-opioid receptors in nuclear membranes and uncoupled opioid binding sites in chromatin.