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Journal of Neuroscience, Vol 13, 104-114, Copyright © 1993 by Society for Neuroscience
Novel opioid binding sites associated with nuclei of NG108-15 neurohybrid cells
M Belcheva, J Barg, J Rowinski, WG Clark, CA Gloeckner, A Ho, XM Gao, DM Chuang and C Coscia
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
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.
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