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A body of evidence has indicated that mu-opioid agonists can inhibit DNA synthesis in developing brain. We now report that kappa-selective opioid agonists (U69593 and U50488) modulate [3H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner, kappa agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the kappa-selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3.5-fold increase was evident. Cell labeling by [3H]thymidine was also inhibited by the kappa-opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbinaltorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of kappa agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that kappa agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.

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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Directly measured and derived geometric and densitometric parameters were obtained by means of the automated image analyzer Quantimet 720-D in Feulgen-stained HeLa cells synchronized by selective mitotic detachment. These data indicate substantial alteration of nuclear morphology during the entire cell cycle, even during the G1 and G2 phases, and the late G1-early S and late S-G2 transitions.