Beta-endorphin is a potent inhibitor of thymidine incorporation into DNA via mu- and kappa-opioid receptors in fetal rat brain cell aggregates in culture

Abstract

Thymidine incorporation into DNA was inhibited dose-dependently by beta-endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by mu (cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr- Pen-Thr amide) or kappa (norbinaltorphimine) antagonists. Complete blockade of the beta-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie-Hofstee analysis revealed that beta-endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, beta-endorphin decreased thymidine incorporation with an IC50 of 0.7 nM. Truncated and N-acetylated beta-endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that beta-endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.

FIG. 1

Antagonist reversal and dose dependency of β-endorphin inhibition of [³H]thymidine incorporation in rat fetal brain cell aggregates. Embryonic (day 15) rat brain cultures were grown for a total of 7 days and were exposed for the last 48 h to various concentrations of β-endorphin, CTOP, and/or nor-BNI. [³H]-Thymidine was included for the final 23 h. Data are mean ± SEM (bars) values from three to six experiments. *p < 0.05, **p < 0.01 for difference from β-endorphin (without antagonists)-treated aggregates.

FIG. 2

Eadie–Hofstee plot of the β-endorphin effect of [³H]-thymidine incorporation in 7-day-old rat fetal brain cell aggregates. Control and β-endorphin-treated cultures (48 h) were exposed for the final 23 h to various concentrations (0.2–3.5 nM) of [³H]thymidine. Data are mean ± SEM (bars) values from three experiments. Thymidine apparent K_m values for control (1.1 ± 0.2 nM) and β-endorphin-treated (1.0 ± 0.3 nM) aggregates do not differ significantly. B_max values for control (477 ± 53 fmol per seeded cell) and β-endorphin-treated (300 ± 28 fmol per seeded cell) aggregates are significantly different (p < 0.05).

FIG. 3

Dose dependency of the effect of β-endorphin and its derivatives on [³H]thymidine incorporation in the presence of protease inhibitors. Aggregate cultures were exposed for 48 h to the indicated concentrations of β-endorphin_1–31, β-endorphin_1–27, N-acetyl-β-endorphin_1–27, or N-acetyl-β-endorphin_1–31, in the presence of 1 μM bestatin, captopril, and phosphoramidon. [³H]-Thymidine was included for the final 23 h. Data are mean ± SEM (bars) values from three to seven experiments; p < 0.05 for difference from β-endorphin treatment alone.