Dynorphins modulate DNA synthesis in fetal brain cell aggregates

Abstract

Previously, opioid peptide analogues, beta-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via kappa- and mu-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 microM, all dynorphins tested and beta-endorphin inhibited [3H]thymidine incorporation into DNA by 20-38% in 7-day rat brain cell aggregates. The putative epsilon-antagonist beta-endorphin (1-27) did not prevent the effect of beta-endorphin, suggesting that the epsilon-receptor is not involved in opioid inhibition of DNA synthesis. The kappa-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a kappa-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete kappa-receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the kappa-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33-86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express kappa-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.

FIG. 1

Effect of enkephalins and endorphins on [³H]thymidine incorporation into 7-day fetal rat brain cell aggregates. Aggregates were prepared from embryonic day 15 fetal rat brain and cultured as described in Materials and Methods. Opioid peptides (1 μM) were added after 5 days together with protease inhibitors for 48 h, and [³H]thymidine was present for the last 24 h of incubation. Control values for thymidine incorporation ranged from 100 to 300 fmol/dish for this and subsequent experiments with 7-day rat aggregates. Data are the mean ± SEM values of three to five experiments. *Significantly different from controls, p < 0.05.

FIG. 2

Effect of dynorphins on [³H]thymidine incorporation into 7-day fetal rat brain cell aggregates. Aggregates were prepared from embryonic day 15 fetal rat brain and grown as described in Materials and Methods and Fig. 1. Dynorphins (1 μM) were added after 5 days together with protease inhibitors, and [³H]thymidine was present for the last 24 h. Data are the mean ± SEM values of three to six experiments. *Significantly different from dynorphin-treated aggregates, p < 0.05.

FIG. 3

Dose dependency of Dyn A (1–13) and Dyn B inhibition of [³H]thymidine incorporation into 7-day fetal rat brain cell aggregates. Cell aggregates were prepared as described in Materials and Methods and Fig. 1. Dynorphins at given concentrations were added after 5 days together with the protease inhibitors, and [³H]thymidine was present for the last 24 h. Data are the mean ± SEM values of three to five experiments. *Significantly different from controls, p < 0.05.

FIG. 4

The κ-opioid antagonist nor-BNI blocks Dyn A and Dyn B inhibition of [³H]thymidine incorporation into 7-day fetal rat brain cell aggregates. Aggregates were prepared from embryonic day 15 fetal rat brain and grown as described in Materials and Methods and Fig. 1. Dynorphins (1 μM) and/or 0.1 μM nor-BNI were added after 5 days together with protease inhibitors, and [³H]thymidine was present for the last 24 h. Data are the mean ± SEM values of three experiments. *Significantly different from control or nor-BNI–treated aggregates, p < 0.05.

FIG. 5

Inhibition of [³H]thymidine incorporation by Dyn B, U69593, and U50488 in 7-day fetal guinea pig brain cell aggregates. Aggregates were prepared from embryonic day 25 guinea pig brains. After 5 days of growth, cultures were treated with 1 μM concentration of κ-opioid receptor agonists and/or 1 μM nor-BNI in the presence of protease inhibitors for the last 48 h, and [³H]thymidine was included for the final 23 h. Data are the mean ± SEM values of three experiments. *Significantly different from control or nor-BNI–treated aggregates, p < 0.01.

FIG. 6

Dynorphins stimulate [³H]thymidine incorporation into 21-day fetal rat brain cell aggregates. Aggregates were prepared from embryonic day 15 fetal rat brain and grown as described in Materials and Methods. Medium was replenished by 50% on every third day of culture. Dynorphins (1 μM) were added after 19 days together with protease inhibitors, and [³H]thymidine was present for the last 24 h. Control values for thymidine incorporation were 16 fmol/dish. Data are the mean ± SEM values of three experiments. *Significantly different from controls and nor-BNI–treated aggregates, p < 0.05.