kappa-Opioid agonist modulation of [3H]thymidine incorporation into DNA: evidence for the involvement of pertussis toxin-sensitive G protein-coupled phosphoinositide turnover

Abstract

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.

FIG. 1

Effects of U69593 on [³H]thymidine incorporation into DNA of rat brain cell aggregates as a function of age (days in culture). Cultures were treated with 1 μM U69593 for the final 48 h, and [³H]thymidine (0.1 μCi/ml) was included in the last 23 h. Data are the means ± SEM of three to five experiments. *p < 0.05, significantly different from untreated controls.

FIG. 2

Dose-dependent effects of κ-opioid agonist U69593 and antagonist norbinaltorphimine on [³H]thymidine incorporation into DNA of rat brain cell aggregates. Cultures were grown for 5 days, then treated with various concentrations of U69593 or norbinaltorphimine in the presence of 1 μM U69593 for the final 48 h. Data are the means ± SEM of three to five experiments. **p < 0.01, significant difference between U69593 and norbinaltorphimine.

FIG. 3

Opioid modulation of [³H]thymidine incorporation into DNA of 7-day rat brain cell aggregates. Cultures were treated with 1 μM DAMGE, 1 μM etorphine, 1 μM U69593, 1 μM U50488, 0.1 μM β-endorphin, or 1 μM DADLE for the final 48 h, and [³H]thymidine (0.1 μCi/ml) was included for the last 23 h. Data are the means ± SEM of three to five experiments. *p < 0.05 and **p < 0.01, significantly different from untreated controls.

FIG. 4

Reversal of the U50488 blockade of [³H]thymidine incorporation into DNA of 7-day rat brain cell aggregates by pertussis toxin. Cultures were exposed to 1 μM κ agonist and/or toxin 48 h prior to being harvested and to [³H]thymidine (0.1 μCi/ml) for the final 23 h. Data are the means ± SEM of three to seven experiments. *p < 0.05, significantly different from untreated controls.

FIG. 5

Concentration-dependent effects of U50488 on LiCl inhibition of [³H]thymidine incorporation into DNA of 7-day rat brain cell aggregates. Cultures were exposed to various concentrations of U50488 and/or to 0.5 mM LiCl 48 h prior to being harvested and to [³H]thymidine (0.1 μCi/ml) for the final 23 h. Data are the means ± SEM of three to seven experiments. *p < 0.05 and **p < 0.01, significantly different from their respective controls (cultures not treated with LiCl).

FIG. 6

Effects of κ-opioid agonist and antagonist on [³H]IP₃ accumulation in rat brain cell aggregates. The effect of U69593 and/or norbinaltorphimine (BINALTO; 1 μM) on [³H]IP₃ accumulation in 7-day (A), 14-day (B), and 21-day cultures (C). Brain cell aggregates were exposed to opioids 30 min prior to being harvested and to myo[³H]inositol for the final 18 h. Data are the means ± SEM of three to five experiments. *p < 0.05 and **p < 0.01, significantly different from untreated controls (CONT).

FIG. 7

Effects of the PKC inhibitor chelerythrine and U69593 on [³H]thymidine incorporation into DNA of rat brain cell aggregates in 7-day (A) and 21-day (B) cultures. Aggregates were exposed to chelerythrine and/or opioid 48 h prior to being harvested and to [³H]thymidine for the final 23 h. Data are the means ± SEM of three to six experiments. *p < 0.05 and **p < 0.01, significantly different from cultures treated only with chelerythrine (CONTROL); *p < 0.05, significantly different from untreated controls.

FIG. 8

Stimulation of [³H]thymidine incorporation into DNA of rat brain mixed glial cell aggregates by U50488. Cultures were exposed to U50488 and/or naltrexone (NALT) 48 h prior to being harvested and to [³H]thymidine (0.1 μCi/ml) for the final 23 h. Incorporation of radioactivity was measured after 7 days of culture. Data are the means ± SEM of three experiments. *p < 0.05, significantly different from all the other treatments, including untreated controls (CONT).