Effects of cycloheximide, alpha-amanitin, and alpha-difluoromethylornithine on thyrotropin-induced increases in the micrococcal nuclease sensitivity of thyroid nuclear chromatin.
Treatment of calf thyroid slices with TSH increases the nuclease sensitivity of nuclear chromatin, i.e. the amount of DNA released from nuclei by mild digestion with DNase I and micrococcal nuclease. Cycloheximide and alpha-amanitin were used to investigate the roles played by protein and RNA synthesis in mediating this effect of TSH; alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, was used to investigate the possible involvement of polyamines. Calf thyroid slices were incubated with or without TSH (50 mU/ml) for 5 h, in the presence or absence of inhibitors. Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 X g. The amount of DNA in 1200 X g supernatants was increased by TSH; this was inhibited by cycloheximide (100 micrograms/ml) and alpha-amanitin (4 micrograms/ml) when these agents were present throughout incubations with TSH. In contrast, alpha-amanitin failed to inhibit the TSH effect when it was added to incubations 30 min or 2 h after the addition of TSH. These results indicate that RNA and protein synthesis play a part in mediating the effect of TSH on the micrococcal nuclease sensitivity of chromatin, and that the RNA synthesis involved takes place within the first 30 min of exposure of thyroid slices to TSH. alpha-Difluoromethylornithine (5 mM) inhibited the TSH-dependent development of micrococcal nuclease sensitivity; however, it also inhibited nuclease digestion when it was added directly to nuclei prepared from fresh thyroid tissue. This observation should serve as a warning against uncritical acceptance of the notion that all effects of alpha-difluoromethylornithine are the result of inhibition of ornithine decarboxylase.
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects* / Q000648:ultrastructure; D003513:Cycloheximide / Q000494:pharmacology*; D004247:DNA / Q000032:analysis; D000518:Eflornithine; D066298:In Vitro Techniques; D008836:Micrococcal Nuclease / Q000494:pharmacology*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D065108:Ornithine Decarboxylase Inhibitors; D013961:Thyroid Gland / Q000187:drug effects*; D013972:Thyrotropin / Q000494:pharmacology*
D000546:Amanitins; D002843:Chromatin; D065108:Ornithine Decarboxylase Inhibitors; D013972:Thyrotropin; D004247:DNA; D003513:Cycloheximide; D009952:Ornithine; D008836:Micrococcal Nuclease; D000518:Eflornithine