Stimulation by thyrotropin, long-acting thyroid stimulator, and dibutyryl 3',5'-adenosine monophosphate of protein and ribonucleic acid synthesis and ribonucleic acid polymerase activities in porcine thyroid in vitro

Abstract

The in vitro incorporation of [³H]uridine into RNA and [³H]leucine into protein in slices of porcine thyroid was studied. Thyrotropin (10-500 mU/ml of medium), when added with [³H]uridine, inhibited incorporation into RNA, but as little as 10 mU of thyrotropin per ml stimulated incorporation of [3H]orotic acid into RNA. Uridine kinase (EC 2.7.1.48) was found to be inhibited in slices incubated with thyrotropin whereas UMP 5' nucleotidase (EC 2.1.3.5) was not. Preincubation of slices with thyrotropin (5-50 mU/ml) led to enhanced incorporation of subsequently added [³H]uridine and [³H]leucine. When slices were preincubated with long-acting thyroid stimulator-IgG (2.5 or 5 mg per ml of medium) incorporation of [³H]uridine and [³H]leucine was similarly enhanced, with the smaller concentration being more effective. Without preincubation these stimulatory effects were mimicked by 1 mM dibutyryl 3',5'-AMP and, to a lesser extent, 1 mM 3',5'-AMP. AMP and ATP also stimulated [³H]uridine incorporation in this system but only after more prolonged periods of incubation than were required for the other nucleotides. RNA polymerase (EC 2.7.7.6) activity measured in isolated thyroid nuclei had two components, one Mg²⁺-stimulated and the other requ ring Mn²⁺ and high salt content [0.4 M (NH₄)₂SO₄]. These activities, and particularly the former, were enhanced if thyroid slices were incubated with thyrotropin (5-100 mU/ml of medium), 2.5 mg or 5.0 mg of long-acting thyroid stimulator-IgG per ml, or 1 mM dibutyryl 3',5'-AMP, before isolatior of the nuclei and measurement of enzyme activities; 1 mM AMP, ADP, or 2',3'-GMP had no influence. Added directly to the nuclei, thyrotropin, long-acting thyroid stimulator-IgG, and dibutyryl 3',5'-AMP had no effect on RNA polymerase activities. These data are seen as affording evidence for mediation by 3',5'-AMP of effects of thyrotropin and long-acting thyroid stimulator on thyroid RNA and protein synthesis, at least in part through an indirect stimulation of nuclear RNA polymerase activities.