Putative Nuclear Triiodothyronine Receptors in Tadpole Erythrocytes: Regulation of Receptor Number by Thyroid Hormone*

Abstract

Putative thyroid hormone (TH) receptors have been detected in the nuclei of red blood cells (RBC) from R. catesbeiana tadpoles, and their binding characteristics have been examined. Nuclear T3 [triiodothyronine] saturation analyses were carried out in vitro in intact RBC suspended in phosphate-buffered amphibian Ringer. After incubation with T3, intact nuclei were obtained by centrifugation after lysing the cells in a sucrose-Tris-HCl buffer containing 0.2% saponin and then adding Triton X-100 (final concentration 0.125%) to the lysed mixture to reduce nonspecific binding to less than 10% of total binding. Scatchard analysis of equilibrium binding data revealed that RBC from premetamorphic (1st yr) tadpoles contained 502 .+-. 39 (SE) T3 binding per sites per nucleus; in prometamorphic (2nd yr) tadpoles the number had increased to 844 .+-. 39. Development was also accompanied by some increase in the affinity of these sites; Kd = 1.8 .+-. 0.39 .times. 10-11 M and 0.95 .+-. 0.108 .times. 10-11 M in pre- and prometamorphic tadpoles, respectively. The number of sites per nucleus in both pre- and prometamorphic tadpole RBC was greatly increased by pretreatment in vivo with either T3 or T4 [thyroxine] for 6-10 days; a comparable number of sites per nucleus (2225 .+-. 65) was observed after maximal stimulation by either hormones at both stages of development. Significant increases in receptor number were observed 10 days after injection of 0.03 nmol T3 or 0.06 nmol T4 into tadpoles weighing 13-15 g; maximal effects were obtained with 0.1 nmol T3 or 1.0 nmol T4. On the basis of these observations and the evidence that more TH is present in pro- than in premetamorphic tadpoles, it is suggested that the spontaneous increase in receptor number in RBC nuclei associated with progression from the pre- to prometamorphic phase is due, at least in part, to increased levels of endogenous TH.