Diego Bellabarba

Human Placental Lhrh Receptor: Agonist and Antagonist Labeling Produces Differences in the Size of the Non-Denatured, Solubilized Receptor

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kavs indifferent from each other but significantly different from the Kavs of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Corticosteroid receptors in the kidney of chick embryo. I. Nature and properties of corticosterone receptor.

Cytosol from kidney of chick embryo (age 16-18 days) contained a corticosterone-binding site with the features of a putative receptor. This receptor was a thermolabile protein, readily digested by proteolytic enzymes, with a sedimentation coefficient of 7-8 S and with an apparent molecular weight greater than 100,000. Simultaneous studies with transcortin (CBG) revealed several differences between the renal- and serum-binding protein pertaining to the effect of temperature, the sedimentation coefficient, the charcoal stripping and, finally, the binding and competition of various steroids for the two proteins. Kinetic analysis showed a rapid association (10 min), which followed second-order reaction kinetics, and a dissociation of pseudo-first-order reaction kinetics with a t1/2 of 168 min at 0 degrees. The analysis of the Scatchard plot showed the presence of a single class of binding sites with an association constant (KA) of 1.3 X 10(8)M-1 and a binding capacity (nmax) of 500-700 fmol/mg protein. We obtained similar results when we used dexamethasone as a ligand. The association (ka) and dissociation (kd) rate constants were respectively 2.9 X 10(6)M-1 sec-1 and 6.86 X 10(-5) sec-1. From their ratio a KA value of 4.2 X 10(10) M-1 was obtained. Studies with various steroids demonstrated that only dexamethasone and, to a lesser degree, progesterone competed for the binding site. These data showed that the kidney of chick embryo possessed one type of receptor for the glucocorticoids, which was similar to the type II described in rat kidney.

Corticosteroid receptors in the kidney of chick embryo. III. Nature, properties, and ontogeny of aldosterone receptor.

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4 degrees revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association (ka) and dissociation (kd) rate constants were, respectively, 4.94 X 10(5) M-1 sec-1 and 8.33 X 10(-6) sec-1. From their ratio a KA value of 5.9 X 10(10) M-1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the KA at equilibrium, obtained from the Scatchard plot, was 3.1 +/- 1.2 X 10(8) M-1, whereas the Nmax was 172 +/- 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.

3,5,3′-Triiodothyronine binding sites in synaptosomes from brain of chick embryo. Properties and ontogeny

In this study we have demonstrated the presence of specific 3,5,3-L-triiodothyronine (T3) binding sites in the synaptosomes of chick embryo cerebral cortex and described their ontogeny. Scatchard analyses of binding data obtained with synaptosomal preparations from 17-day-old embryos revealed two T3 binding sites. The first site (N1) had a high affinity and low capacity since its dissociation constant (Kd) was 68 +/- 1.3 nM T3 (mean +/- S.D.; n = 3-5) and its maximal binding capacity (Bmax) was 8.63 +/- 1.59 ng T3/mg of protein, whereas the second site (N2) had a higher Kd of 5.04 +/- 0.5 microM T3 and a larger Bmax of 405 +/- 49 ng T3/mg of protein. The relative affinity of the synaptosomal fraction for T3 and other analogs was the following: T3 greater than T4 (thyroxine) greater than D-T3 (3,5,3-D-triiodothyronine) = TRIAC (triiodothyroacetic acid) greater than rT3 (reverse T3). Gel chromatography of the [125I]T3 labeled fraction revealed a partially saturable peak with an estimated MW of more than 100 kDa. The ontogenic pattern showed a progressive increase of Kd and Bmax of N1, occurring mainly between the 12 and 19 days of incubation, and a marked fall, particularly of the Bmax, after hatching. The second site did not show any important variation during the embryogenesis. These data indicate the existence of specific T3 binding sites in synaptosomes from cerebral cortex of chick embryo, whose properties and ontogeny are completely different from those of the nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticosteroid receptors in the kidney of chick embryo II. Ontogeny of corticosterone receptor and cellular development

In this study the ontogeny of cytosol receptors for corticosterone in the chick embryo kidney was examined and then this ontogenic profile was compared with that of an index of cellular development, i.e., the enzyme ornithine decarboxylase (ODC). The corticosterone receptor concentration (nmax) increased by sevenfold from Day 12 to Day 15 of embryogenesis and then declined to its lowest level by the time of hatching (Day 21). Similar results were obtained when dexamethasone was used as ligand, except that the baseline values at Day 9 and 21 were higher than those found with corticosterone. An identical ontogenic profile was obtained when the results were corrected for the endogenous glucocorticoids present in the cytosol. This increase of the corticosterone receptor occurred simultaneously with the enhanced adrenal corticoid synthesis. The ODC also showed a marked increase and a rapid fall during chick embryogenesis, but the enzyme activity was at its maximum when the corticosterone receptor number was still low (Days 12 and 13) and quickly decreased by the time the receptors had reached their highest levels (Days 14 and 15). The lowest level of ODC was observed immediately before hatching. These results indicate that during chick embryogenesis adrenal corticoids may induce the development of the corticosteroid receptor and that such development may cause a suppression of ODC activity. This suppressive effect of glucocorticoids could represent a mechanism of hormonal action on the kidney.

Triiodothyronine Nuclear Receptors in Liver, Brain and Lung of Neonatal Rats

In this study we have examined the effect of neonatal hypothyroidism and triiodothyronine (T3) replacement therapy on the maximal binding capacity (MBC) and the affinity of T3 receptors prepared from liver, brain and lungs. Rats were radiothyroidectomized at birth and administered T3 or its placebo starting at 8 days of age. The thyroid state in normal, hypothyroid and hypothyroid T3-treated rats was assessed by serum determination of thyroxine, T3 and by the measurement of the hepatic alpha-glycerophosphate dehydrogenase. The animals were sacrificed at 8, 16 or 24 days of age and the T3 binding was estimated in isolated nuclei and in salt nuclear extracts. The MBC of the T3 receptors was higher in the hypothyroid rats at all ages when it was determined in isolated nuclei, but not when was measured in nuclear extracts. At 8 days, the MBC had risen twofold or more in the receptors from brain (1.0 +/- 0.37 vs. 0.4 +/- 0.1 ng T3/mg DNA in controls) and from lungs (0.6 +/- 0.25 vs. 0.3 +/- 0.15 ng T3/mg DNA in controls), but was only slightly elevated in the hepatic receptor (0.62 +/- 0.08 vs. 0.48 +/- 0.15 ng T3/mg DNA in controls). At 16-24 days, the highest value of MBC was observed in the hepatic receptor (0.83 +/- 0.04 vs. 0.41 +/- 0.1 ng T3/mg DNA in controls) followed by the brain receptor (0.65 +/- 0.03 vs. 0.35 +/- 0.02 ng T3/mg DNA in controls), and that of lung (0.39 +/- 0.07 vs. 0.20 +/- 0.03 ng T3/mg DNA in controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of thyroid hormones by nuclei of target tissues during the chick embryo development

In the present studies we have compared the ontogeny of the binding of thyroxine (T4) and triiodothyronine (T3) to isolated nuclei from various target tissues of chick embryo. We observed a marked difference between the patterns of Satchard plots, maximal binding capacities (MBC) and association constants (Ka) of T4 and those of T3. Scatchard plots revealed that T4 and T3 had different binding sites. In liver, brain and lung MBCs and Kas of T3 and T4 were rather similar at day 9, but during the following days (12-19) T3 MBCs and Kas showed small changes, whereas T4 MBC markedly increased (4-5-fold) and T4 Ka significantly declined. In liver, for instance, T3 MBC = 395 +/- 19 (day 9) and 489 +/- 66 fmol/mg protein (day 19); T4 MBC = 631 +/- 6.5 (day 9) and 2201 +/- 516 fmol/mg protein (day 19); T4 Ka = 1.92 +/- 0.01 (day 9) and 0.56 +/- 0.21 X 10(8) M-1 (day 19). These data indicate that, during chick embryogenesis, nuclei of target tissues contain multiple T4 binding proteins, but only a single T3 binding site.

Glucocorticoid receptors in chick embryos: properties and ontogeny of the nuclear renal receptor

A putative nuclear receptor for glucocorticoids was identified in the kidney of chick embryo. This receptor was a thermolabile protein which was readily digested by proteolytic enzymes. Its sedimentation coefficient on sucrose density gradient was 3.5S and its MW approximated, according to the Svedberg formula, at 98,500 Da. Binding assays, performed with crude or purified nuclei, and nuclear extracts showed that the latter preparation was the most suitable for the binding studies since it yielded a Bmax of 8-11% with a very low nonspecific binding (1% or less). Scatchard plots performed at various days of embryogenesis revealed a single class of binding sites with an association constant (Ka) of 0.12 +/- 0.06 X 10(9) M-1 (mean +/- SD; n = 5) and a maximal binding capacity (Nmax) that rose from 3.9 +/- 1.2 fmol/micrograms DNA at Day 13 of age to 13.2 +/- 2.2 fmol/micrograms DNA at Day 16 and then rapidly fell to 1.8 +/- 1.1 fmol/micrograms DNA before hatching (means +/- SD; n = 5). Competition studies with various steroids showed that only glucocorticoids and, to a lesser degree, progesterone had an affinity for the receptor. These results demonstrate that this nuclear-binding protein had physiochemical properties similar to those attributed to other glucocorticoid receptors in target cells.

Ontogeny of Thyroid during Embryogenesis: Evidence of Two Thyroxine Binding Sites

Evidences gathered over the past ten years indicate that triiodothyronine (T3) is the thyroid hormone responsible for the metabolic effect (1). However, there are particular situations in which little or no T3 is produced by the organism and, therefore, it is not clear how the thyroid action will be expressed. One of these situations is the fetal or neonatal period, in humans and in various experimental animals. Very low amounts of T3 are produced during this time, since the 5’-monodeiodinase is not yet fully developed (2–4). Therefore, thyroxine (T4) is the only thyroid hormone present in large quantities during a period of great hormonal need because of the rapid maturation of several target tissues. Such a situation is particularly evident in chick embryo where most of the target organs, particularly the nervous system, develop between 7–12 days (5), when the concentration of T3 in the serum is very low (6).