Jean-Guy Lehoux

Dehydroepiandrosterone protects low density lipoproteins against peroxidation by free radicals produced by γ-radiolysis of ethanol–water mixtures

Oxidized low density lipoproteins (LDL) are believed to play a central role in the events that initiate atherosclerosis. Antioxidants have been shown to decrease the oxidation of LDL, leading to the diminution of atherosclerosis. Since it is well-known that decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, we studied the modulation of the oxidation of LDL by DHEA. LDL were obtained from 10 healthy subjects and oxidized by free radicals produced by gamma-radiolysis of ethanol-water mixtures. The formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), the vitamin E content, as well as the incorporation of 4-[14C]DHEA in LDL and the chemotactic effect of oxidized LDL in the presence of DHEA towards monocytes, were investigated. It was found that DHEA was able to inhibit the oxidation of LDL by reducing over 90% of the conjugated dienes and TBARS formation, as well as by reducing the vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes. Our results suggest that DHEA exerts its antioxidative effect by protecting the endogenous vitamin E of LDL.

A comparative study of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in vertebrate adrenocortical tissues.

Abstract Our study compares the activities in vitro of HMG-CoA reductase in hamster, rabbit, rat, chicken, and frog adrenocortical tissues. Microsomal and mitochondrial preparations obtained by differential centrifugation were incubated with [3- 14 C]HMG-CoA and a NADPH generating system, and the [ 14 C]mevalonic acid formed was isolated on a silica gel column and by thin-layer chromatography. The identity of the product was confirmed by formation of the benzhydrylamide derivative followed by crystallization to constant isotope ratio. The following adrenocortical HMG-CoA reductase activities were found (nanomoles of mevalonic acid formed per milligram of protein per 30 min): hamster microsomal fraction, 19.3; hamster mitochondrial fraction, 15.1; rabbit microsomal fraction, 2.8; rat microsomal fraction, 0.35; chicken microsomal fraction, 7.4; chicken mitochondrial fraction, 3.4; and frog microsomal fraction, 0.14. Our results suggest that the ability of various adrenocortical tissues to synthetize cholesterol is related to the level of HMG-CoA reductase activity present.

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)

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.

Hydroxymethylglutaryl coenzyme A reductase activity in hamster adrenal mitochondria purified on a sucrose gradient.

Abstract Hamster adrenal homogenates were fractionated by differential centrifugation to obtain crude mitochondrial and microsomal pellets. The mitochondria were further purified on a linear sucrose density gradient. The crude mitochondrial fraction was separated into three bands on the gradient. One of the bands (band 3, D 20 20 = 1.165) contained all the measurable cytochrome C oxidase activity. Band 3 also contained the highest specific activity of HMG-CoA reductase corresponding to a 1.9 fold enrichment compared to the crude mitochondrial pellet. The evidence presented supports the possibility that a part of the HMG-CoA reductase activity in hamster adrenals is associated with mitochondria.

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).