The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)

A proposed EPR approach to evaluating agonist binding site of a peptide receptor

Angiotensin II (Ang II) and its transmembrane AT(1) receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC(1)-Ang II), as well as an inactive control (TOAC(4)-Ang II) analogs were mixed in solution with various synthesized AT(1) fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT(1) molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes.

2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human A1 adenosine receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5- dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro 4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino- 3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA¹AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist- A₁AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH₂)_n- bridge, n ) 3 < n ) 4 < n ) 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA₁AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

Projections to the preoptic area from the paraventricular nucleus, acuate nucleus and the bed nucleus of the stria terminalis are unlikely to be involved in stress-induced suppression of GnRH secretion in sheep

Stress compromises reproductive function and the major physiological system activated during stress is the hypothalamo-pituitary-adrenal axis. Corticotrophin-releasing hormone and arginine vasopressin (AVP), which are produced in neurones of the paraventricular nucleus (PVN), drive the hypothalamo-pituitary-adrenal axis and are also implicated in the suppression of the reproductive axis. We used retrograde tracing and Fos labelling to map the projections from the PVN to the preoptic area (POA) where most gonadotrophin releasing hormone (GnRH) neurones are found. Fluorogold (FG) injections were made into the POA of gonadectomised male and female sheep (n = 5/sex), the animals were stressed and the brains recovered for histochemistry. All animals responded to stress with an increase in the number of Fos-labelled nuclei in the PVN. Few retrogradely labelled cells of the PVN were activated by stress. Dual labelling showed that very few FG-labelled cells also stained for corticotrophin-releasing hormone, none for AVP or enkephalin. Dual labelling for FG and Fos in the bed nucleus of the stria terminalis (BNST) and the arcuate nucleus showed that no FG-labelled cells in the BNST and only few in the ARC were activated by stress. No sex differences were observed in the activation of FG-labelled cells in any of the nuclei examined. We conclude that, although cells of the PVN, BNST and/or arcuate nucleus may affect reproduction via the GnRH cells of the POA, this is unlikely to involve direct input to the POA. If cells of these regions are involved in GnRH suppression during stress, this may occur via interneuronal pathways.

Estradiol enables cortisol to act directly upon the pituitary to suppress pituitary responsiveness to GnRH in sheep

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

ß2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the β2-adrenoceptor agonist (β2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.

Sex difference in the effect of cortisol on the LH response of the pituitary to exogenous GnRH in hypothalamo-pituitary disconnected gonadectomised sheep

We have used the hypothalamo-pituitary disconnected (HPD) sheep model to investigate direct pituitary actions of cortisol to suppress LH secretion in response to exogenous GnRH. We previously observed that, during the non-breeding season, treatment with cortisol did not suppress the LH response to GnRH in HPD gonadectomised rams or ewes.1 In the present experiment, we tested the effect of cortisol on the LH response to exogenous GnRH in gonadectomised HPD sheep during the breeding season. Using a cross-over design, HPD gonadectomised Romney Marsh rams (n = 6) and ewes (n = 5) received a saline or cortisol (250 μg/kg/h) infusion for 30 h on each of two days, one week apart. All animals were treated with 125 ng i.v. injections of GnRH every 2 h during a 6 h control period preceding the infusion and during the infusion. Jugular blood samples were taken during the control period and the first 6 h and last 6 h of the infusion (over 3 LH pulses). Mean plasma concentrations of LH and LH pulse amplitudes, driven by programmed GnRH injections, were similar in gonadectomised rams and ewes and there were no significant effects of saline infusion between the control periods or the saline infusion in either sex. The amplitude of LH pulses was significantly (P < 0.05) reduced in rams during the first 6 h of the cortisol infusion compared to the control period, but there were no effects of the cortisol infusion in ewes. These data show that, in the absence of sex steroids, there is a sex difference in the mechanism by which cortisol acts at the pituitary to reduce LH secretion in response to exogenous GnRH in HPD gonadectomized sheep during the breeding season. We conclude that the effect of cortisol to reduce secretion of LH involves an action on the pituitary, at least in gonadectomised rams.

Agonist allosteric enhancers at A1 adenosine receptors

Pharmaceutical agents that may be protective to the heart during ischemia were targeted in this project. Analogs to a lead compound containing potential cardioprotective properties were prepared. In the pharmacological evaluation five compounds were found to be more potent than the lead compound.

Short term aerobic exercise training in young males does not alter sensitivity to a central serotonin agonist

An increase in the concentration of serotonin in the brain has been shown to cause fatigue during exercise in humans and experimental animals. This type of fatigue is referred to as central fatigue and is likely to be mediated by the concentration of serotonin as well as serotonin receptor sensitivity. Serotonin (5-HT) receptor antagonism in humans and experimental animals has been shown to improve endurance performance. A previous report has shown decreased receptor sensitivity in athletes compared to sedentary controls. It is unclear whether this is due to a training adaptation or if individuals are predisposed to enhanced athletic performance due to their inherent decreased receptor sensitivity. The present study investigated changes in 5-HT receptor sensitivity in response to aerobic exercise. Subjects completed 3 × 30 min of stationary cycling at 70% of their peak aerobic power (V̇O2,peak) for 9 weeks. Serotonin receptor sensitivity was assessed indirectly by measuring the neuroendocrine response following administration of a serotonin agonist (buspirone hydrochloride). The neuroendocrine response following administration of a placebo was also investigated in a blind crossover design. A group of sedentary control subjects was also recruited to control for seasonal variations in central receptor sensitivity. The training caused a significant increase in V̇O2,peak (3.1 ± 0.16 to 3.6 ± 0.15 l min−1, P < 0.05) and endurance capacity (93 ± 8 to 168 ± 11 min, P < 0.05), but there was no change (P > 0.05) in the neuroendocrine response in the presence of a serotonin agonist. However, one-quarter of the subjects in the training group demonstrated decreases in receptor sensitivity. These results suggest that despite increases in V̇O2,peak and endurance performance, there was no measurable change in 5-HT receptor sensitivity in the presence of a serotonin agonist. In addition, it is possible that changes in receptor sensitivity may take longer to occur, that the training stimulus used in the present investigation was inadequate and/or that changes occurred in receptor subtypes that were not probed by the agonist used in the present investigation.

Endurance training in Wistar rats decreases receptor sensitivity to a serotonin agonist

There is mounting evidence that increased brain serotonin during exercise is associated with the onset of CNS-mediated fatigue. Serotonin receptor sensitivity is likely to be an important determinant of this fatigue. Alterations in brain serotonin receptor sensitivity were examined in Wistar rats throughout 6 weeks of endurance training, running on a treadmill four times a week with two exercise tests per week to exhaustion. Receptor sensitivity was determined indirectly as the reduction in exercise time in response to a dose of a serotonin (1A) agonist, m-chlorophenylpiperazine (m-CPP). The two groups of controls were used to examine (i) the effect of the injection per se on exercise performance and (ii) changes in serotonin receptor sensitivity associated with maturation. In the test group, undrugged exercise performance significantly improved by 47% after 6 weeks of training (4518 ± 729 to 6640 ± 903 s, P=0.01). Drugged exercise performance also increased significantly from week 1 to week 6 (306 ± 69–712 ± 192 s, P = 0.04). Control group results indicated that the dose of m-CPP alone caused fatigue during exercise tests and that maturation was not responsible for any decrease in receptor sensitivity. Improved resistance to the fatiguing effects of the serotonin agonist suggests desensitization of central serotonin receptors, probably the 5-HT1A receptors. Endurance training appears to stimulate an adaptive response to the fatiguing effects of increased brain serotonin, which may enhance endurance exercise performance.

Homogenização da coorte folicular pela administração de estradiol em ciclos de estimulação ovariana controlada com antagonista de GnRH protocolo de doses múltiplas

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