Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.     

Modulatory Effects of Thyroxine Treatment on Central and Peripheral Benzodiazepine Receptors in the Rat

The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up-regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress.     

Enhancement of γ-Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors

We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro

Epidermal cells were dissociated from tails of the bullfrog tadpole, Rana catesbeiana, and cultured to investigate their response to steroid and thyroid hormones. Charcoal-treated serum (CTS) was used in the growth medium when cells were to be grown in the absence of steroid and thyroid hormones. The cells could be maintained for 2 weeks with a small increase in cell number in medium that contained CTS (CTS medium). Addition of cortisol to CTS medium increased both cellular attachment to the culture dishes and the proliferation of the attached cells with an optimum concentration of 5 X 10(-7) M. The cells remained viable and attached for at least a week. Cortisol stimulated the rate of protein synthesis 1.8-fold but did not alter the rate of DNA synthesis. The cells did not proliferate in the medium containing triiodothyronine (T3) and detached themselves from the dish within 5 days, which occurred in a dose-dependent manner with a maximum effect at 10(-8) M. It drastically decreased the rate of DNA synthesis but did not influence the rate of protein synthesis. These responses of cells to cortisol and T3 may reflect growth and death of tail epidermal cells in vivo at metamorphosis.     

Single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat.

We studied the effects of single (45 min) and repeated (ten daily 45-min sessions) microwave exposures (2450-MHz, 1 mW/cm2, average whole-body SAR of 0.6 W/kg, pulsed at 500 pps with pulse width of 2 microseconds) on the concentration and affinity of benzodiazepine receptors in the cerebral cortex, hippocampus, and cerebellum of the rat. We used a receptor-binding assay with 3H-flunitrazepam as ligand. Immediately after a single exposure, an increase in the concentration of receptor was observed in the cerebral cortex, but no significant effect was observed in the hippocampus or cerebellum. No significant change in binding affinity of the receptors was observed in any of the brain-regions studied. In rats subjected to repeated exposures, no significant change in receptor concentration was found in the cerebral cortex immediately after the last exposure, which may indicate an adaptation to repeated exposures. Our data also show that handling and exposure procedures in our experiments did not significantly affect benzodiazepine receptors in the brain. Because benzodiazepine receptors in the brain are responsive to anxiety and stress, our data support the hypothesis that low-intensity microwave irradiation can be a source of stress.     

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.     

Changes in the characteristics of low affinity GABA binding sites elicited by Ro15-1788

3H-GABA binding was studied in cortical membranes from cerebral cortex of handling-habituated and naive rats after the in vitro addition of Ro15-1788. At low concentrations (10(-8), 10(-9) M) Ro15-1788 increased the total number of low affinity 3H-GABA binding sites in brain tissue from naive rats but failed to modify 3H-GABA binding in tissue from handling-habituated ones. On the contrary, Ro15-1788 at higher concentrations (10(-5), 10(-6)M) decreased the total number of low affinity 3H-GABA binding sites in tissue from handling-habituated rats but failed to modify 3H-GABA binding in tissue from naive animals. Ro15-1788 (10(-7)M) failed to modify significantly low affinity 3H-GABA binding in membranes from both naive and handling-habituated rats. However, this concentration abolished the effect of beta-carbolines and diazepam on 3H-GABA binding in membranes from naive and handling-habituated rats, respectively. The changes in the affinity of 3H-GABA binding were inversely related to the changes in the number. The results suggest that: a) the action "in vitro" of Ro15-1788 on low affinity 3H-GABA binding depends from its concentration at the benzodiazepine recognition sites; b) the benzodiazepine recognition site has a modulatory role in the control of the function of GABA-ergic receptor. Our data might explain the conflicting results obtained with this compound "in vivo".     

I-125 labelled thyroid hormones in cultured mammalian nerve tissue

Summary Cultures of embryonic mouse and rat spinal cord-dorsal root ganglion combinations, and of newborn hamster and rat cerebellum were treated with 5×10^-6M I-¹²⁵ thyroxine and I-¹²⁵ triiodothyronine. Autoradiographic studies on glutaraldehyde-osmium tetroxide fixed tissue indicated that these hormones were located predominantly in neurons, but could be found in glia cells as well. This pattern was observed in cultures treated for 2 hours to 4 days. Some of the effects of thyroid hormones on the developing nervous system were therefore linked to a direct action of these hormones on nerve cells.Furthermore, the label was seen not only over the cytoplasm, as might be expected from past biochemical studies, but was also present over the nucleus, particularly the nucleolus. This suggested a possible role for these hormones in the process of translation.This work was supported by NIH Grant NB06735, The Nancy Louise Trynor Memorial Grant for Research on Multiple Sclerosis 433-D-10, and United States Public Health Service Grant No. NB-07849-02.Post-Doctoral Fellow, Department of Pathology, Yale University.Kennedy Scholar of the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, New York.     

Interactions between nitrogen oxide-containing compounds and peripheral benzodiazepine receptors

Nitrogen oxide-containing compounds displaced the peripheral benzodiazepine ligand [³H]Ro5-4864 from guinea pig membrane preparations. Sodium nitroprusside (SNP) was the most potent (IC₅₀ = 5.61 ± 1.72 × 10⁻⁵ M). Moreover, its ability to bind to these receptors showed marked tissue variability (heart> kidney cerebral cortex). When tested on rat atrium, SNP by itself had no effect on basal inotropy or the increase in inotropy induced by (−)-S-BAY K 8644. In contrast, Ro5-4864 potentiated the marked increase in inotropy induced by (−)-S-Bay K 8644, and SNP completely abolished the potentiation of inotropy observed with Ro5-4864. Since peripheral benzodiazepine receptors are associated with calcium mobilization in the heart, these findings may indicate that some of the clinical effects of nitric oxide-generating drugs could be mediated by these receptors.     

Brain benzodiazepine binding sites in ethanol dependent and withdrawal states

The brain benzodiazepine system has been implicated to be important in both the mechanism, and treatment of ethanol related syndromes. In this report evidence is presented which indicates that "peripheral type" benzodiazepine binding sites are probably more relevant than "central type" receptors for the neurochemical consequences of ethanol dependence and withdrawal states. Utilizing radioreceptor binding techniques 20-50% increases in the binding of [3H]RO-5-4864 (a "peripheral type" ligand) to brain membranes derived from rat cerebral cortex, cerebellum and hippocampus are observed in ethanol dependent rats. These increases persist for 3 days after cessation of ethanol. The number of [3H]RO-5-4864 binding sites in cerebellum returns to normal during 4-7 days after ethanol withdrawal. In all brain areas examined no changes were observed in the "central type" benzodiazepine receptor as judged by [3H]-ethyl-Beta-carboline-3-carboxylate, BCCE binding. Scatchard analysis revealed that the number of [3H]RO-5-4864 binding sites is increased in each brain area while the affinity was unchanged.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic caffeine on brain adenosine receptors: Regional and ontogenetic studies

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [³H] diethylphenylxanthine, the benzodiazepine receptor, using [³H] diazepam and the adenosine uptake site, using [³H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogency of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20–30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Oxygen-Induced Modifications of Benzodiazepine Receptors and D2 Dopamine Receptors in the Rat Under Hyperoxia

Peripheral-type benzodiazepine Receptors (PBR) in the kidney and Central-type Benzodiazepine Receptors (CBR) in the cerebral cortex were not affected in rats exposed to chronic hyperoxia (85% O₂, ATA, 6 days). Nevertheless, cortical CBR showed a significant decrease (29%) after hyperbaric hyperoxia (100% O₂, 3.5 ATA, 2h) in rats at a preconvulsive stage, with no concomitant alteration of kidney PBR. A similar down-regulation of striatal D₂ dopamine receptors was noticed (27%) - after hyperbaric hyperoxia— without any modification of cortical PBR. On the contrary, an up regulation of liver PBR was obtained in the same conditions (20%). It is likely that receptors implicated in neurotransmission are particularly down regulated or altered under hyperbaric hyperoxia.     

The nature of thyroid hormone receptors. Intracellular functions of thyroxine-binding prealbumin

The effect of tyroxin-binding prealbumin (TBPA) of blood serum on the template activity of chromatin was studied. It was found that the values of binding constants of TBPA for T3 and T4 are 2 X 10(-11) M and 5 X 10(-10) M, respectively. The receptors isolated from 0.4 M KCl extract of chromatin and mitochondria as well as hormone-bound TBPA cause similar effects on the template activity of chromatin. Based on experimental results and the previously published comparative data on the structure of TBPA, nuclear, cytoplasmic and mitochondrial receptors of thyroid hormones as well as on translocation across the plasma membrane and intracellular transport of TBPA, a conclusion was drawn, which suggested that TBPA is the "core" of the true thyroid hormone receptor. It was shown that T3-bound TBPA caused histone H1-dependent conformational changes in chromatin. Based on the studies with the interaction of the TBPA-T3 complex with spin-labeled chromatin, a scheme of functioning of the thyroid hormone nuclear receptor was proposed.     

Benzodiazepine Enhancement of γ-Aminobutyric Acid-Mediated Chloride Ion Flux in Rat Brain Synaptoneurosomes

Benzodiazepine agonists such as Ro 11-6896 [B10(+)], diazepam, clonazepam, and flurazepam were found to enhance muscimol-stimulated 36Cl- uptake into rat cerebral cortical synaptoneurosomes. The rank order of potentiation was B10(+) greater than diazepam greater than clonazepam greater than flurazepam. These benzodiazepines had no effect on 36Cl-uptake in the absence of muscimol. Further, the inactive enantiomer, Ro 11-6893 [B10(-)], and the peripheral benzodiazepine receptor ligand Ro 5-4864 did not potentiate muscimol-stimulated 36Cl- uptake at concentrations up to 10 microM. In contrast, the benzodiazepine receptor inverse agonists ethyl-beta-carboline-3-carboxylate and 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylic acid methyl ester inhibited muscimol stimulated 36Cl- uptake. Benzodiazepines and beta-carbolines altered the apparent K0.5 of muscimol-stimulated 36Cl- uptake, without affecting the Vmax. The effects of both benzodiazepine receptor agonists and inverse agonists were reversed by the benzodiazepine antagonists Ro 15-1788 and CGS-8216. These data further confirm that central benzodiazepine receptors modulate the capacity of gamma-aminobutyric acid receptor agonists to enhance chloride transport and provide a biochemical technique for studying benzodiazepine receptor function in vitro.     

Effect of long term diazepam administration on testicular benzodiazepine receptors and steroidogenesis.

We evaluated the effect of acute and chronic diazepam administration on testicular peripheral type benzodiazepine receptors (PBZD-R), serum testosterone and LH levels and the "in vitro" androgen production in response to Ro 5-4864, a PBZD-R agonist. The chronic diazepam treatment induced a significant fall in plasma testosterone concentration while LH levels remained unchanged. The number of PBZD-R was reduced by 37% and low concentrations (10(-8)-10(-6) M) of Ro 5-4864 failed to stimulate "in vitro" androgen production. The acute diazepam administration caused a significant increase in plasma testosterone levels while no changes were observed in LH concentrations and testicular PBZD-R. These results further suggest a modulatory role of PBZD-R on testicular steroidogenic activity.