Eduardo Ortí

Regulation of Glucocorticoid Receptors in Brain by Corticosterone Treatment of Adrenalectomized Rats

Binding of (3H)-corticosterone in cytosol of hippocampus and hypothalamus has been measured in adrenalectomized (ADX) rats in the presence or absence of corticosterone replacement therapy (suspended shortly before receptor analysis). Corticosterone pellet implantation into female rats or oral corticosterone administration in salinized drinking water given to males for 3 weeks reduced (3H)-corticosterone binding by half in the hippocampus. This reduction was observed whether corticosterone or dexamethasone was employed as competitor to determine nonspecific binding, thus eliminating transcortin as the cause of the corticosterone effect on binding. Scatchard analysis of binding data revealed that the reduction was mostly due to decreased number of receptors. Animals pretreated with corticosterone had a reduction in thymus weight, indicating further the biological effectiveness of the treatment. Further, serum corticosterone in ADX rats pretreated with corticosterone (but with therapy suspended for 24 h) was very low and similar to that of untreated ADX rats. Uptake studies after injection of (3H)-corticosterone intravenously into ADX rats showed that the injected hormone was absent from blood and brain tissues 1 day later, ruling out (in addition to the measurement of serum corticosterone) that the reduction in binding was due to occupation of receptor sites by exogenous corticosterone remaining after withdrawal from therapy. It is suggested that down-regulation of glucocorticoid receptors in brain follows the chronic corticosterone administration. These data are discussed in relation with evidence for down-regulation of other classes of steroid receptors in several tissues, and the consequence that changes in receptor binding in brain may have on the feedback mechanism of corticoids at the central level.

Adrenocorticoid action in the spinal cord: some unique molecular properties of glucocorticoid receptors.

Summary1.Glucocorticoid hormones affect several functions of the spinal cord, such as synaptic transmission, biogenic amine content, lipid metabolism, and the activity of some enzymes (ornithine decarboxylase, glycerolphosphate dehydrogenase), indicating that this tissue is a target of adrenal hormones.2.Corticosterone, the main glucocorticoid of the rat, is detected at all regional levels of the spinal cord, and cold stress increases this steroid, predominantly in the cervical regions.3.Intracellular glucocorticoid receptors have been found in the spinal cord, with higher concentrations in the cervical and lumbar enlargements. Prima facie, these receptors presented biochemical, stereospecifical, and physicochemical properties similar to those of receptors found in other regions of the nervous system. The prevalent form in the spinal cord is the type II receptor, although type I is also present in small amounts.4.The type II glucocorticoid receptor of the spinal cord shows an affinity lower (Kd 3.5 nM) than that of the hippocampal type II site (Kd 0.7 nM) when incubated with [3H]dexamethasone. This condition may impair the nuclear translocation of the spinal cord receptor.5.Another peculiar property of spinal cord type II site is a greater affinity for DNA-cellulose binding than the hippocampal receptor during heat-induced transformation. Also, the spinal cord receptor shows resistance to the action of RNAse A, an enzyme which increases DNA-cellulose binding of the hippocampal receptor, indicating that both receptors may be structurally different.6.Therefore, it is possible that a different subclass of type II, or “classical glucocorticoid receptor,” is present in the spinal cord. This possibility makes the cord a useful system for studying diversity of glucocorticoid receptors of the nervous system, especially the relationship between receptor structure and function.

Glucocorticoid Regulation of Glycerol Phosphate Dehydrogenase and Ornithine Decarboxylase Activities in the Spinal Cord of the Rat

Abstract: We examined the effects of glucocorticoids on induction of glycerol phosphate dehydrogenase (GPDH) and ornithine decarboxylase (ODC) in the spinal cord of rats. After a single subcutaneous dose of 5 mg/kg of dexametha‐sone (DEX) phosphate, GPDH activity was maximally increased at 20 h with the effect still persisting for 46 h, in contrast to ODC activity, which was already stimulated at 4 h. The enzyme induction was accompanied by a reduction in number of cytosolic glucocorticoid receptors already at 1 h after DEX treatment, with replenishment at 22 h. A dose‐response curve for DEX demonstrated that the minimal effective dose (0.2 mg/kg) for enzyme induction also reduced the number of cytosolic receptors because of occupation/depletion. The effects were specific for natural and synthetic glucocorticoids, as GPDH and ODC activities were not stimulated by aldosterone, testosterone, estradiol, or progesterone. ODC was induced in the cervical region of the spinal cord as well as in the horse tail plus filum terminale, whereas GPDH responded in the former but not the latter region. Previous work has demonstrated that glucocorticoid receptors are slightly more concentrated in the cervical spinal cord. It is suggested that glucocorticoid induction of these two predominantly glial enzymes occurs by a steroid receptor‐mediated event, as postulated in other regions of the nervous system. In view of the short latency required for induction of ODC, we also examined the effect of inhibitors of transcription and translation. Whereas cycloheximide reduced the stimulatory effect of DEX, a paradoxical stimulation was obtained when DEX and dactinomycin (actinomy‐cin D) were given concomitantly. It is suggested that the inductive responses of GPDH and ODC to glucocorticoids may be different. Considering that GPDH induction has been shown by other laboratories to represent a genomic effect of adrenal steroids, different levels of control may account for the stimulation of ODC in the spinal cord.

Heterogeneity and properties of transformation of corticosteroid receptors in spinal cord and hippocampus.

The central nervous system contains two classes of corticoid receptors, named types I and II following terminology accepted for the kidney. Phenotypically, type I sites are differentiated into a corticosterone (CORT)-preferring species (Ia) and a mineralocorticoid receptor (Ib). These populations were tentatively compared in the spinal cord and hippocampus. Using [3H]dexamethasone (DEX) and selective blockage of sites, we have observed that type II receptors were comparable in both tissues, while Ia was almost exclusive of the hippocampus. Saturation analysis using [3H]DEX demonstrated that type Ia was a low affinity receptor (Kd approximately equal to 2-5 nM) while type II was a higher affinity site (KdII less than KdI). Using [3H]CORT, or [3H]aldosterone (ALDO), as ligand, preferential labeling of type I sites was achieved, always showing higher concentrations in the hippocampus. Therefore, [3H]DEX seems a ligand of choice to visualize types Ia and II receptors. Another difference noted between the spinal cord and hippocampus, pertained to the sensitivity towards the enzyme RNAse A, which increases heat-induced transformation of the bound receptor, according to the results of DNA cellulose affinity chromatography. In these experiments, type I sites of both spinal cord and hippocampus, plus type II of hippocampus, showed sensitivity toward the enzyme, whereas type II of the spinal cord was refractory to RNAse A enhancement of transformation. These results indicate that the dynamics of transformation is different among receptors showing similar affinity and competition, suggesting further heterogeneity due to receptors themselves, or to tissue factors regulating their biochemical properties.

Dynamic aspects of glucocorticoid receptors in the spinal cord of the rat

Abstract: In spite of biochemical and autoradiographic evidence for glucocorticoid binding sites in the spinal cord (SC), events occurring after the preliminary step of hormone binding were not studied. In this investigation, we have examined the transformation (activation) of the cytosolic receptor coupled to [3H]dexamethasone (DEX) and the in vivo interaction of adrenal hormone [corticosterone (CORT)] with purified nuclei from the SC, in addition to the CORT content of the SC before and after stress. Binding of [3H]DEX in the SC was 40% lower than in the hippocampus (HC), although the KD values were comparable. Transformation of [3H]DEX‐receptor complexes in the cytosol was demonstrated by diethylaminoethane‐cellulose chromatography, by DNA‐cellulose binding, and by a combined minicolumn procedure including hydroxyapatite in addition to the last two techniques for separation of transformed, nontransformed, and meroreceptor complexes. In all these situations, SC glucocorticoid binding sites behaved similarly to those in the HC. Nuclear uptake of a tracer dose of [3H]CORT was much lower in the SC than in the HC; nuclear retention of CORT was more easily detected by radioimmunoassay after injection of 1 mg of CORT into adrenalectomized rats. Substantial amounts of CORT, which increased in level after stress, were measured in five regions in the SC, with higher concentrations in the cervical regions. These studies suggest that although SC and HC receptors show similar properties in vitro, differences emerged at the level of nuclear uptake in vivo, in that glucocorticoid action in the SC was similar to that in the optic nerve, where receptors seem to be localized mostly in glial cells.

Evidence of high affinity, stereoselective binding sites for [3H]-aldosterone in the spinal cord

Cytosol from the spinal cord (SC) of adrenalectomized rats was incubated with a range of [3H]-aldosterone (ALDO) concentrations and the results analyzed according to Scatchard. High affinity (Kd less than or equal to 1 nM) as well as low affinity (Kd 30-195 nM) sites were measured; the low affinity site was abolished by co-incubation with the pure antiglucocorticoid RU 28362. [3H]-ALDO in concentrations shown to bind to the high affinity site only, was completely displaced by the spirolactone, RU 26752, but not by RU 28362; however, the latter compound competed for 40% of the sites when incubated with a higher (10 nM) concentration of [3H]-ALDO, binding approximately one-half to each receptor type. The high affinity site was distributed uniformly in four sections of the SC, with significantly lower levels in the region corresponding to the filum terminale and horse tail and slightly higher in the cervical and lumbar enlargements. The high affinity site acquired increased affinity for DNA-cellulose after heat-induced transformation, with reduced capacity to bind to DEAE-cellulose. These results suggest that the SC contains, in addition to glucocorticoid receptors, binding molecules of high affinity and stereoselectivity for mineralocorticoids, and that, under appropriate conditions, present increased affinity for DNA acceptor sites.

Site-Specific Effects of the Nonsteroidal Anti-Inflammatory Drug Lysine Clonixinate on Rat Brain Opioid Receptors

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 × 10–4 mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

Regulatory factors of glucocorticoid binding in early and term rat placenta.

We have measured by an exchange procedure the binding of [3H]dexamethasone in cytosol of early (10-13 days) and late (19-22 days) placentas from pregnant rats. Binding was 3-fold higher in late placentas both in the presence of Na2MoO4. We then studied some possible regulatory factors in order to explain differences in binding at both gestational ages. The activity of enzymes compromising the phosphorylation (acid and alkaline phosphatases) or stability (protease) of the receptor were normal or lower in early as opposed to late placenta, discarding these enzymes as leading regulatory factors. Cyclic nucleotides were also studied, in view that they regulate steroid binding in uterus and placenta. Both basal and epinephrine-stimulated production of cAMP were higher in early placenta. cAMP (but not cGMP) inhibited [3H]dexamethasone binding by reducing the number of sites without changing the Kd. Moreover, addition of epinephrine in concentrations that maximally stimulated cAMP, inhibited subsequent binding of [3H]dexamethasone in cytosol. We suggest that cAMP may be a modulator of glucocorticoid binding at the early stages of placental development. The significance of this mechanism may be understood in terms of the opposing effects of cAMP and glucocorticoids on placental progesterone production.

A comparison of the glucocorticoid receptor system in the spinal cord and hippocampus.

Studied under in vivo conditions, uptake of [3H]corticosterone (CORT) by purified cell nuclei of the hippocampus was much higher than in the spinal cord, although the latter may contain in cytosol up to 50% of glucocorticoid receptors found in cytosol of hippocampus. Experiments were undertaken to explain these differences. First, the in vivo affinity of receptors for exogenous CORT was comparable in both tissues. Second, an inhibitor of translocation, although present, was not preferentially concentrated in the spinal cord as compared to the hippocampus. However, the sensitivity towards RNAase A, an enzyme that increased binding to DNA-cellulose (taken as a measure of increased affinity for nuclear components), was preserved in the hippocampus but absent in the cord. We discuss the possibility that refractoriness to RNAase A may play a role in the reduced nuclear uptake of [3H]CORT shown by the spinal cord in vivo, but also consider possible that heterogeneity of receptor types binding CORT in the spinal cord and hippocampus may account for the differences observed in both tissues.

Steps of glucocorticoid action in normal and diabetic rat placenta

This investigation examined the effects of Streptozotocin diabetes in pregnancy on several parameters of glucocorticoid action in the rat placenta. Pregnant diabetic rats showed reduced body weight, increased adrenal weight and serum corticosterone concentrations. Glucocorticoid receptors in placental cytosol of labyrinthine zone, measured in the absence of MoO4Na2 were similar in control and diabetic rats, but after addition of MoO4Na2 receptor number were moderately, but significantly reduced in diabetic placentas (P less than 0.01). No changes in affinity were detected in saturation analysis. Furthermore, transformation of the receptor assessed by its capacity for binding to DNA-cellulose, was enhanced in diabetic animals, suggesting increased efficiency of the receptor-bound hormone. Since the function of the glucocorticoid receptor of rat placenta may be the inhibition of local progesterone production (Heller and De Nicola, J. steroid Biochem. 19 (1983) 1339-1343), we determined progesterone synthesis in vitro and found that diabetic placentas synthesized significantly less progesterone than control tissue (P less than 0.05). Lastly, we found that the metabolism of corticosterone to 11-dehydrocorticosterone, while declining in control placentas as pregnancy advanced, it was sustained in diabetic pregnancy. It is suggested that diabetic rat placentas showed increased activity towards the glucocorticoid receptor, resulting in reduction in progesterone synthesis and sustained catabolism of corticosterone. The latter may possibly constitute a compensatory mechanism to protect the fetal compartment from high levels of maternal glucocorticoids.