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

1. 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.     

Dynamic Aspects of Glucocorticoid Receptors in the Spinal Cord of the Rat

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.     

The dioxin receptor: characterization of its DNA-binding properties

The binding of the rat hepatic dioxin and glucocorticoid receptors to the polyanionic matrices heparin-Sepharose and DNA-cellulose in vitro and to cell nuclei in vivo was studied under various conditions. In a non-liganded and non-activated state both receptors eluted from heparin-Sepharose at a low ionic strength and were not retained on DNA-cellulose. Following ligandation and activation in vitro both receptors showed an increased affinity for heparin-Sepharose and were retained on DNA-cellulose. In analogy to these in vitro data, it was found that a high salt concentration (0.4 M KCl) was required to extract in vivo liganded dioxin receptor from purified nuclear preparations in contrast to that previously reported for non-liganded nuclear receptors. Limited proteolysis of both dioxin and glucocorticoid receptors resulted in molecular species of similar binding properties with regard to DNA-cellulose and heparin-Sepharose. We conclude that, in addition to the dioxin and glucocorticoid receptors showing considerable similarities in their physicochemical properties, they may also share a similar structural organization with regard to functional domains.     

Regulation of 1,25(OH)2D3 (calcitriol) receptor by vitamin B6

In vitamin B6-deficient rats the concentration of in vivo occupied nuclear and total cellular receptors of 1.25(OH)2D3 increases 1.3-1.7 times, whereas the binding of in vitro occupied receptors to DNA-cellulose increases by 40%. Pyridoxal-5'-phosphate (PLP) added in vitro to solubilized receptors of 1.25(OH)2D3 lowers the ligand binding by 15-25% but causes no dissociation of hormone-receptor complexes formed in vivo. The association of in vitro occupied receptors of 1.25(OH)2D3 with DNA-cellulose is suppressed by PLP (3.5-4.5-fold). It has been shown for the first time that vitamin B6 is a physiological regulator of 1.25(OH)2D3 receptor binding by chromatin and DNA which diminish the concentration of occupied receptors and thus suppress the hormonal response.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.     

Differential activation and blockade of excitatory amino acid receptors in the mammalian and amphibian central nervous systems

1. Experiments were conducted in vitro on isolated spinal cords of frogs and immature rats and in vivo on cat spinal neurones. 2. The concept of two major types of excitatory amino acid receptors present in these preparations is summarized, one type (NMDA receptors) being activated specifically by N-methyl-D-aspartate (NMDA) and blocked by specific antagonists such as D(-)-2-amino-5-phosphonovalerate (APV), and a second type (non-NMDA receptors) characterized by insensitivity to specific NMDA antagonists. This second type may be comprised of two sub-types activated selectively by the agonists quisqualate and kainate. The putative transmitters L-glutamate and L-aspartate have mixed action on both NMDA and non-NMDA receptors. The major action of both transmitter candidates is considered to be on non-NMDA receptors, but the proportion of the composite responses mediated by NMDA receptors (at least for spinal neurones) appears to be greater for L-aspartate than for L-glutamate. 3. The preference of NMDA and non-NMDA receptors for a range of agonists is discussed. Some newer agonists are considered, in addition to several known agonists not previously discussed in terms of NMDA- and non-NMDA-receptor preference. Structure-activity relations of agonists are discussed. 4. The actions of some new amino acid antagonists are reported. Some of these have useful kainate and quisqualate blocking activity, in addition to their ability to block NMDA induced responses. 5. Evidence is presented suggesting that excitatory amino acid receptors are involved in both polysynaptic and monosynaptic excitation in the spinal cord, NMDA receptors mediating polysynaptic excitation and non-NMDA receptors monosynaptic excitation. 6. The unusual effect is reported of L-2-amino-4-phosphonobutyrate, which potently blocks spinal synaptic excitation in the absence of depressant action on excitatory amino acid-induced responses.     

The action of quinolinate in the rat spinal cord in vitro

The responses of dorsal horn neurones to the excitatory amino acids quisqualate, kainate, N-methyl-D-aspartate (NMDA), and quinolinate have been examined in an in vitro preparation of the rat spinal cord. The antagonism of these responses by iontophoretically applied D-(-)-2-amino-5-phosphonovalerate (DAPV), kynurenate, and acridinate was tested, and the results were compared with data obtained from the spinal cord in vivo. The pattern of antagonism was similar in both preparations, although the potencies of agonists and antagonists were found to be significantly greater in vitro. The antagonism of amino acid induced firing of neurones was also recorded during the application of DAPV and kynurenate in the bathing medium. Dose-response curves and IC50 values were determined for these antagonists against all four agonists. The responses to quinolinate were antagonized differently from those to NMDA, quisqualate, or kainate, suggesting that quinolinate does not act specifically through the NMDA receptor as it does in other regions, nor does it appear to act via two or more of the three archetypal amino acid receptors. These findings suggest that a fourth amino acid receptor responsible for quinolinate's action in the spinal cord may exist.     

Nociceptin/orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions

Nociceptin and its receptor are present in dorsal spinal cord, indicating a possible role for this peptide in pain transmission. The majority of functional studies using behavioral and electrophysiological studies have shown that nociceptin applied at spinal level produces antinociception through pre- and post-synaptic mechanisms. The spinal inhibitory effect of nociceptin is not sensitive to antagonists of opioid receptors such as naloxone. Thus, nociceptin-induced antinociception is mediated by a novel mechanism independent of activation of classic opioid receptors. This has raised the possibility that agonists of the nociceptin receptor may represent a novel class of analgesics. Supporting this hypothesis, several groups have shown that intrathecal nociceptin alleviated hyperalgesic and allodynic responses in rats after inflammation or partial peripheral nerve injury. Electrophysiological studies have also indicated that the antinociceptive potency of spinal nociceptin is maintained or enhanced after nerve injury. It is concluded that the predominant action of nociceptin in the spinal cord appears to be inhibitory. The physiological role of nociceptin in spinal nociceptive mechanisms remains to be defined. Moreover, further evaluation of nociceptin as a new analgesic calls the development of non-peptide brain penetrating agents.     

Amino acid pharmacology in a mature rat spinal cord preparation in vitro

1. It has been shown that a relatively large intact piece of mature mammalian spinal cord can be maintained in vitro if suitable experimental conditions are employed. 2. The preparation as described gives robust and reproduceable reflex responses in both dorsal and ventral horns (ventral horn activity has been maintained for over 36 hr in vitro). 3. The action of antagonists at both excitatory and inhibitory amino acid receptors give qualitatively predictable results from known in vivo experiments with the added advantage of fine quantitative control. 4. A significant advantage of such an adult preparation over more common immature spinal cord preparation is the ability to clearly separate low threshold reflexes, such as those described in this article, from reflexes evoked by small diameter non-myelinated afferents.     

Inhibition of hippocampal field potentials by GYKI 52466 in vitro and in vivo.

GYKI 52466 is a specific antagonist of the neuronal excitation mediated by the non-NMDA type excitatory amino acid receptors, at several sites in the central nervous system. The experiments presented here show that the drug has a dose-dependent, slowly developing, long-lasting and reversible inhibitory action on the field potentials recorded from the CA1 region of the rat hippocampus, in vitro. Its action is similar to that of the well-known non-NMDA receptor blocker, CNQX. When the stimulus intensity-dependence of the population spikes was investigated, both drugs shifted the input-output curves in a parallel manner, while the maximum responses were only slightly depressed at the doses applied. With i.v. application, GYKI 52466 also inhibited the hippocampal field potentials recorded from the CA1 region of anesthetized rats dose-dependently. The inhibition was relatively weak compared to the effect found in earlier studies in the spinal cord, by the same doses. Four mg/kg i.v., a doses which is able to block spinal reflexes completely, caused an only about 20% depression of the recorded responses in the hippocampal CA1 area.     

Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord

Abstract: In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo , we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [³H]quinuclidinyl benzilate. After a 24-h exposure to carbachol given every 4 h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.     

UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: behavioral and electrophysiological studies in mice

Nociceptin/orphanin FQ (N/OFQ) modulates various biological functions, including nociception, via selective stimulation of the N/OFQ peptide receptor (NOP). Here we used the NOP selective antagonist UFP-101 to characterize the receptor involved in the spinal antinociceptive effects of N/OFQ evaluated in the mouse tail withdrawal assay and to investigate the mechanism underlying this action by assessing excitatory postsynaptic currents (EPSC) in laminas I and II of the mouse spinal cord dorsal horn with patch-clamp techniques. Intrathecal (i.t.) injection of N/OFQ in the range of 0.1-10 nmol produced a dose dependent antinociceptive effect, which was prevented by UFP-101, but not by naloxone. In contrast the antinociceptive effect of the mu-opioid peptide receptor agonist endomorphin-1 was blocked by naloxone but not by UFP-101. Moreover, N/OFQ and endomorphin-1 induced a significant antinociceptive effect in wild type mice while in mice knockout for the NOP receptor gene only endomorphin-1 was found to be active. In mouse spinal cord slices 1 microM N/OFQ reduced EPSC to 60+/-4% of control values. This inhibitory effect was reversed in a concentration dependent manner by UFP-101 (pA2 value 6.44). The present results demonstrate that N/OFQ-induced spinal antinociception in vivo and inhibition of spinal excitatory transmission in vitro are mediated by receptors of the NOP type.     

Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp.

Narp (neuronal activity-regulated pentraxin) is a secreted immediate-early gene (IEG) regulated by synaptic activity in brain. In this study, we demonstrate that Narp possesses several properties that make it likely to play a key role in excitatory synaptogenesis. Narp is shown to be selectively enriched at excitatory synapses on neurons from both the hippocampus and spinal cord. Overexpression of recombinant Narp increases the number of excitatory but not inhibitory synapses in cultured spinal neurons. In transfected HEK 293T cells, Narp interacts with itself, forming large surface clusters that coaggregate AMPA receptor subunits. Moreover, Narp-expressing HEK 293T cells can induce the aggregation of neuronal AMPA receptors. These studies support a model in which Narp functions as an extracellular aggregating factor for AMPA receptors.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Characteristics of central binding sites for [3H] DAMGO in spontaneously hypertensive rats

The binding of [3H] DAMGO, a highly selective ligand for mu-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. [3H] DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (Bmax value) and apparent dissociation constant (Kd value) of [3H] DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of [3H] DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the Kd values in the two strains did not differ. On the other hand, the Bmax value of [3H] DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the Kd values in the two strains were similar. It is concluded that SHR rats have higher density of mu-opiate receptors in hypothalamus and midbrain but lower density in amygdala in comparison with WKY rats, and that such differences in the distribution of mu-opiate receptors may be related to the elevated blood pressure in SHR rats.     

Binding of corticosteroid receptors to rat hippocampus nuclear matrix.

In rat hippocampus, the mineralocorticoid receptor and the glucocorticoid receptor bind corticosterone with high affinity. We have studied the association of these receptors with the nuclear matrix both after in vivo and in vitro administration of radiolabelled corticosterone to hippocampus cells. It was found that in vivo 100% and in vitro 60% of the corticosterone that specifically bound to rat hippocampus nuclei was attached to the nuclear matrix. A selective glucocorticoid receptor agonist did not compete for corticosterone binding. This indicates that this binding was mediated by the mineralocorticoid receptor rather than the glucocorticoid receptor.     

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.