RESUMEN
BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Neuronas/fisiología , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos , Hipocampo/citología , Inmunosupresores/farmacología , Ratones , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Prostaglandina-E Sintasas , Ratas , Tacrolimus/farmacologíaRESUMEN
The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.
Asunto(s)
Apoptosis , Citosol/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Transporte Activo de Núcleo Celular , Apoptosis/efectos de los fármacos , Factor Inductor de la Apoptosis/metabolismo , Caspasa 9/metabolismo , Línea Celular , Dineínas/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas de Unión al GTP rac/genéticaRESUMEN
Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.
Asunto(s)
Caveolina 1/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Mamarias Experimentales/patología , Acetato de Medroxiprogesterona/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caveolina 1/genética , Femenino , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Regiones Promotoras Genéticas , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
The mineralocorticoid receptor (MR) is primarily localized in the cytoplasm of the cell in the absence of ligand. The first step in the genomic-dependent mechanism of action of mineralocorticoids is the binding of steroid to the MR, which in turn triggers MR nuclear translocation. The regulation of hormone-binding to MR is complex and involves a multifactorial mechanism, making it difficult to determine the optimal structure of a steroid for activating the MR and promoting its nuclear translocation. Here we review the structure-activity relationship for several pregnanesteroids that possess various functional groups, and suggest that a flat conformation of the ligand rather than the presence of particular chemical groups is a critical parameter for the final biological effect in vivo. We also discuss how the MR undergoes differential conformational changes according to the nature of the bound ligand, which in turn affects the dynein-dependent retrograde rate of movement for the steroid/receptor complex.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Pregnenodionas/administración & dosificación , Pregnenodionas/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pregnenodionas/química , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
We have demonstrated previously that a planar conformation of the molecular frame is required for steroids to acquire optimal sodium-retaining activity and binding properties to the mineralocorticoid receptor (MR). One of the most active sodium-retaining compounds tested in those studies was 11, 19-oxidoprogesterone. Despite its biological potency, the relative affinity of 11,19-oxidoprogesterone for the MR is 5-fold lower than that of 21-deoxycorticosterone and 10-fold lower than aldosterone. Such a discrepancy may be assigned to uncommon biopharmacological properties of this synthetic steroid or an unusual molecular mechanism of action. In this work, we studied the biopharmacological and mechanistic features of 11,19-oxidoprogesterone. We show that both the pharmacokinetic properties of 11,19-oxidoprogesterone and its ability to transform and translocate the MR into the nucleus are undistinguishable from aldosterone. However, the capability of the serine/threonine phosphatase inhibitor tautomycin to impair nuclear translocation of the aldosterone-MR complex is not observed for the 11,19-oxidoprogesterone-MR complex. In addition, the binding properties of both steroids are differentially affected by modification of crucial lysyl residues of the MR. Kinetic studies performed on the aldosterone-MR complex in the presence of low concentrations of oxidopregnane suggest that 11,19-oxidoprogesterone may bind to the MR in a different binding site from the aldosterone binding pocket. Consistent with this postulate, a biologically inactive dose of 0.6 ng of oxidopregnane is able to potentiate the mineralocorticoid effect of a suboptimal dose of aldosterone.
Asunto(s)
Riñón/efectos de los fármacos , Progesterona/análogos & derivados , Progesterona/farmacología , Piranos , Receptores de Mineralocorticoides/metabolismo , Sodio/metabolismo , Compuestos de Espiro , Aldosterona/farmacología , Animales , Antifúngicos/farmacología , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Antagonismo de Drogas , Proteínas HSP90 de Choque Térmico/metabolismo , Riñón/metabolismo , Lisina/química , Masculino , Tasa de Depuración Metabólica , Antagonistas de Receptores de Mineralocorticoides , Conformación Molecular , Progesterona/antagonistas & inhibidores , Progesterona/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/química , Receptores de Esteroides/química , Espironolactona/farmacologíaRESUMEN
In vitro studies have demonstrated that cysteine groups present in most of the steroid receptors play an essential role in the steroid binding process as well as in the ability of this superfamily of signaling proteins to function as transcription factors. However, there is poor experimental evidence, if any, which demonstrates that under conditions of oxidative stress the steroid receptors in general, and the mineralocorticoid receptor in particular, are affected in vivo in a similar fashion as has been described for cell-free systems or cells in culture. In the present work we report that when mice are exposed to oxidative stress by treatment with L-buthionine-(S,R)-sulfoximine (L-(S,R)-BSO), a glutathione depleting agent, the aldosterone-dependent mineralocorticoid biological response (measured as sodium retention and potassium elimination) was diminished in a directly proportional manner with respect to the depletion of renal glutathione. Accordingly, the steroid binding capacity of the mineralocorticoid receptor was also abrogated, whereas the receptor protein level remained unchanged. The harmful effects observed in mice after glutathione depletion were efficiently prevented by co-treatment with glutathione monoethyl ester. Similar inhibition in the steroid binding capacity was also generated in vitro by receptor alkylation and receptor oxidation, an effect which was prevented in the presence of reducing agents. Since the glutathione deficit generated in vivo by treatment with L-(S,R)-BSO did not significantly affect other renal proteins which are known to be required for the mineralocorticoid mechanism of action, we suggest that in renal cells a low redox potential exerts drastic and uncompensated inhibition of the receptor-mediated mineralocorticoid biological response. This effect was ascribed to the loss of steroid binding capacity of oxidized receptor, most likely by modification of essential cysteines as supported by experiments where a decreased number of reactive thiols and reduced covalent binding of thiol-reactive ligand were evidenced on immunopurified receptor after in vivo treatment with L-(S,R)-BSO.
Asunto(s)
Antimetabolitos/farmacología , Butionina Sulfoximina/farmacología , Glutatión/análogos & derivados , Glutatión/metabolismo , Riñón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Glutatión/antagonistas & inhibidores , Glutatión/farmacología , Humanos , Riñón/metabolismo , Ratones , Mineralocorticoides/metabolismo , Oxidación-Reducción/efectos de los fármacos , Esteroides/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon steroid binding. Thus toxic effects observed with these metal ions may be a consequence of modifications of those essential groups. Our results support the notion that toxicity of metals on renal mineralocorticoid function may be predicted according to their chemical hardness.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Riñón/efectos de los fármacos , Metales/farmacología , Antagonistas de Receptores de Mineralocorticoides , Esteroides/metabolismo , Aminoácidos/química , Animales , Riñón/metabolismo , Masculino , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/químicaRESUMEN
The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol-acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, deltaHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes deltaHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.
Asunto(s)
Glucocorticoides/metabolismo , Hidroxiprogesteronas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Apoptosis/efectos de los fármacos , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Genes Reporteros , Glucocorticoides/química , Glucocorticoides/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Hidroxiprogesteronas/química , Hidroxiprogesteronas/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Cinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Modelos Moleculares , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Timo/efectos de los fármacos , Timo/metabolismo , TransfecciónRESUMEN
The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as 'transformation'. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient (S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4-5.4S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [3H]ALDO-MR complex and the percentage of transformed 5.4S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form (Ki(app)=7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.
Asunto(s)
Riñón/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Piranos , Receptores de Mineralocorticoides/metabolismo , Compuestos de Espiro , Aldosterona/metabolismo , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Animales , Antifúngicos/farmacología , Núcleo Celular/metabolismo , Celulosa/análogos & derivados , Celulosa/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía por Intercambio Iónico , Citoplasma/metabolismo , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Riñón/enzimología , Cinética , Levamisol/farmacología , Masculino , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Conformación Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/química , TemperaturaRESUMEN
UNLABELLED: The following in vitro glucocorticoid (GC) parameters of progesterone (P), 1-ene progesterone (deltaP), 11beta-hydroxyprogesterone (HOP), 11beta-1-ene progesterone (deltaHOP) and dexamethasone (Dexa) were assayed in the presence or absence of bovine calf serum (BCS): binding to thymus cytosol, dissociation of the glucocorticoid receptor (GR)-heat shock protein 90 (hsp90) complex (diss.), tyrosine aminotransferase (TAT) induction in hepatocytes and the inhibition of 3H-uridine and 35S-methionine uptake by thymocytes. Without BCS, steroids were in most cases active in this general order: Dex > deltaHOP > HOP > deltaP > P. BCS abolished all activities in P and deltaP, but left them unaltered in all other steroids, except diss. in HOP, which diminished intermediately. Binding of P, deltaP, HOP and deltaHOP to GR and CBG paralleled their in vivo activating effects on glycogen deposition. CONCLUSIONS: in this steroid series, BCS, but not CBG, inhibits GC responses of P and deltaP. 11-Beta hydroxylation frees those molecules from the inhibitory effects of BCS.
Asunto(s)
Hígado/metabolismo , Progesterona/análogos & derivados , Progesterona/farmacología , Timo/metabolismo , Adrenalectomía , Animales , Sangre , Bovinos , Células Cultivadas , Corticosterona/metabolismo , Citosol/metabolismo , Dexametasona/farmacología , Inducción Enzimática , Proteínas HSP90 de Choque Térmico/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Glucógeno Hepático/biosíntesis , Masculino , Metionina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Relación Estructura-Actividad , Timo/efectos de los fármacos , Transcortina/metabolismo , Tirosina Transaminasa/biosíntesis , Uridina/metabolismoRESUMEN
In the rat, the conformationally highly bent steroid 21-hydroxy-6, 19-oxidoprogesterone efficiently displaces [3H]corticosterone from thymus-glucocorticoid receptors and blocks type II receptors in kidney cytosols but competes with neither [3H]aldosterone for kidney-mineralocorticoid receptors nor [3H]progesterone for uterus-progesterone receptors. It evokes Na+ retention only at very high doses (approximately 100 microg/100 g of rat weight) and is unable to induce tyrosine aminotransferase or to increase glycogen deposits in rat liver. When coincubated with corticosterone or dexamethasone, 2.5 microM 21OH-6OP inhibits 80% of tyrosine aminotransferase induction. It may therefore be used experimentally as an antiglucocorticoid virtually lacking mineralocorticoid or glucocorticoid properties as well as affinity for mineralocorticoid or progesterone receptors.
Asunto(s)
Progesterona/análogos & derivados , Receptores de Glucocorticoides/antagonistas & inhibidores , Aldosterona/metabolismo , Androstanoles/metabolismo , Animales , Riñón/metabolismo , Masculino , Progesterona/química , Progesterona/metabolismo , Progesterona/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Espironolactona/metabolismo , Relación Estructura-Actividad , Timo/metabolismo , Transcortina/metabolismo , TritioRESUMEN
11 beta-hydroxyprogesterone (HOP) and 11-ketoprogesterone (KP) are reversible components of a shuttle pair whose interconversion in rat liver is catalyzed by isoform-1 of 11 beta-hydroxysteroid dehydrogenase. Kidneys also produce this interconversion. The present study was carried out to investigate the shuttle pair and its components in the rat. As in corticosterone/11-dehydrocorticosterone, oxidation is more effective at an alkaline pH, while reduction prevails at a neutral pH. Moreover, both reactions are inhibited by the detergent 3-[(3-cholamido propyl)-dimethylammonio]-1-propane-sulphonate (CHAPS). However, at variance with the 11-ketosteroids cortisone (E) and 11-dehydrocorticosterone (A) thought to be "inactive," KP has slight direct Na(+)-retaining properties, and it, as well as HOP, induces glucocorticoids (11 beta-hydroxycorticoids) to retain sodium. 11-ketoprogesterone exhibits 17 times better affinity for native type 1 mineralocorticoid receptor than HOP and a 3-fold affinity for partially purified (transcortin free) mineralocorticoid receptor. However, KP, in contrast to HOP, binds only weakly to transcortin, not at all to glucocorticoid receptor, and requires reduction at C11 for tyrosine aminotransferase (TAT) induction.
Asunto(s)
Hidroxiprogesteronas/metabolismo , Progesterona/análogos & derivados , 11-beta-Hidroxiesteroide Deshidrogenasas , Adrenalectomía , Animales , Unión Competitiva , Ácidos Cólicos , Inducción Enzimática , Concentración de Iones de Hidrógeno , Hidroxiesteroide Deshidrogenasas/metabolismo , Masculino , Progesterona/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Sodio/metabolismo , Transcortina/metabolismo , Tirosina Transaminasa/biosíntesisRESUMEN
The purpose of this work is to review stability and activation properties of type I receptor, in order to explain the reasons for its extreme in vitro instability. We demonstrate that the treatment of rat kidney cytosol with H2O2 prevents aldosterone binding, DNA/steroid-receptor complex interactions, and prevents the receptor thermal inactivation. In contrast, exogenous sulfhydryl reducing reagents are necessary to insure maximum binding of mineralocorticoid receptor and DNA/steroid-receptor interaction. However, the presence of beta-mercaptoethanol in thermal induced incubations reverts the H2O2 protection. We also demonstrate that contaminations with free or sequestered iron are harmful for both, receptor binding capacity (in a reversible form) and for hormone-receptor/DNA binding properties (in a partially reversible form). We propose a sulfhydryl oxidative mechanism for type I mineralocorticoid receptor inactivation in which iron contaminants might accelerate this process by oxidative catalysis. We also demonstrate that when thiol groups are blocked by specific reagents such as N-ethyl-maleimide or dithionitrobenzoic acid, type I sites loose binding capacity, but the protein is protected from oxidation as well as inactivation.