RESUMO
Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroiddehydrogenase 2 activity in a cell line derived from distal nephronpolarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroiddehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the responsedepending also on substrate concentration. The stimulation was blocked by thespecific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.
Assuntos
Animais , Cães , /metabolismo , Ativação Enzimática , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Serotonina/farmacologia , Linhagem Celular , Néfrons/enzimologia , Comunicação ParácrinaRESUMO
Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroiddehydrogenase 2 activity in a cell line derived from distal nephronpolarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroiddehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the responsedepending also on substrate concentration. The stimulation was blocked by thespecific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.(AU)
Assuntos
Animais , Cães , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Serotonina/farmacologia , Linhagem Celular , Néfrons/enzimologia , Comunicação ParácrinaRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Pregnenodionas/administração & dosagem , Pregnenodionas/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pregnenodionas/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Rim/efeitos dos fármacos , Progesterona/análogos & derivados , Progesterona/farmacologia , Piranos , Receptores de Mineralocorticoides/metabolismo , Sódio/metabolismo , Compostos de Espiro , Aldosterona/farmacologia , Animais , Antifúngicos/farmacologia , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Antagonismo de Drogas , Proteínas de Choque Térmico HSP90/metabolismo , Rim/metabolismo , Lisina/química , Masculino , Taxa de Depuração Metabólica , Antagonistas de Receptores de Mineralocorticoides , Conformação Molecular , Progesterona/antagonistas & inibidores , Progesterona/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/química , Receptores de Esteroides/química , Espironolactona/farmacologiaRESUMO
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.
Assuntos
Glucocorticoides/metabolismo , Hidroxiprogesteronas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/genética , Genes Reporter , Glucocorticoides/química , Glucocorticoides/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Hidroxiprogesteronas/química , Hidroxiprogesteronas/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Timo/efeitos dos fármacos , Timo/metabolismo , TransfecçãoRESUMO
Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.
Assuntos
Endotelina-1/farmacologia , Zona Glomerulosa/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Aldosterona/biossíntese , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Bovinos , Células Cultivadas , Colesterol/metabolismo , Diglicerídeos/biossíntese , Endotelina-1/metabolismo , Masculino , Ouabaína/farmacologia , Peptídeos Cíclicos/farmacologia , Ratos , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Verapamil/farmacologia , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacologia , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacosRESUMO
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.
Assuntos
Fígado/metabolismo , Progesterona/análogos & derivados , Progesterona/farmacologia , Timo/metabolismo , Adrenalectomia , Animais , Sangue , Bovinos , Células Cultivadas , Corticosterona/metabolismo , Citosol/metabolismo , Dexametasona/farmacologia , Indução Enzimática , Proteínas de Choque Térmico HSP90/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Glicogênio Hepático/biossíntese , Masculino , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade , Timo/efeitos dos fármacos , Transcortina/metabolismo , Tirosina Transaminase/biossíntese , Uridina/metabolismoRESUMO
Up to now, only glucocorticoids were thought to act on the renal proximal Na+/H+ exchanger. Using fluorimetric techniques we studied the kinetics of Na+/H+ exchange in brush border vesicles from ADX rats treated with increasing doses of corticosterone (B) and 18-hydroxycorticosterone (18OHB). Significant linear correlations were obtained when the Vmax of each treatment were plotted against log doses. 18OHB exhibits a slightly higher sensitivity than B and log-dose responses were steeper for 18OHB than for B treated rats. Differences between both treatments were highly significant at the 4.8 microg/100 g level, corresponding to the physiological blood level of 18OHB. Physiological doses of both steroids elicited equal Na+/H+ exchange-responses. 18OHB is not a glucocorticoid since even 88 microg/100 g did not promote hepatic glycogen deposition while the same dose of B increases glycogen deposits 3.5-fold. These results demonstrate the importance of the Na+/H+ exchanger as a mediator between corticoid action and H+ transport and that of the non-glucocorticoid 18OHB in this process.
Assuntos
18-Hidroxicorticosterona/farmacologia , Corticosterona/farmacologia , Túbulos Renais Proximais/metabolismo , Adrenalectomia , Animais , Proteínas de Transporte/metabolismo , Relação Dose-Resposta a Droga , Glicogênio/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microvilosidades/metabolismo , Ratos , Ratos Sprague-Dawley , Simportadores de Cloreto de Sódio-PotássioRESUMO
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.
Assuntos
Progesterona/análogos & derivados , Receptores de Glucocorticoides/antagonistas & inibidores , Aldosterona/metabolismo , Androstanóis/metabolismo , Animais , Rim/metabolismo , Masculino , Progesterona/química , Progesterona/metabolismo , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Espironolactona/metabolismo , Relação Estrutura-Atividade , Timo/metabolismo , Transcortina/metabolismo , TrítioRESUMO
3 Beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones and is usually considered to be mainly microsomal, although there is a dual distribution of the enzyme in toad interrenals. The present study demonstrates that in the testicular tissue, as in interrenals of Bufo arenarum H., 3 beta HSD/I is both mitochondrial and microsomal. The conversion of dehydroepiandrosterone to androstenedione takes place only in microsomes while pregnenolone is converted to progesterone in both microsomes and mitochondria. Kinetic constants of 3 beta HSD/I were determined by the oxidation of pregnenolone and dehydroepiandrosterone. The preferred substrate of the microsomal 3 beta HSD/I enzyme was dehydroepiandrosterone (K(m) = 0.17 microM and 0.53 microM for dehydroepiandrosterone and pregnenolone, respectively) not only during the breeding season but also in the non-breeding period (K(m) = 0.49 microM and 2.9 microM for dehydroepiandrosterone and pregnenolone, respectively).