RESUMO
Several reports suggest putative interactions between valproic acid (VPA) treatment and the hypothalamus-pituitary-adrenal axis. Given that VPA alters mitochondrial functions, an action of this drug on a mitochondrial process such as steroid synthesis in adrenal cells should be expected. In order to disclose a putative action of VPA on the adrenocortical cell itself we evaluated VPA effects on regulatory steps of the acute stimulation of steroidogenesis in Y1 adrenocortical cells. This study demonstrates that VPA increases progesterone production in non-stimulated cells without inducing the levels of Steroidogenic Acute Regulatory (StAR) protein, which facilitates cholesterol transport. This result suggests that VPA increases mitochondrial cholesterol transport through a StAR-independent mechanism and is further supported by the fact that in isolated mitochondria VPA stimulates exogenous cholesterol metabolization to progesterone. VPA also reduces the cAMP-mediated increase of the StAR protein, mRNA levels, promoter activity and progesterone production. In summary, the present data show that VPA can alter steroid production in adrenal cells by a complex mechanism that mainly involves an action on cholesterol access to the inner mitochondrial membrane. The VPA-mediated increase of basal steroidogenesis could be linked to the increase of basal cortisolemia described in patients under VPA treatment.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Colesterol/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Valproico/farmacologia , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Córtex Suprarrenal/metabolismo , Animais , Anticonvulsivantes/toxicidade , Transporte Biológico , Linhagem Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , AMP Cíclico/agonistas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Mitocôndrias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ácido Valproico/toxicidadeRESUMO
Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.
Assuntos
Glândulas Suprarrenais/enzimologia , Ácido Araquidônico/metabolismo , Células Intersticiais do Testículo/enzimologia , Tioléster Hidrolases/metabolismo , Acetil-CoA Hidrolase/metabolismo , Animais , Colesterol/metabolismo , Humanos , Masculino , Mitocôndrias/enzimologia , Esteroides/biossínteseRESUMO
Although the role of arachidonic acid (AA) in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl-CoA thioesterase (MTE-I). We have shown that recombinant MTE-I hydrolyses arachidonoyl-CoA to release free AA. An acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2-mediated pathway. Inhibition of ACS4 and MTE-I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH-stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl-CoA synthetase and the acyl-CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2-mediated pathway that involves a hormone-induced acyl-CoA synthetase and a hormone-regulated acyl-CoA thioesterase.
Assuntos
Ácido Araquidônico/fisiologia , Hormônios/metabolismo , Transdução de Sinais/fisiologia , Esteroides/biossíntese , Acil Coenzima A/antagonistas & inibidores , Animais , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Membranas Intracelulares/metabolismo , Masoprocol/farmacologia , Mitocôndrias/enzimologia , Palmitoil-CoA Hidrolase/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , RNA Mensageiro/antagonistas & inibidores , Triazenos/farmacologiaRESUMO
Several stimuli, including stress conditions, promote the activation of MAP kinases family members (ERK1/2, JNK, p38). In turn, these enzymes regulate several cellular functions. Given that MAPK activation requires the phosphorylation of these proteins, their inactivation depends on the activity of specific phosphatases. MAPK phosphatase-1 (MKP-1), a phosphatase specifically involved in the inactivation of MAPK family members, is induced by mitogenic stimuli and stress conditions. Here we describe the effect of heat shock (HS), 10 min, 45 degrees C, on MAPKs activities and MKP-1 mRNA and protein levels in Y1 adrenocortical cells. Western blot analysis performed with antibodies against the phosphorylated forms of ERK1/2 and JNK revealed that HS produced the rapid activation of these kinases. Their inactivation was also a rapid event and occurred together with the increase of MKP-1 protein levels detected by Western blot analysis. In addition, the effect of HS on MKP-1 protein levels seems to be exerted at the transcriptional level, since the amount of its mRNA in heat shocked cells was higher than in nonheated cells. Comparison of the temporal profiles of MKP-1 protein induction and MAPKs phospho-dephosphorylation suggests that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS.
Assuntos
Córtex Suprarrenal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transtornos de Estresse por Calor/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Córtex Suprarrenal/patologia , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Proteínas Imediatamente Precoces/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Choque/metabolismo , Fatores de TempoRESUMO
The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Androgênios/biossíntese , Gonadotropina Coriônica/farmacologia , Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Animais , Arsenicais/farmacologia , Colesterol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Fosfoproteínas/genética , Proteínas Tirosina Fosfatases/antagonistas & inibidores , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transdução de Sinais , Fatores de Tempo , Células Tumorais CultivadasRESUMO
A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 microM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 +/- 30, ACTH + PAO = 145 +/- 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Esteroides/biossíntese , Animais , Arsenicais/administração & dosagem , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Camundongos , Organofosfonatos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Esteroides/antagonistas & inibidores , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Our recent reports indicate that protein tyrosine phosphorylation is an obligatory component of the mechanism of action of ACTH in its stimulatory action of corticosteroid production in adrenal zona fasciculata (ZF). The role of protein tyrosine phosphatase (PTP) activity in the regulation of steroidogenesis by LH/chorionic gonadotropin (CG) was tested using cell-permeable PTP inhibitors. Thus, PTP inhibition blocks LH- and 8-bromo-cAMP-stimulated testosterone production by Leydig cells without affecting 22(R)OH-cholesterol-supported steroidogenesis, similar results to those obtained in the adrenal ZF/ACTH system, leading us to propose that PTP action is an obligatory and common step in the cascade triggered by both hormones. Then, we continued the study testing whether LH modulates PTP activity in MA-10 cells, a Leydig cell line. In this regard, we observed by an in-gel PTP assay two PTPs of 110 and 50 kDa that are activated by hormone and 8-bromo-cAMP activation of the cells. Moreover, there is a transient increase by the second messenger in total PTP activity that correlates with the higher activity displayed by the 110 and 50 kDa proteins in the in-gel assay. In accordance with these results, analysis of tyrosine phosphorylated proteins showed the LH-induced dephosphorylation of proteins of 120, 68 and 50 kDa. The results of this study indicate that PTPs play an important role in the regulation of Leydig cell functions and that there exists a cross talk between serine/threonine phosphorylation and tyrosine dephosphorylation mediated by hormone-activated cAMP-dependent protein kinase and PTPs. These results are the first evidence of PTP having a role in LH/CG-stimulated steroidogenesis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gonadotropinas Hipofisárias/metabolismo , Células Intersticiais do Testículo/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Testosterona/biossíntese , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Análise de Variância , Animais , Linhagem Celular , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Gonadotropina Coriônica/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hormônio Luteinizante/metabolismo , Masculino , Oxigenases/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Ratos , Ratos Wistar , Vanadatos/farmacologiaRESUMO
In adrenal cortex, ACTH regulation of steroidogenesis depends on PKA-dependent serine/threonine phosphorylation and also on the activity of protein tyrosine phosphatases (PTPs). In addition, ACTH increases total PTPs involving at least three soluble PTPs (50, 82 and 115 kDa). Serine/threonine phosphorylation of these enzymes themselves could be a regulatory mechanism of their activity since the increase of total PTP activity is dependent on PKA-activation. In this report we analyzed the effect of in vitro phospho-dephosphorylation processes on the activity displayed by the ACTH-activated PTP of 115 kDa. Using an in-gel PTP assay we demonstrate that dephosphorylation catalyzed by potato acid phosphatase (PAP) reduces the activity of the 115 kDa PTP present in ZF from ACTH-treated animals and PKA-mediated phosphorylation reverses this effect.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fosfatase Ácida/farmacologia , Animais , Ativação Enzimática , Masculino , Fosforilação , Ratos , Ratos Wistar , Zona Fasciculada/enzimologiaRESUMO
It has been well established that arachidonic acid (AA) and its metabolism to leukotrienes plays an obligatory role in steroid production. The release of AA is regulated by hormone stimulation and protein phosphorylation. We have cloned a cDNA of a phosphoprotein with a molecular mass of 43 kDa (p43), purified from the cytosol of stimulated adrenal glands. This protein acts as intermediary in the stimulation of steroid synthesis through AA release, and has been found to be a member of a recently described acyl-CoA thioesterase family. In view of the mandatory role of this protein in the activation of AA-mediated steroidogenesis, the term Arachidonic acid-Related Thioesterase Involved in Steroidogenesis (ARTISt), is proposed for p43. The present study describes the production of the recombinant protein by cDNA expression in Escherichia coli and its functional characterization. Recombinant acyl-CoA thioesterase was capable to release AA from the respective acyl-CoA, and this activity was affected by well-recognized inhibitors of AA release and metabolism: 4-bromophenacyl bromide (BPB) and nordihydroguariaretic acid (NDGA). In addition, the inhibition of acyl-CoA thioesterase activity by NDGA correlates with the inhibition of steroid synthesis produced by this compound in adrenal cortex cells. Moreover, the recombinant protein was phosphorylated in vitro by PKA. These results provide the first evidence linking acyl-CoA thioesterases with the regulation of steroidogenesis, and support a regulatory role for acyl-CoA thioesterases in steroidogenic tissues, suggesting an alternative pathway for AA release in signal transduction.
Assuntos
Ácido Araquidônico/metabolismo , Esteroides/biossíntese , Tioléster Hidrolases/fisiologia , Acetofenonas/farmacologia , Acil Coenzima A/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Masoprocol/farmacologia , Proteínas Mitocondriais , Palmitoil-CoA Hidrolase , Fosfolipases A/antagonistas & inibidores , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismoRESUMO
Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , AMP Cíclico/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Hormônio Adrenocorticotrópico/metabolismo , Animais , Arsenicais/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Peso Molecular , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/química , Ratos , Ratos Wistar , Transdução de Sinais , Frações Subcelulares/enzimologia , Vanadatos/farmacologia , Zona Fasciculada/enzimologiaRESUMO
We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur J. Biochem. 224, 709-716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues.
Assuntos
Ácido Araquidônico/metabolismo , Palmitoil-CoA Hidrolase/genética , Fosfoproteínas/genética , Esteroides/biossíntese , Tioléster Hidrolases/genética , Zona Fasciculada/metabolismo , Sequência de Aminoácidos , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Feminino , Glucocorticoides/farmacologia , Proteínas Mitocondriais , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Ratos Wistar , Zona Fasciculada/química , Zona Fasciculada/efeitos dos fármacosRESUMO
The steady state level of most cellular phosphoproteins is dependent on the relative catalytic activities of intracellular protein kinases and phosphatases. In adrenal cortex, ACTH acts through PKA activation and Ser/Tre phosphorylation. Phosphatases involved in this pathway are not completely described, particularly the role of phosphotyrosine protein phosphatase (PTP) activity on ACTH action. We investigated potential changes in PTPs activity in adrenal gland upon in vivo and in vitro PKA activation. In vivo ACTH stimulates cytosolic PTP activity (2-fold). Similar effect is detected by in vitro stimulation. In accordance with the effects of ACTH on PTP activity, cell permeable PTP inhibitors block ACTH stimulation on adrenal zona fasciculata (ZF) cells: ACTH (1 nM) = 108.2 +/- 3.5 ng corticosterone/10(5) cells vs. ACTH + phenylarsine oxide (2 nM) = 60 +/- 4 (P < 0.001) and ACTH + pervanadate (10 mM) = 68 +/- 2 (P < 0.01). These results are reproduced when cells are stimulated with cAMP. The inhibition is not observed when steroidogenesis is supported by 22(R)OH cholesterol. We describe, for the first time, a hormonal regulation of PTP activity. According to the effect of PTP inhibitors on steroid production activated by ACTH we propose that PTP activation is a crucial event in hormone action in the steroidogenic pathway. We also propose that PTP activity is located after PKA activation and prior to cholesterol transport to the inner mitochondrial membrane.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/enzimologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Arsenicais/farmacologia , Corticosterona/antagonistas & inibidores , Corticosterona/biossíntese , Citosol/enzimologia , Combinação de Medicamentos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Hidroxicolesteróis/farmacologia , Ratos , Vanadatos/farmacologia , Zona Fasciculada/citologiaRESUMO
Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.
Assuntos
Ácido Araquidônico/fisiologia , Células Intersticiais do Testículo/metabolismo , Lipoxigenase/metabolismo , Hormônio Luteinizante/antagonistas & inibidores , Testosterona/biossíntese , Acetofenonas/farmacologia , Animais , Bucladesina/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Quinacrina/farmacologia , Ratos , Ratos WistarRESUMO
We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , DNA Complementar/química , Fosfoproteínas/genética , Zona Fasciculada/química , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/química , Fosfoproteínas/farmacologia , Fosforilação , Progesterona/biossíntese , RatosRESUMO
It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confirming the role of mitochondrial proteins in intramitochondrial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 +/- 0.5 vs. 13.0 +/- 1.0 ng P4/tube for CHx-ACTH-treated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 +/- 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.
Assuntos
Cicloeximida/farmacologia , Citosol/metabolismo , Mitocôndrias/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/metabolismo , Esteroides/biossíntese , Zona Fasciculada/ultraestrutura , Animais , Ácido Araquidônico/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Citosol/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Progesterona/biossíntese , Ratos , Ratos Wistar , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismoRESUMO
Arachidonic acid (AA) and the lipooxygenase products have been shown to play an obligatory role in the mechanism of action of LH and ACTH, at a point after cAMP-dependent phosphorylation. We have demonstrated the presence of a phosphoprotein (p43) that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue, an effect that is blocked by phospholipase A2 inhibitors. In this report we demonstrate that p43 exhibits autoproteolytic activity that is regulated by ACTH. Protein purified from ACTH-treated animals exhibited degradation in some of the isoforms resolved on two dimensional gel electrophoresis. Proteinase inhibitors (PMSF and 1,10 phenantroline) inhibited steroid synthesis induced by ACTH and 8-Br-cAMP in intact cells. Addition of exogenous AA reverted in part that inhibition. Here we present evidence for a hormone-regulated proteolytic activity of p43 and for the inhibition of steroidogenesis by proteinase inhibitors acting prior to the release of arachidonic acid.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Fosfolipases A/metabolismo , Proteínas/metabolismo , Esteroides/biossíntese , Tioléster Hidrolases , Animais , Ativação Enzimática , Hidrólise , Proteínas Mitocondriais , Palmitoil-CoA Hidrolase , Fosfolipases A2 , Fosforilação , RatosRESUMO
The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.
Assuntos
Corticosteroides/biossíntese , Citosol/metabolismo , Proteínas Quinases/metabolismo , Zona Fasciculada/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Linhagem Celular , Citosol/enzimologia , Masculino , Mitocôndrias/metabolismo , Fosforilação , Progesterona/biossíntese , Ratos , Zona Fasciculada/enzimologiaRESUMO
Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.