RESUMO
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
Treatment of intact isolated rat testis interstitial cells with S-adenosylmethionine as methyl donor, increases substantially the number of LH human CG receptors (100-200%) without modifying the equilibrium dissociation constant. The increase in binding capacity was associated with an augmentation in the sensitivity of the rat testis interstitial cells to produce testosterone in response to LH, suggesting a functional role of the unmasked receptors. The amount of S-adenosylmethionine necessary to obtain an increase in LH binding capacity and preserve cell viability was 25-50 micrograms/ml per 1.6 X 10(7) cells. 10 mM MgCl2 in addition to the Mg2+ present in the medium was necessary to maintain cell viability. 3H-labelled methyl groups were incorporated mainly into the lipid fraction (208 fmol/10(6) cells) when 3H-S-adenosylmethionine was incubated with the cells for 2 h at 30 degrees C. Our results are consistent with the conclusion that early action of LH may involve an activation of methyltransferase activity, phospholipid methylation, an increase in LH binding capacity and an increase in receptor function.
Assuntos
Hormônio Luteinizante/fisiologia , Lipídeos de Membrana/fisiologia , Receptores do LH/fisiologia , Testículo/fisiologia , Animais , Membrana Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Gonadotropina Coriônica/metabolismo , Técnicas In Vitro , Masculino , Metilação , Ratos , S-Adenosilmetionina/farmacologia , Testosterona/biossínteseRESUMO
We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.
Assuntos
Agonistas Adrenérgicos/farmacologia , Ácidos Graxos/metabolismo , Coração/efeitos dos fármacos , Tioléster Hidrolases/metabolismo , Animais , Anticorpos/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Isoproterenol/farmacologia , Masoprocol/farmacologia , Proteínas Mitocondriais , Miocárdio/enzimologia , Palmitoil-CoA Hidrolase/metabolismo , Perfusão , Fenilefrina/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Tioléster Hidrolases/análise , Tioléster Hidrolases/imunologiaRESUMO
We have investigated the effect of the proteinase inhibitors 1,10-phenantroline (OP) and phenylmethylsulfonyl fluoride (PMSF) on steroidogenesis in rat adrenal cortex. Both PMSF and OP inhibited adrenocorticotropin (ACTH)- and 8-Br cAMP-induced stimulation of corticosterone synthesis. On the contrary, arachidonic acid-induced stimulation of corticosterone synthesis was only slightly inhibited by PMSF and unchanged by OP. Intra- and extracellular cAMP levels were determined by radioimmunoassay. While PMSF did not affect neither the intra- nor the extracellular cAMP levels, OP decreased the intra- and extracellular levels of unstimulated as well as ACTH-stimulated cells. The site of action of the proteinase inhibitors was also studied by recombination of mitochondria with the different subcellular fractions in vitro. Addition of PMSF abolished the stimulation achieved by in vitro activation of cytosol by cAMP and PKA. On the other hand, OP completely inhibited the activation of mitochondria. Our results provide evidence for the involvement of proteinases in ACTH-induced stimulation of steroidogenesis in adrenal cortex both prior to the release of arachidonic acid and at the level of cholesterol transport from the outer to the inner mitochondrial membrane.
Assuntos
Corticosterona/biossíntese , AMP Cíclico/metabolismo , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Zona Fasciculada/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Hormônio Adrenocorticotrópico/antagonistas & inibidores , Hormônio Adrenocorticotrópico/farmacologia , Animais , Ácido Araquidônico/farmacologia , Hidroxicolesteróis/metabolismo , Técnicas In Vitro , Cinética , Masculino , Fenantrolinas/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Pregnenolona/metabolismo , Progesterona/metabolismo , Ratos , Ratos Wistar , Zona Fasciculada/citologia , Zona Fasciculada/efeitos dos fármacosRESUMO
Germline mutations in specific hot spot-codons of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2). Clinical RET gene testing has been routine for the last 10 years in some countries. In Argentina, RET testing excluding MEN 2B was always reported with a mutation at codon 634, with one exception: we described a novel mutation T > C transition at codon 630 (C630R), the family to which we extend the study in the present report. This family comprised 29 members in four generations including 6 individuals affected with medullary thyroid cancer (MTC), positive for the C630R mutation and normal adrenaline/ noradrenaline and ionized calcium/parathyroid hormone levels. Two asymptomatic mutation carriers aged 5 and 11 years underwent total thyroidectomy. The histopathologic examination showed C-cell hyperplasia and microcarcinoma foci, while preoperative basal calcitonins were normal for both. Our report emphasizes the importance of testing for non-hot spot RET mutations in apparently mutation negative MEN 2 families. Furthermore, it would appear that C630R mirrors C634R in penetrance (100% in this family) and in early age of onset of MTC, although paradoxically, no pheochromocytomas and hyperparathyroidism have developed. In addition to recommending RET testing before 5 years of age; we also can postulate that codon 630 may be the key point along the extracellular domain, important in the tissue-specific penetrance.
Assuntos
Carcinoma Medular/genética , Mutação em Linhagem Germinativa/genética , Proteínas Oncogênicas/genética , Penetrância , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Idade de Início , Criança , Pré-Escolar , Saúde da Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
LH receptor internalization was studied with an antireceptor monoclonal antibody (aLHR) which induces Leydig cells to produce testosterone. To follow receptor-mediated aLHR internalization, cells were incubated with aLHR at 10 C for 3 h to generate an aLHR complex; this was followed by a second incubation with fluorescent labeled antimouse immunoglobulin at 34 C, a temperature which allows internalization. Within 15 min at 34 C, cytoplasmic fluorescent staining was detectable; this staining was strongly visible after 60 min. At no time was nuclear staining observable. Employing such an approach, it has also been possible to follow the fate of unoccupied receptors when cells are stimulated with a submaximal dose of LH. The results show that LH interactions with 20% of its receptors produces microaggregation, patching, capping, and internalization of free receptor sites. The results further demonstrate that cells with receptors in the state of capping are less sensitive to a second LH stimulation, suggesting that in this state receptors are no longer coupled to the adenylate cyclase system.
Assuntos
Hormônio Luteinizante/fisiologia , Receptores de Superfície Celular/metabolismo , Adenilil Ciclases/metabolismo , Animais , Anticorpos Monoclonais , AMP Cíclico/metabolismo , Resistência a Medicamentos , Corantes Fluorescentes , Imunoeletroforese , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/imunologia , Masculino , Camundongos , Ratos , Receptores do LH , Temperatura , Testosterona/biossíntese , Fatores de TempoRESUMO
The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.
Assuntos
Androgênios/metabolismo , Castração , Epididimo/metabolismo , Proteínas/metabolismo , Testosterona/farmacologia , Animais , Sítios de Ligação , Citoplasma/metabolismo , Citosol/metabolismo , Di-Hidrotestosterona/metabolismo , Epididimo/anatomia & histologia , Epididimo/efeitos dos fármacos , Cinética , Masculino , Tamanho do Órgão , Ligação Proteica , Ratos , Receptores de Superfície Celular , Fatores de TempoRESUMO
We studied the involvement of major histocompatibility (MHC) class I antigens on the mechanism of LH/hCG receptor activation. For this purpose we investigated the effects of anti-MHC class I antibodies on hormone-receptor interaction, signal transduction, and MHC class I antigen-receptor interaction. Monoclonal antibodies against MHC class I antigen were able to stimulate testosterone production in mouse Leydig cells with the same potency as LH. This biological effect depends on the concentration of antibody used and could be abolished by a LH antagonist. There is a perfect parallelism, for each monoclonal antibody, between the specificity for a particular haplotype and the response of the target cells from the strains carrying such a haplotype. The same antibodies were able to precipitate the soluble LH/hCG receptors, as both a hormone-receptor complex and a free receptor. The results suggest that bound hormone triggers an association of the MHC class I antigen with the LH/hCG receptor, resulting in activation of the target cell.
Assuntos
Antígenos HLA/imunologia , Hormônio Luteinizante/imunologia , Receptores do LH/imunologia , Animais , Anticorpos , Anticorpos Monoclonais , Células Intersticiais do Testículo/imunologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Receptores do LH/fisiologia , Testosterona/biossínteseRESUMO
The effects of L-arginine on corticosterone production, cGMP, and nitrite levels were examined in zona fasciculata adrenal cells. L-Arginine significantly decreased both basal and ACTH-stimulated corticosterone production. This effect was still evident when steroidogenesis was induced by 8-bromo-cAMP and 22(R)-hydroxycholesterol, but not in the presence of exogenously added pregnenolone. L-Arginine increased cGMP and nitrite levels,; these effects were blocked by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl-ester. Transport of L-[3H]arginine was rapid, saturable, and monophasic, with an apparent Km of 163+/-14 microM and a maximum velocity of 53+/-6 pmol/min x 10(5) cells. The basic amino acids L-lysine and L-ornithine, but not D-arginine or the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl-ester and N(G)-nitro-L-arginine, impaired L-arginine uptake. Taken together, these results suggest that steroidogenesis in zona fasciculata adrenal cells may be negatively modulated by L-arginine-derived nitric oxide.
Assuntos
Arginina/farmacologia , Óxido Nítrico Sintase/fisiologia , Zona Fasciculada/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Arginina/antagonistas & inibidores , Arginina/farmacocinética , Corticosterona/biossíntese , GMP Cíclico/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Hidroxicolesteróis/farmacologia , Lisina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ornitina/farmacologia , Pregnenolona/farmacologia , Ratos , Ratos Wistar , Zona Fasciculada/citologia , Zona Fasciculada/metabolismoRESUMO
Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corticosteroides/biossíntese , AMP Cíclico/metabolismo , Lipoxigenase/biossíntese , Zona Fasciculada/metabolismo , Zona Glomerulosa/metabolismo , Hormônio Adrenocorticotrópico/farmacocinética , Aldosterona/metabolismo , Angiotensina II/farmacocinética , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Bioensaio , Masculino , Mitocôndrias/efeitos dos fármacos , Progesterona/biossíntese , RatosRESUMO
This work describes the stimulation by a peptide hormone of an individual cell in a prescribed region of its plasma membrane. When Leydig cells were stimulated via a section of membrane tightly sealed to an electrode containing LH, a very localized area exhibited the morphological change known as 'rounding up', which is a cyclic AMP-dependent protein kinase-mediated response. This localized stimulation did not produce a wider response through intracellular, intermembranous or extracellular signals. Each individual cell responded to peptide stimulation gradually, with an increase over time and with dose. In contrast, when the stimulation was accomplished using a non-hydrolysable cyclic AMP analogue in the patch electrode, a general response throughout an individual cell was produced. Locally stimulated peptide hormone receptors, adenylate cyclases and cyclic AMP-dependent protein kinases appear to be closely associated so that second messenger production and the effects it mediates are compartmentalized.
Assuntos
Compartimento Celular , Membrana Celular/metabolismo , Hormônio Luteinizante/farmacologia , Proteínas Quinases/metabolismo , Membrana Celular/ultraestrutura , Eletrodos , Cinética , Tumor de Células de Leydig , Microscopia Eletrônica de Varredura , Sistemas do Segundo Mensageiro , Células Tumorais CultivadasRESUMO
The present study was designed to investigate the role of nitric oxide (NO) in the regulation of adrenocortical function. Different NO donors, such as sodium nitroprusside (SNP), S-nitroso-L-acetyl penicillamine, diethylamine/NO complex sodium salt and diethylenetriamine NO adduct, significantly decreased corticosterone production both in unstimulated and in corticotropin-stimulated zone fasciculata adrenal cells, in a dose-dependent manner. The effect of SNP was reversed by ferrous hemoglobin. A selective inhibitor of NO synthase, L-NG-nitro-arginine significantly increased corticosterone secretion. The effect of SNP was not mediated by cGMP as permeable cGMP analogs did not reproduce its inhibitory effect. SNP significantly inhibited the steroidogenesis stimulated by 8Br-cAMP and 22(R)-OH-cholesterol, but was ineffective when corticosterone was produced in the presence of exogenously added pregnenolone. Moreover, the conversion of [3H]cholesterol to [3H]pregnenolone and the production of pregnenolone or progesterone (assessed by RIA) were significantly decreased by SNP. Taken together, these results suggest that NO may be a negative modulator of adrenal zona fasciculata steroidogenesis.
Assuntos
Corticosterona/biossíntese , Óxido Nítrico/fisiologia , Nitroprussiato/farmacologia , Penicilamina/análogos & derivados , Vasodilatadores/farmacologia , Zona Fasciculada/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Células Cultivadas , Dietilaminas/farmacologia , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Penicilamina/farmacologia , Pregnenolona/biossíntese , Pregnenolona/farmacologia , Progesterona/biossíntese , Ratos , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina , Estimulação Química , Zona Fasciculada/efeitos dos fármacosRESUMO
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
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
Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Heme Oxigenase (Desciclizante)/biossíntese , Sulfonamidas , Córtex Suprarrenal/efeitos dos fármacos , Animais , Bilirrubina/farmacologia , Western Blotting/métodos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Heme Oxigenase (Desciclizante)/análise , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Isoquinolinas/farmacologia , Proteínas de Membrana , Metaloporfirinas/farmacologia , Camundongos , Pregnenolona/análise , Protoporfirinas/farmacologia , RNA Mensageiro/análise , Estimulação Química , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-dependent protein phosphokinase of rat interstitial cells was characterized by ion-exchange chromatography and sucrose density gradient centrifugation. The 0.2 M NaCl fraction from DEAE-Sephadex showed a small 2.9-S peak of basal enzyme activity, and a large 6.5-S peak of cyclic AMP-dependent protein kinase activity; fractions eluted from DEAE-Sephadex with 0.3-0.5 M NaCl contained a major 3.8-S peak of cyclic AMP-dependent enzyme activity. Activation of protein kinase in cell extracts by cyclic AMP, and in intact interstitial cells by trophic hormone, caused a major shift of enzyme activity to the 2.9-S cyclic AMP-dependent form which was eluted from DEAE-Sephadex by 0.2 M NaCl. These results are consistent with the presence of two distinct protein kinase holoenzymes, with a common 2.9-S catalytic subunit. During hormonal activation of protein kinase in dispersed interstitial cells by 10-10 M human chorionic gonadotropin (hCG), conversion to the 2.9-S catalytic subunit was observed between 2 and 30 min of incubation. Protein kinase activity was correlated with cyclic AMP production, and full enzyme activation occurred at the time of maximum intracellular cyclic AMP concentration. The presence of two forms of cyclic AMP-dependent protein kinase in the Leydig cell provides a potential mechanism whereby progressive occupancy of gonadotropin receptors could evoke a series of discrete target cell responses.
Assuntos
AMP Cíclico/farmacologia , Isoenzimas/metabolismo , Células Intersticiais do Testículo/enzimologia , Proteínas Quinases/metabolismo , Animais , Gonadotropina Coriônica/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Cinética , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Ratos , Xantinas/farmacologiaRESUMO
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.
Assuntos
Gonadotropina Coriônica/fisiologia , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/fisiologia , Testículo/metabolismo , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Intersticiais do Testículo/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Receptores do LH/metabolismo , Testículo/ultraestruturaRESUMO
In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.
Assuntos
Bradicinina/análogos & derivados , Isoenzimas/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Receptores da Bradicinina/fisiologia , Transdução de Sinais/fisiologia , Veias Umbilicais/enzimologia , Anti-Inflamatórios não Esteroides/farmacologia , Bradicinina/farmacologia , Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Humanos , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/fisiologia , Proteínas de Membrana , Técnicas de Cultura de Órgãos , Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Receptores da Bradicinina/agonistas , Transdução de Sinais/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
The Human Variome Project (HVP) is an international effort aiming systematically to collect and share information on all human genetic variants. It has been working for years in collaboration with local scientific societies by establishing systems to collect every genetic variant reported in a country and to store these variants within a database repository: LOVD (Argentinian chapter: ar.lovd.org). Formally established in 2017 in the Argentinian Node, up to June 2019 we collected more than 25,000 genetic variants deposited by 17 different laboratories. Nowadays the HVP country nodes represent more than 30 countries. In Latin America there are four country nodes: Argentina, Brazil, Mexico and Venezuela; the first two interacted recently launching the LatinGen database. In the present work we want to share our experience in applying the HVP project focusing on its organization, rules and nomenclature to reach the goal of sharing genetic variants and depositing them in the Leiden Open Variation Database. Contributing laboratories are seeking to share variant data to gain access all over the country. It is one of our goals to stimulate the highest quality by organizing courses, applying current nomenclature rules, sponsoring lectures in national congresses, distributing newsletter to serve the Argentinian genomics community and to stimulate the interaction among Latin America countries.
El Proyecto Varioma Humano (HVP) es un esfuerzo internacional que tiene como objetivo recopilar y compartir sistemáticamente información sobre todas las variantes genéticas humanas. Hemos estado trabajando durante tres años en colaboración con sociedades científicas locales, mediante el establecimiento de sistemas para recolectar todas las variantes genéticas reportadas en el país y almacenarlas dentro de la base de datos LOVD (capítulo argentino: ar.lovd.org). En el año 2017 fue establecido formalmente el Nodo Argentino del HVP, habiéndose recolectado más de 25.000 variantes genéticas depositadas por 17 laboratorios diferentes hasta junio de 2019. Hoy en día existen al menos 30 nodos del HVP, correspondientes a diferentes países. En América Latina hay cuatro nodos: Argentina, Brasil, México y Venezuela; Los dos primeros interactuaron recientemente lanzando la base de datos LatinGen. En el presente trabajo queremos compartir nuestra experiencia en la aplicación del proyecto HVP centrándonos en su organización, reglas y nomenclatura para alcanzar el objetivo de compartir variantes genéticas y depositarlas en la base de datos de variaciones abiertas de Leiden (LOVD). Es uno de nuestros objetivos estimular la más alta calidad mediante la organización de cursos, aplicación de las reglas de nomenclatura actuales, patrocinio de conferencias en congresos nacionales, distribución de boletines informativos para la comunidad de genómica argentina, y estimulación de la interacción entre los países de América Latina.