Luiz Fernando de Souza

Retinol and retinoic acid modulate catalase activity in Sertoli cells by distinct and gene expression-independent mechanisms

Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed that retinol induces oxidative stress on different cellular types. Catalase (EC 1.11.1.6; CAT) is a hydrogen peroxide metabolizing enzyme, and its activity and expression is widely used as an index to measure oxidative stress and perturbations in the cellular redox state. The aim of this study was to investigate the effects of retinol and its major biologically active metabolite, all-trans retinoic acid (RA), on CAT regulation. For this purpose, cultured Sertoli cells (a physiological target of vitamin A) were treated with retinol or RA. Retinol (7 microM, 14 microM) and RA (100 nM, 1 microM) enhanced intracellular reactive species production and increased CAT activity after 24 h of treatment. Retinol increased CAT immunocontent but did not alter CAT mRNA expression, while the increase in CAT activity by RA was not related to alterations in immunocontent or mRNA expression. In vitro incubation of purified CAT with retinol or RA did not alter enzyme activity.

Extracellular purines from cells of seminiferous tubules

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.

Retinol increases catalase activity and protein content by a reactive species-dependent mechanism in Sertoli cells.

Vitamin A (retinol) is widely used as an antioxidant in therapeutic interventions and dietary supplementations. However, the redox properties of retinoids have been the subject of intense debate in the last few years, as recent works observed deleterious effects caused by retinol supplementation in clinical trials. In the present work, we show that retinol treatment (7 microM, 24 h) led to catalase (EC 1.11.1.6; CAT) activation in cultured Sertoli cells by increasing its protein content in a reactive species-dependent manner. Retinol treatment also increased cell lipoperoxidation, assessed by determination of thiobarbituric acid-reactive substances (TBARS), and intracellular reactive species production, determined by the real-time dihydrochlorofluorescein (DCFH-DA) assay. However, no alterations on CAT mRNA expression (assessed by RT-PCR) were observed, indicating an effect independent of CAT gene-transcription regulation. Importantly, all the effects induced by retinol were inhibited by the antioxidant Trolox, a hydrophilic analogue of alpha-tocopherol. These results show for the first time that retinol increases CAT activity by a redox-dependent modulation of its protein content in a cell culture model. CAT activity or expression are widely used as indexes of oxidative stress in biological systems; since no changes in CAT mRNA expression were detected in these conditions, the use of CAT gene-transcription activation when assessing oxidative stress should be re-evaluated.

Mesenchymal Stem Cell-Conditioned Medium Triggers Neuroinflammation and Reactive Species Generation in Organotypic Cultures of Rat Hippocampus

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: modulation by A2A and A2B adenosine receptors.

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1microg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10microM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10microM) and Jun N-terminal kinase (JNK; SP600125, 10microM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

High glucose increases RAW 264.7 macrophages activation by lipoteichoic acid from Staphylococcus aureus

BACKGROUND Type 2 diabetes mellitus is associated with an increased risk of cardiovascular diseases and accelerated atherosclerosis, which has been associated to hyperglycemia and chronic inflammation. Activated macrophages are described to participate in atherosclerosis due to foam cell formation and pro-inflammatory mediators production. Bacterial infections are described to accelerate atherosclerosis, moreover, gram-positive and negative bacterial DNA was described in atherosclerotic plaques. METHODS We studied the glucose modulation of RAW 264.7 macrophages activation by the gram-positive bacterial antigen lipoteichoic acid (LTA), evaluating nitrite production, tumor necrosis factor alpha secretion and matrix metalloproteinase 9 activity. RESULTS High glucose increased macrophages activation by LTA, evidenced by exacerbated nitric oxide and tumor necrosis factor alpha production, as well matrix metalloproteinase 9 secretion. CONCLUSIONS These effects could contribute to atherosclerotic risk parameters, like atherome plaque instability, and participate in chronic inflammation present in type 2 diabetes.

Extracellular inosine is modulated by H2O2 and protects sertoli cells against lipoperoxidation and cellular injury.

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.

Extracellular inosine participates in tumor necrosis factor-alpha induced nitric oxide production in cultured Sertoli cells

Recent reports have described purinergic modulation of tumor necrosis factor-alpha (TNF-α) signaling in neutrophils and astrocytes. In Sertoli cells, both TNF-R1 and TNF-R2 TNF-α receptors are present and this cytokine modulates many functions of these cells related to the maintenance of spermatogenesis. Sertoli cells express distinct purinoreceptors and previous work has shown that these cells secrete extracellular nucleotides and their metabolites. In this work, we studied the possible role of extracellular purines in TNF-α signaling in cultured Sertoli cells. This cytokine increased inosine concentration from 30 min to 6 h, with no effect at 24 h. Both TNF-α and inosine increased nitrite accumulation and nitric oxide synthase activity. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor, abolished the TNF-α induced inosine increase, nitrite accumulation and nitric oxide synthase activity. These results suggest that extracellular inosine acts as intermediary in TNF-α stimulated nitric oxide production in cultured Sertoli cells.

Effects of follicle-stimulating hormone and vitamin A upon purinergic secretion by rat Sertoli cells

Follicle-stimulating hormone (FSH) and vitamin A (retinol) are two of the main regulators of the male reproductive system. Recently, it has been described that extracellular purines can affect some important reproductive-related functions in Sertoli cells and germinative cells, by activating specific purinergic receptors. In this work, we report that both FSH and retinol are able to induce changes in the levels of extracellular purines of cultured rat Sertoli cells. FSH induced an increase in adenosine, mainly caused by enhanced ecto-ATPase activity, while retinol increased xanthine and hypoxanthine levels, and decreased uric acid concentration by an unknown mechanism. These data indicate that purinergic signaling may be involved in the control and/or regulation of some of the reproductive-related actions of these hormones. (Mol Cell Biochem 278: 185–194, 2005)

Planejamento integrado de uso da terra: uma disciplina integradora no ensino da agronomia na UFRGS

A disciplina Planejamento Integrado de Uso da Terra, do Curso de Agronomia da Universidade Federal do Rio Grande do Sul, associa os conhecimentos da area de solos com os demais do Curso de Agronomia, interligando-os e dando-lhes consistencia e significado. A disciplina vale-se do planejamento integrado para reunir o conhecimento agronomico num projeto de exploracao e desenvolvimento sustentavel de uma ou mais propriedades rurais por meio de uma sequencia de atividades, assim definidas: caracterizacao regional, levantamento dos recursos naturais existentes na propriedade, levantamento do seu uso atual, diagnostico do sistema produtivo e elaboracao de uma proposta de exploracao sustentavel da propriedade, que e apresentada para a comunidade local. Os resultados desta experiencia demonstram que a disciplina tem sido muito proveitosa no treinamento e aperfeicoamento dos estudantes, tendo recebido suporte e apoio por parte do corpo discente, dos agricultores e das organizacoes envolvidas no processo de planejamento agricola.