Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Avalia����o da prote����o conferida pela via de sinaliza����o do Nrf2 num modelo experimental de sobrecarga de ferro in vivo

O ferro �� encontrado em praticamente todos os seres vivos, sendo um cofator para prote��nas que desempenham fun����es essenciais �� vida. Nos mam��feros, a maioria do ferro est�� incorporada na hemoglobina ou armazenado no f��gado, ligado �� ferritina. �� absorvido pelos enter��citos, sendo a principal forma de controlo dos seus n��veis. A sobrecarga de ferro pode levar a hemocromatose, podendo ser t��xica para v��rios ��rg��os. O fator de transcri����o Nrf2 �� importante na ativa����o de genes citoprotetores em situa����es de stress oxidativo/eletrof��lico, colocando-se a hip��tese de que poder�� estar envolvido na resposta �� progress��o de doen��a devido �� sobrecarga de ferro. Com o objetivo de determinar se a via do Nrf2 representa uma prote����o contra a toxicidade do ferro a n��vel hep��tico, foram realizadas duas experi��ncias nas quais murganhos C57BL/6 (B6) e Nrf2-/- machos foram alimentados com dieta standard ou com dieta enriquecida em ferro carbonilo (FeC) (0,5% ou 2,0%). Os resultados demonstram sobrecarga de ferro nos animais que receberam dieta enriquecida, sendo que os que receberam FeC 2,0% apresentaram n��veis mais elevados de ferro hep��tico e s��rico, bem como da satura����o da transferrina. Os murganhos Nrf2-/- s��o mais suscet��veis a esta acumula����o, mostrando evid��ncias patol��gicas mais graves, nomeadamente necrose hepatoc��tica e infiltra����o de c��lulas inflamat��rias. A dele����o do Nrf2 associado a uma dieta suplementada com FeC 2,0% parece n��o ser suficiente para o desenvolvimento de fibrose hep��tica. O estudo da express��o de genes e prote��nas do metabolismo do ferro mostrou que os animais B6 e Nrf2-/- s��o igualmente capazes de responder �� sobrecarga de ferro, sugerindo que a sua diferente suscetibilidade �� toxicidade do ferro n��o se dever�� a uma regula����o ineficiente da homeostasia do Fe. A dieta com FeC 2,0% aumentou a express��o de dois genes alvo do Nrf2, Nqo1 e Gsta1, o que n��o se verificou com os genes e prote��nas GCLC e GCLM. A express��o de genes pr��-inflamat��rios n��o mostrou evid��ncias de inflama����o nestes animais. Foi demonstrado que os animais Nrf2-/- s��o mais suscet��veis �� toxicidade do ferro, concluindo-se que a via do Nrf2 �� ativada em resposta a uma dieta contendo quantidades excessivas de FeC e que confere prote����o contra a acumula����o de ferro em murganhos B6.

���Papel dos recetores purin��rgicos no crescimento e temodela����o do tecido cavernoso���

A adenosina �� um nucle��sido ub��quo envolvido na regula����o de controlo do t��nus vascular do tecido cavernoso, desempenhando um papel importante na fisiopatologia da Disfun����o Er��til (DE) resistente aos f��rmacos relaxantes musculares cl��ssicos. Apesar da import��ncia comprovada dos recetores da adenosina na fisiopatologia da DE no homem, pouca informa����o �� conhecida no que diz respeito �� express��o e localiza����o dos recetores purin��rgicos no Tecido Cavernoso de Ratazana (TCR). Neste trabalho avaliou-se o fen��tipo dos recetores purin��rgicos respons��veis pela regula����o do t��nus do tecido er��til de ratazana por imunofluoresc��ncia indireta aplicada �� microscopia confocal em co-culturas de c��lulas endoteliais e musculares lisas do TCR. Para al��m da caracteriza����o imunofenot��pica, desenvolveu-se uma t��cnica que permite diferenciar funcionalmente em tempo real (por microscopia confocal funcional) c��lulas musculares lisas e c��lulas endoteliais isoladas de TCR em co-cultura marcadas com a sonda fluorescente Fluo-4NW. Esta t��cnica permite distinguir cada um dos subtipos celulares mediante o padr��o e a magnitude das oscila����es dos n��veis intracelulares de Ca2+ ([Ca2+]i) em resposta ao ATP (agonista P2) e �� fenilefrina (PE, agonista ��-adren��rgico). Nas c��lulas musculares lisas, observou-se uma resposta mais acentuada ao agonista ��-adren��rgico, PE, e uma resposta menos significativa ao ATP. O contr��rio foi observado relativamente ��s c��lulas endoteliais. A incuba����o das c��lulas musculares lisas e endoteliais com ATP (300 ��M) causou um aumento dos n��veis de [Ca2+]i. O efeito do ATP (300 ��M) parece envolver a ativa����o de recetores dos subtipos P2X1 e P2X3 sens��veis ao bloqueio com NF023 (3��M) e A317491 (100 nM), respetivamente. J�� o aumento dos n��veis [Ca2+]i produzido pelo ADP (300 ��M) parece envolver a ativa����o de recetores P2Y1, P2Y12 e P2Y13 mediante o antagonismo produzido pelos antagonistas MRS 2179 (0,3��M), AR-C66096 (0,1 ��M) e MRS 2211 (10��M), respetivamente. Os dois tipos celulares expressam imunorreatividade contra recetores A2A, A2B, P2X1, P2X3, P2Y1, P2Y12 e P2Y13.

Influence of Soil Chemistry and Plant Physiology in the Phytoremediation of Cu, Mn, and Zn

Different anthropogenic sources of metals can result from agricultural, industrial, military, mining and urban activities that contribute to environmental pollution. Plants can be grown for phytoremediation to remove or stabilize contaminants in water and soil. Copper (Cu), manganese (Mn) and zinc (Zn) are trace essential metals for plants, although their role in homeostasis in plants must be strictly regulated to avoid toxicity. In this review, we summarize the processes involved in the bioavailability, uptake, transport and storage of Cu, Mn and Zn in plants. The efficiency of phytoremediation depends on several factors including metal bioavailability and plant uptake, translocation and tolerance mechanisms. Soil parameters, such as clay fraction, organic matter content, oxidation state, pH, redox potential, aeration, and the presence of specific organisms, play fundamental roles in the uptake of trace essential metals. Key processes in the metal homeostasis network in plants have been identified. Membrane transporters involved in the acquisition, transport and storage of trace essential metals are reviewed. Recent advances in understanding the biochemical and molecular mechanisms of Cu, Mn and Zn hyperaccumulation are described. The use of plant-bacteria associations, plant-fungi associations and genetic engineering has opened a new range of opportunities to improve the efficiency of phytoremediation. The main directions for future research are proposed from the investigation of published results.

Vitamin D Intake and Cardiometabolic: Risk Factors in Adolescents

Background: A growing body of research suggests that vitamin D might play an important role in overall health. No data exist on vitamin D intake for the Azorean adolescent population. The purpose of this study was to assess vitamin D intake and investigate a possible association between vitamin D intake and cardiometabolic risk factors in Azorean adolescents. Methods: A cross-sectional school-based study was conducted on 496 adolescents (288 girls) aged 15���18 years from the Azorean Islands, Portugal. Anthropometric measurements (waist circumference and height), blood pressure (systolic), and plasma biomarkers [fasting glucose, insulin, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs)] were measured to assess metabolic risk. Homeostasis model assessment (HOMA), TC-to-HDL-C ratio, and waist-to-height ratio were calculated. For each of these variables, a Z-score was computed by age and sex. A metabolic risk score was constructed by summing the Zscores of all individual risk factors. High risk was considered when the individual had ��� 1 standard deviation(SD) of this score. Vitamin D intake was assessed with a semiquantitative food frequency questionnaire. Participants were classified into quartiles of vitamin D intake. Logistic regression was used to determine odds ratios for high cardiometabolic risk scores after adjusting for total energy intake, pubertal stage, fat mass percentage, and cardiorespiratory fitness. Results: Mean (SD) vitamin D intake was 5.8 (6.5) mg/day, and 9.1% of Azorean adolescents achieved the estimated average requirement of vitamin D (10 mg/day or 400 IU). Logistic regression showed that the odds ratio for a high cardiometabolic risk score was 3.35 [95% confidence interval (CI) 1.28���8.75] for adolescents in the lowest vitamin D intake quartile in comparison with those in the highest vitamin D intake quartile, even after adjustment for confounders. Conclusion: A lower level of vitamin D intake was associated with worse metabolic profile among Azorean adolescents.

Iron Metabolism: From Health to Disease

Background Iron is vital for almost all living organisms by participating in a wide range of metabolic processes. However, iron concentration in body tissues must be tightly regulated since excessive iron may lead to microbial infections or cause tissue damage. Disorders of iron metabolism are among the most common human diseases and cover several conditions with varied clinical manifestations. Methods An extensive literature review on the basic aspects of iron metabolism was performed, and the most recent findings on this field were highlighted as well. Results New insights on iron metabolism have shed light into its real complexity, and its role in both healthy and pathological states has been recognized. Important discoveries about the iron regulatory machine and imbalances in its regulation have been made, which may lead in a near future to the development of new therapeutic strategies against iron disorders. Besides, the toxicity of free iron and its association with several pathologies has been addressed, although it requires further investigations. Conclusion This review will provide students in the fields of biochemistry and health sciences a brief and clear overview of iron physiology and toxicity, as well as imbalances in the iron homeostasis and associated pathological conditions.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel ��-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.