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Silva-Filho J.L.,Federal University of Rio de Janeiro | Souza M.C.,Institute Tecnologia em Farmacos | Henriques M.G.,Institute Tecnologia em Farmacos | Morrot A.,Federal University of Rio de Janeiro | And 5 more authors.
Archives of Biochemistry and Biophysics | Year: 2015

Abstract Angiotensin II (Ang II) plays an important role in the regulation of the T-cell response during inflammation. However, the cellular mechanisms underlying the regulation of lymphocytes under physiologic conditions have not yet been studied. Here, we tested the influence of Ang II on T-cell migration using T cells from BALB/c mice. The results obtained in vivo showed that when Ang II production or the AT1 receptor were blocked, T-cell counts were enhanced in blood but decreased in the spleen. The significance of these effects was confirmed by observing that these cells migrate, through fibronectin to Ang II via the AT1 receptor. We also observed a gradient of Ang II from peripheral blood to the spleen, which explains its chemotactic effect on this organ. The following cellular mechanisms were identified to mediate the Ang II effect: upregulation of the chemokine receptor CCR9; upregulation of the adhesion molecule CD62L; increased production of the chemokines CCL19 and CCL25 in the spleen. These results indicate that the higher levels of Ang II in the spleen and AT1 receptor activation contribute to migration of naive T cells to the spleen, which expands our understanding on how the Ang II/AT1 receptor axis contributes to adaptive immunity. © 2015 Elsevier Inc. All rights reserved. Source


Silva-Filho J.L.,Federal University of Rio de Janeiro | Souza M.C.,Institute Tecnologia em Farmacos | Henriques M.D.G.,Institute Tecnologia em Farmacos | Morrot A.,Federal University of Rio de Janeiro | And 6 more authors.
Molecular Immunology | Year: 2011

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT1 and AT2, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4+ and CD8+ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10-8M Ang II did not change any of the parameters evaluated. However, 10-6M losartan, an AT1 receptor antagonist: (i) reduced the percentage of CD25+ and CD69+ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10-6M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10-8M). None of these responses was modified by 10-7M PD123319, an AT2 antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT1 receptor activation. Moreover, AT1 receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT2 receptor expression did not change after T cell receptor triggering. © 2011 Elsevier Ltd. Source


Ribeiro M.C.,Federal University of Rio de Janeiro | Costa-Alves M.S.,Federal University of Rio de Janeiro | Wengert M.,Federal University of Rio de Janeiro | Meyer-Fernandes J.R.,Federal University of Rio de Janeiro | And 6 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2012

Background: The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes. Methods: We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-32P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation. Results: This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl2 was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN3) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN3. The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg- 1 x min- 1 and K0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia. Conclusion: Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia. General Significance: This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia. © 2012 Elsevier B.V. Source

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