Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor

Baus, Erika; Urbain, Jacques; Léo, Oberdan; Andris, Fabienne

doi:10.1016/0022-1759(94)90281-x

Cited by 22 publications

(23 citation statements)

References 15 publications

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“…Previously, fluorescent indicator such as fura-2 and fluo-3 have been used as model anions for identifying and studying the mechanisms of organic anion transport activity in different cell types [3,10,17,21,38]. However, the use of fluo-3 alone poses problems, since fluorescence intensity increases more than 40-fold upon calcium binding (K D =0.4 µM) [42].…”

Section: Discussionmentioning

confidence: 99%

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Abrahamse

Rechkemmer²

2001

Pfl�gers Archiv European Journal of Physiology

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The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.

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Section: Discussionmentioning

confidence: 99%

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Abrahamse

Rechkemmer²

2001

Pfl�gers Archiv European Journal of Physiology

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“…Cells were cultured in RPMI-1640 medium containing penicillin, streptomycin, glutamine, and 10% fetal calf serum (FCS), subsequently referred to as complete medium. Cells were loaded with Fluo 3-AM (Sigma, St. Louis, MO), a Ca 2ϩ -sensitive fluorescent dye (24,25). Briefly, cells were washed twice in Hanks' balanced salt solution, pH 7.0, with 5% FCS, resuspended in the same buffer at a concentration of 2 ϫ 10 6 cells/ml, and incubated for 30 min at 37°C with 5 mol/l Fluo 3-AM in the presence of 0.01% Pluronic F-127 detergent (Sigma).…”

Section: Selection Of Patientsmentioning

confidence: 99%

Autoantibody Response to CD38 in Caucasian Patients With Type 1 and Type 2 Diabetes

Mallone¹,

Ortolan

Baj

et al. 2001

Diabetes

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Insulin secretion is one of the functions mediated by CD38, a nonlineage pleiotropic cell surface receptor. The molecule is the target of an autoimmune response, because serum autoantibodies (aAbs) to CD38 have been detected in diabetic patients. In the healthy Caucasian population, the CD38 gene is bi-allelic (86% CD38*B and 14% CD38*A), whereas an Arg 140 Trp mutation has been identified in Japanese diabetic patients. We investigated the relationship between CD38 and diabetes in Caucasian patients by characterizing anti-CD38 aAbs in terms of prevalence and function (agonistic/nonagonistic activity) and by exploring the potential influence of the CD38 genetic background. A novel enzymatic immunoassay, using recombinant soluble CD38 as the target antigen, was developed for the analysis of anti-CD38 aAb titers. Sera from 19.15% of type 1 and 16.67% of type 2 diabetic patients were positive. The majority of anti-CD38 aAbs (57.14%) displayed agonistic properties, i.e., they demonstrated the capability to trigger Ca 2؉ release in lymphocytic cell lines. In agreement with these functional features, the presence of anti-CD38 aAbs in type 2 diabetic patients was associated with significantly higher levels of fasting plasma C-peptide and insulin, as compared with anti-CD38 -counterparts. No diabetic subject carrying the Arg 140 Trp mutation and no preferential association between diabetes or aAb status and the CD38*A allele was found in the study population. These results show the significance of anti-CD38 aAbs as a new diagnostic marker of ␤-cell autoimmunity in diabetes. Moreover, the prevalent agonistic activity of these aAbs suggests that they could mediate relevant effects on target cells by means of Ca 2؉ mobilization. Diabetes 50:752-762, 2001

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“…Levels of intracellular calcium were measured with the calcium-binding dye fluo-3 (12). Directly after the isolation or after 24 h incubation at 37°C in a 5% CO 2 , humidified atmosphere, purified human CD4 ϩ T cells were washed twice in calcium-and magnesium-free HBSS and incubated at a concentration of 5 ϫ 10 6 cells/ml with 0.25 mM sulfinpyrazone (Sigma), 100 g/ml pluronic acid F-127 (Molecular Probes, Leiden, The Netherlands), and 5 M fluo-3 (Molecular Probes), for 30 min at 37°C.…”

Section: Calcium Measurementsmentioning

confidence: 99%

“…P2Rs can be divided in two families: the ionotropic P2X, which are ligandgated cation channels; and the metabotropic P2Y, which are part of the superfamily of G-protein-coupled receptors. At this stage, eight genuine human P2YRs have been identified and characterized: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 (4), P2Y 12 (5,6), and P2Y 13 (7) as well as the uridine 5Ј-diphosphoglucose receptor which is structurally related to P2Y 12 (8). In particular, extracellular nucleotides exert numerous actions, mediated by several P2Y and P2XRs, on monocytes and macrophages, dendritic cells, lymphocytes, and granulocytes (reviewed in Ref.…”

mentioning

confidence: 99%

Extracellular Adenine Nucleotides Inhibit the Activation of Human CD4+ T Lymphocytes

Duhant

Schandené²,

Bruyns

et al. 2002

The Journal of Immunology

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ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE2, inhibit human CD4+ T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4+ T cells. The rank order of potency of the various nucleotides was: adenosine 5′-O-(3-thiotriphosphate) (ATPγS) ≈ 2′- and 3′-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ≫ dATP, 2-propylthio-β,γ-dichloromethylene-d-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATPγS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPγS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-γ; expression of CD25; and proliferation after activation of CD4+ T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4+ T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.

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Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor

Cited by 22 publications

References 15 publications

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Autoantibody Response to CD38 in Caucasian Patients With Type 1 and Type 2 Diabetes

Extracellular Adenine Nucleotides Inhibit the Activation of Human CD4+ T Lymphocytes

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