Images were captured with a Q Imaging model Retiga 13001 fast cooled monochromatic digital camera (12-bit; QImaging) every 30 s (exposure time = 30 ms, gain = 0.5). most ATP-induced ethidium uptake since this was drastically reduced Terlipressin by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1?/? mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1?/? mice, in which levels of P2X7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP. 0.05, ** 0.01. The number of experiments is indicated in each bar. The pharmacological characterization of Panx1 channels was further confirmed using T cells derived from C57BL/6 mice with the Panx1 gene deleted (Panx1?/?) (Fig. S2). In these cells, the ATP-induced Etd uptake was significantly reduced in both CD4+ and CD8+ T-cell Terlipressin populations as compared with that of WT cells (Fig.?8). Notably, the small population of CD4+ lymphocytes that presented a high dye uptake and were insensitive to Panx channel blockers was still present in CD4+ T cells Panx1?/? mice (Fig.?8A, B, and C), suggesting that this T cell small population expresses a different Etd uptake pathway activated by extracellular ATP that was not further characterized in this work. In contrast CD8+ T cells derived Terlipressin from Panx1?/? mice presented a low to absent Etd uptake rate (Fig.?8A, D, and E). Open in a separate window Figure?8. The absence of Panx1 reduces the ATP-induced Etd uptake in T cells. (A) Representative microphotographs showing the CD4+ (green) and CD8+ (red) reactivity in T cells derived from popliteal lymph nodes (PLN) obtained from wild type GRK7 or Panx1?/? mice before or after addition of 1mM ATP. The three bright cells present in WT cells untreated with ATP (basal) were dead (calibration bar: 20 m). Etd uptake kinetics from PLN derived T cells in (B) CD4 or (D) CD8 positive cells obtained from wild type () or Panx1() mice before and after the addition of extracellular ATP. Bars represent the comparison of dye uptake rates in T cells of WT (black) and Panx1?/? (white) mice induced by different concentrations of ATP (100C3,000 M) in (C) CD4+ or (E) CD8+ T cells. Each bar corresponds to the mean SEM of 4 different mice, * 0.05, ** 0.01. Previously, it has been demonstrated that total CD4+ cells can be subdivided into three subpopulations with characteristic Etd fluorescence intensities induced by extracellular ATP,41 suggesting that each subtype presents different levels of pore activity and/or different uptake pathways. In addition, it has been observed that regulatory T cells (CD4+CD25+) and memory T cells (CD4+CD44highCD45RBlow) have higher membrane permeability to Etd than conventional T cells do.41,42 Indeed, in Etd uptake studies performed by FACS analysis we found that conventional T cells treated with ATP exhibit 3 distinct populations with different Etd uptake, one with very low or null Etd uptake (called 1), a second one with medium Etd uptake values (called 2) and a third one with the highest Etd uptake (called 3) (Fig. S2). However, CD4+ T cells obtained from Panx1?/? mice exhibited a great reduction in subpopulation 3 and, while subpopulation 2 was absent (Fig. S2) suggesting that all cells of subpopulation 2 and almost two thirds of subpopulation 3 express Panx1. In addition, Etd uptake of CD8+ T cells obtained from Panx1?/? mice was completely absent (Fig. S2), suggesting that all CD8+ T cells express Panx1, which constitutes the only pathway linked to P2X7Rs. This is also the case of most CD4+ cells. However, one third of subpopulation 3 expresses an Etd uptake pathway independent of Panx1. We evaluated whether Panx1 channels of T cells also serve as membrane pathways for ATP. The evaluation of ATP release via Panx1 channel activated through P2X7Rs is difficult to measure because the use of exogenous ATP to activate the purinergic receptors increases the signal-to-noise ratio and thus interferes with the detection of ATP released from the cells. In this way, since Panx1 channels open in response to an increase in intracellular free Ca2+ concentration ([Ca2+]i)13 we tested whether T cells treated with a calcium ionophore 4Br-A23187 (2.5 M) for 5 min show ATP release to the extracellular solution. We found that the calcium ionophore prominently increased the extracellular.
Images were captured with a Q Imaging model Retiga 13001 fast cooled monochromatic digital camera (12-bit; QImaging) every 30 s (exposure time = 30 ms, gain = 0
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