Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, Buenos Aires, Argentina; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal de Rio de Janeiro, Rio de Janeiro, Brasil; INPeTAm Instituto Nacional de Ciência e Tecnologia em Pesquisa Translacional em Saúde e Ambiente na Reigião Amazônica, Rio de Janeiro, Brasil.
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal de Rio de Janeiro, Rio de Janeiro, Brasil; INPeTAm Instituto Nacional de Ciência e Tecnologia em Pesquisa Translacional em Saúde e Ambiente na Reigião Amazônica, Rio de Janeiro, Brasil.
PLoS One. 2014 May 23;9(5):e96216. doi: 10.1371/journal.pone.0096216. eCollection 2014.
In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration.
在人类红细胞（h-RBCs）中，各种刺激会导致 [cAMP] 的增加，从而引发 ATP 的释放。细胞外 ATP 积累的模式（ATPe 动力学）取决于 ATP 释放和外切 ATP 酶活性对 ATPe 的降解。在这项研究中，我们评估了处于不同感染阶段（环状、滋养体和裂殖体阶段）的疟原虫感染的 h-RBC 原代培养物中的 ATPe 动力学。包含异丙肾上腺素（β-肾上腺素能激动剂）、福司可林（腺苷酸激酶激活剂）和罂粟碱（磷酸二酯酶抑制剂）的“3V”混合物用于诱导 cAMP 依赖性 ATP 释放。r-RBCs（环状感染 RBCs）、t-RBCs（滋养体感染 RBCs）和 s-RBCs（裂殖体感染 RBCs）的 ATPe 动力学显示 [ATPe] 迅速达到峰值，然后是较慢的时间依赖性下降。在所有红细胞内阶段，ΔATP1（刺激后 1 分钟测量的 [ATPe] 与基础 [ATPe] 之间的差异）与寄生虫血症呈非线性增加（从 2%增加到 12.5%）。在 3V 暴露下，寄生虫血症为 94%的 t-RBCs（t94-RBCs）的 ΔATP1 值比 h-RBCs 高 3.8 倍，表明 ATP 释放上调。预先暴露于 100 µM 卡波氯铵、100 nM 甲氟喹或 100 µM NPPB 将 h-RBCs 的 ΔATP1 降低至 83-87%，将 t94-RBCs 的 ΔATP1 降低至 63-74%。在 t94-RBCs 中，外切 ATP 酶活性在低纳摩尔浓度（300-900 nM）和 500 µM 外源性 ATPe 浓度下分别增加了约 400 倍，而 t94-RBCs 中的细胞内 ATP 浓度为 h-RBCs 的 65%。在 t94-RBCs 中，一氧化氮（NO）的产生比 h-RBCs 高约 7 倍，并用 L-NAME 预处理部分抑制。在含有 L-NAME 的培养基中，h-RBCs 的 ΔATP1 值比无 L-NAME 时高 2.7 倍，t94-RBCs 的 ΔATP1 值比无 L-NAME 时高 4.2 倍。结果表明，疟原虫感染 h-RBCs 强烈激活了这些细胞中的 Pannexin 1 介导的 ATP 释放。几种过程部分抵消了 ATPe 积累：上调的 ATPe 降解、增强的 NO 产生和细胞内 ATP 浓度降低。
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