Departamento de Investigación en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Calzada de Tlalpan 4502, 14080, Mexico City, Mexico.
Laboratorio HLA, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Calzada de Tlalpan 4502, 14080, Mexico City, Mexico.
Purinergic Signal. 2019 Mar;15(1):69-76. doi: 10.1007/s11302-019-09644-7. Epub 2019 Jan 13.
Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sensitized guinea pigs were used for determining the extracellular metabolism (as assessed by inorganic phosphate production) of ATP, ADP, AMP, or adenosine, and for detecting possible changes in the expression (qPCR and Western blot) of major ectonucleotidases (NTPDase1, NTPDase3, and NPP1) and purinoceptors (P2X, P2X, P2Y, and P2Y). Contrary to our hypothesis, we found that leukocytes from allergic animals produced higher amounts of inorganic phosphate after stimulation with ATP and ADP, as compared with leukocytes from non-sensitized animals. Although at first glance, this result suggested that sensitization caused higher efficiency of ectonucleotidases, their mRNA and protein expressions were unaffected. On the other hand, after sensitization, we found a significant increase in the protein expression of P2X and P2Y, two purinoceptors known to be responsible for ATP release after activation. We concluded that allergic sensitization increased the amount of ATP hydrolyzed by ectonucleotidases, the latter probably not due to the enhanced efficiency of its enzymatic breakdown, but rather due to an increased release of endogenous ATP or other nucleotides, partly mediated by enhanced expression or P2X and P2Y receptors.
在哮喘患者的支气管肺泡灌洗液中发现 ATP 水平升高，并且患有这种疾病的患者而不是健康受试者在雾化 ATP 后会发展为支气管痉挛。由于控制细胞外 ATP 的有害作用的主要机制是其酶水解，因此我们假设过敏敏化伴随着这种水解作用的功能降低。在本研究中，使用致敏和非致敏豚鼠的外周血白细胞来确定 ATP、ADP、AMP 或腺苷的细胞外代谢（通过无机磷酸盐产生来评估），并检测主要核苷酸酶（NTPDase1、NTPDase3 和 NPP1）和嘌呤受体（P2X、P2X、P2Y 和 P2Y）表达的可能变化（qPCR 和 Western blot）。与我们的假设相反，我们发现与非致敏动物的白细胞相比，过敏动物的白细胞在受到 ATP 和 ADP 刺激后产生更多的无机磷酸盐。尽管乍一看，这一结果表明敏化导致核苷酸酶的效率更高，但它们的 mRNA 和蛋白表达不受影响。另一方面，致敏后，我们发现 P2X 和 P2Y 两种嘌呤受体的蛋白表达显著增加，这两种受体在激活后已知负责 ATP 释放。我们得出结论，过敏敏化增加了外核苷酸酶水解的 ATP 量，后者可能不是由于其酶促分解的效率提高，而是由于内源性 ATP 或其他核苷酸的释放增加，部分由增强的表达或 P2X 和 P2Y 受体介导。
