Ectonucleotidase activities in Sertoli cells from immature rats
Ectonucleotidase activities in Sertoli cells from immature rats
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DOI：
10.1590/s0100-879x2001001000003
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发表时间：
2001-10-01
影响因子：
2.3
通讯作者：
Bernard, EA
中科院分区：
文献类型：
--
作者：
Casali, EA;da Silva, TR;Bernard, EA
支持细胞已被证明是细胞外嘌呤如ATP和腺苷的靶标。这些嘌呤通过两种嘌呤受体亚型P-2 Y2和P-A1在支持细胞中引起反应。嘌呤受体的信号通常通过外核苷酸酶的作用而终止。为了证明这些酶的活性，我们培养大鼠睾丸支持细胞四天，然后将它们用于不同的测定。通过使用比色法测量释放的Pi来估计ATP、ADP和AMP水解。通过HPLC测定腺苷脱氨酶活性（EC 3.5.4.4）。孵育40分钟后细胞未被破坏，认为酶活性位于细胞外。ATP和ADP水解显着增加二价阳离子的反应介质中。竞争图表明只有一个酶位点负责ATP和ADP的水解。该结果表明，作用于Sertoli细胞表面上三磷酸核苷和二磷酸核苷降解的酶是真正的ATP二磷酸水解酶（EC 3.6.1.5）（ATP和ADP的比活性分别为113 +/- 6和21 +/- 2 nmol Pi mg（-1）min（-1））。外-5 '-核苷酸酶（EC 3.1.3.5）和外腺苷脱氨酶活性（AMP的比活性分别为32 +/- 2 nmol Pi mg（-1）min（-1）和1.52 +/- 0.13 nmol腺苷mg（-1）min（-1）显示能够终止嘌呤的作用，并可能与生精小管内核苷酸和核苷的细胞外水平的生理控制相关。
Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P-2Y2 and P-A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1) respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.