Losenkova Karolina, Paul Marius, Irjala Heikki, Jalkanen Sirpa, Yegutkin Gennady G
MediCity Research Laboratory, University of Turku, Turku, Finland.
Department of Otorhinolaryngology, Head and Neck Surgery, Turku University Hospital and Turku University, Turku, Finland.
Methods Mol Biol. 2020;2041:107-116. doi: 10.1007/978-1-4939-9717-6_7.
Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).
细胞外ATP及其他嘌呤的病理生理学和治疗潜力研究是一个重要且发展迅速的领域。细胞的整体反应由多种因素决定，包括内源性ATP的释放、不同类型核苷酸和腺苷选择性受体的共表达，以及控制嘌呤能信号持续时间和强度的外切酶的具体组成。目前的研究结果支持存在一个广泛的嘌呤转化外切酶网络，这些酶在哺乳动物组织中以不同程度共表达，并且在底物特异性方面具有相似性。在此，我们描述了一种在同一组织切片中同时检测外切核苷酸酶和组织非特异性碱性磷酸酶（TNAP）活性的组织化学方法。将该技术进一步用于人腭扁桃体冰冻切片染色，揭示了关键外切酶在某些扁桃体结构内的选择性分布，包括生发中心和结缔组织（外切5'-核苷酸酶/CD73），以及滤泡间区域（TNAP和NTPDase1/CD39）。
