New method to simultaneously characterize the expression and in situ activity of ecto-nucleotidase in human tissues

Abstract

Introduction: Extracellular nucleotides, such as ATP, and nucleosides, such as adenosine, act as autocrine and paracrine molecules that have multiple roles in virtually all organs and tissues, including female reproductive organs. Extracellular ATP and adenosine levels are regulated by the action of ecto-nucleotidases that hydrolyse ATP to adenosine. The aim of the present study was to set up a new method to simultaneously localize the cellular distribution and in situ activity of ecto-nucleotidases in tissue sections. We used this method to characterize the expression of ecto-nucleotidases in human oviducts.

Material and Methods: Cryosections of non-pathological human oviducts were obtained from salpingectomy at the Service of Gynecology of Bellvitge Hospital. Samples were incubated with the following primary antibodies against human enzymes: anti-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39), anti-NTPDase2, and anti-placental alkaline phosphatase (PLAP). In situ activity reactions were performed on the same slides using the Wachstein/Meisel lead phosphate method with ATP or ADP as substrate. For alkaline phosphatase activity, the BCIP/NBT revealing reagent was used. The sections were then incubated with the appropriate Alexa Fluor-conjugated secondary antibodies and mounted with Prolong Gold antifade with DAPI medium.

Results: NTPDase1 was expressed in the smooth muscle and endothelial cells, coinciding with localization of ADPase activity. NTPDase2 was largely expressed and active (ATPase activity) in ciliated cells and in connective tissue. PLAP was immunodetected and active in luminal epithelium.

Conclusions: We found that this new method is specific, sensitive, and useful with diferent tissues. Our results show that ecto-nucleotidases are abundantly present in human oviducts where these enzymes work in concert to metabolize extracellular ATP to adenosine. This study contributes to knowledge of purinergic signaling by ecto-nucleotidases in the female reproductive system.