Studies of the Extracellular ATP-Adenosine Pathway in Human Urinary Tract Epithelial Cells

Abstract

Aims: Extracellular ATP may be metabolized to AMP and adenosine by the ectonucleotidases CD39 and CD73 and, in this study, we characterized the pathways for adenosine formation in human urinary tract epithelial cells. Methods: Bladder (RT4) and kidney (A498) epithelial cells were grown in cell culture and the expression of CD39 and CD73 was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. High-performance liquid chromatography was used to determine adenosine formation in cell medium. Results: RT-PCR and immunohistochemistry revealed a high CD73 and a low CD39 expression in human urinary tract epithelial cells, whereas neutrophils had a higher CD39 than CD73 expression. Adenosine was produced when the cells were exposed to 5′-AMP (substrate for CD73), but not when exposed to 5′-ATP (substrate for CD39). A pronounced inhibition of 5′-AMP-induced adenosine formation by the CD73 inhibitor AMP-CP confirmed the involvement of CD73. Adenosine production from 5′-ATP was slightly increased (p < 0.05) when epithelial cells were cocultured with neutrophils. Conclusions: The data demonstrate that adenosine formation from extracellular ATP is negligible in urinary tract epithelial cells due to low CD39 expression in this cell type. However, the epithelial cells express CD73 and are able to convert extracellular AMP to adenosine.

Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder

“…Despite the distribution of ectonucleotidase subtypes may present specificities among species and differ within the various layers of the bladder wall, immunofluorescence studies in the mouse showed that ecto-5′-nucleotidase/CD73 is present exclusively in the detrusor smooth muscle together with ecto-NTPDase1/CD39 [37]. Data from a previous study demonstrated that adenosine formation from extracellular ATP is negligible in isolated epithelial cells from the human urinary tract [40]. This layout suggests that biosynthesis of adenosine from released ATP is positioned to favor a more important role of the nucleoside in suburothelial and detrusor muscle layers as compared to the urothelium where ecto-5′-nucleotidase/CD73 is almost absent if one excludes the basal cell layer [37]; see also preliminary results in the human bladder in [41].…”

P2Y Receptor Modulation of ATP Release in the Urothelium

“…The basal and intermediate cells of the mouse bladder urothelium express NTPDase 3 [29]. Similarly, the human urothelial cell line RT4 used in the current study expresses NTPDase 3 and 5 [12] but not NTPDase 1 [12, 30]. NTPDase 1, 2, and 3 are known to face the extracellular environment and catalyse the breakdown of extracellular ATP [29].…”

Purinergic signalling in the urinary tract in health and disease

“…Bradykinin increases NGF mRNA expression in the human urothelial cell line (UROtsa) and stretch-induced ATP release [532]. It was claimed that adenosine formation from extracellular ATP was negligible in human urinary tract urothelial cells due to low CD39 expression, but the cells express CD73, which converts extracellular AMP to adenosine [492]. Expression of P2X3 receptors has been described on suburothelial myofibroblasts of the normal human urinary bladder [438].…”