Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase |
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Authors: | Lucia Semianrio-Vidal Catharina van Hesuden Govindasamy Mugesh Eduardo Rodolfo Lazarowski |
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Institution: | (1) Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina School of Medicine, 7017 Thurston-Bowles Building, CB 7248, Chapel Hill, NC 27599-7248, USA;(2) Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, India; |
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Abstract: | Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that
protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution
of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release
and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted
nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In
the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase
and adenyl kinase activities. Addition of radiotracer γ
32P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of 32P]-inorganic phosphate (32Pi). Inclusion of UDP in the incubation medium resulted in conversion of γ
32P]ATP to 32P]UTP, while inclusion of AMP resulted in conversion of γ
32P]ATP to 32P]ADP. Ebselen markedly reduced 32P]UTP formation but displayed negligible effect on 32Pi or 32P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of
ATP (IC50 = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549
cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V
max of the reaction (K
i = 7.6 ± 3 μM), having negligible effect on K
M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK. |
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Keywords: | Nucleoside diphosphokinase Ebselen Extracellular nucleotides Nucleotide release Lung epithelial cells |
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