Production of soluble Neprilysin by endothelial cells

Don Monath Sanjaya Kuruppu; Niwanthi Wickramasekara Rajapakse; Dmitriy Minond; Alexander Ian Smith

doi:10.1016/j.bbrc.2014.01.158

Production of soluble Neprilysin by endothelial cells

Don Monath Sanjaya Kuruppu, Niwanthi Wickramasekara Rajapakse, Dmitriy Minond, Alexander Ian Smith

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Abstract

A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40muM) assay, we determined the activity of recombinant human NEP (rhNEP; 12ng), and NEP in the media of endothelial cells (10 v/v; after 24h incubation with cells) to be 9.35+/-0.70 and 6.54+/-0.41mumols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32muM, respectively. Treatment of cells with TPI2155-14 (15muM) and TPI2155-17 (4.3muM) resulted in a significant decrease in NEP activity in media (62.37+/-1.43 and 38.30+/-4.70, respectively as a of control; P

Original language	English
Pages (from-to)	423 - 427
Number of pages	5
Journal	Biochemical and Biophysical Research Communications
Volume	446
Issue number	2
DOIs	https://doi.org/10.1016/j.bbrc.2014.01.158
Publication status	Published - 2014

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10.1016/j.bbrc.2014.01.158

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title = "Production of soluble Neprilysin by endothelial cells",

abstract = "A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40muM) assay, we determined the activity of recombinant human NEP (rhNEP; 12ng), and NEP in the media of endothelial cells (10 v/v; after 24h incubation with cells) to be 9.35+/-0.70 and 6.54+/-0.41mumols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32muM, respectively. Treatment of cells with TPI2155-14 (15muM) and TPI2155-17 (4.3muM) resulted in a significant decrease in NEP activity in media (62.37+/-1.43 and 38.30+/-4.70, respectively as a of control; P",

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T1 - Production of soluble Neprilysin by endothelial cells

AU - Kuruppu, Don Monath Sanjaya

AU - Rajapakse, Niwanthi Wickramasekara

AU - Minond, Dmitriy

AU - Smith, Alexander Ian

PY - 2014

Y1 - 2014

N2 - A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40muM) assay, we determined the activity of recombinant human NEP (rhNEP; 12ng), and NEP in the media of endothelial cells (10 v/v; after 24h incubation with cells) to be 9.35+/-0.70 and 6.54+/-0.41mumols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32muM, respectively. Treatment of cells with TPI2155-14 (15muM) and TPI2155-17 (4.3muM) resulted in a significant decrease in NEP activity in media (62.37+/-1.43 and 38.30+/-4.70, respectively as a of control; P

AB - A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40muM) assay, we determined the activity of recombinant human NEP (rhNEP; 12ng), and NEP in the media of endothelial cells (10 v/v; after 24h incubation with cells) to be 9.35+/-0.70 and 6.54+/-0.41mumols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32muM, respectively. Treatment of cells with TPI2155-14 (15muM) and TPI2155-17 (4.3muM) resulted in a significant decrease in NEP activity in media (62.37+/-1.43 and 38.30+/-4.70, respectively as a of control; P

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Production of soluble Neprilysin by endothelial cells

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Control of proteases in infectious, degenerative and cardiovascular disease

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Production of soluble Neprilysin by endothelial cells

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Control of proteases in infectious, degenerative and cardiovascular disease

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