Nitric oxide inhibits the production of soluble endothelin converting enzyme-1

Research output: Contribution to journalArticleResearchpeer-review

Abstract

This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 microM), NOS substrate L-Arg (200-1,000 microM), a L-Arg transport inhibitor (L-Lys; 10 microM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 microM). The effect of L-Arg (1,000 microM) was also tested in the presence of L-Lys (10 microM), L-Gln (100 microM) and L-NAME (10-100 microM). Ultracentrifugation (100,000xg, 4 degrees C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P <0.05, unpaired t test, n = 4) in the presence of DETA (75.31 +/- 3.59; 800 microM) and L-Arg (60.97 +/- 9.22; 1,000 microM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 microM; 99.19 +/- 0.58) and L-Gln (100 microM; 104.41 +/- 0.65). In addition, the presence of L-Lys (10 microM) significantly blocked the L-Arg (1,000 microM)-induced reduction in soluble ECE-1 levels (122.38 +/- 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.
Original languageEnglish
Pages (from-to)49 - 54
Number of pages6
JournalMolecular and Cellular Biochemistry
Volume396
Issue number1-2
DOIs
Publication statusPublished - 2014

Cite this

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title = "Nitric oxide inhibits the production of soluble endothelin converting enzyme-1",
abstract = "This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 microM), NOS substrate L-Arg (200-1,000 microM), a L-Arg transport inhibitor (L-Lys; 10 microM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 microM). The effect of L-Arg (1,000 microM) was also tested in the presence of L-Lys (10 microM), L-Gln (100 microM) and L-NAME (10-100 microM). Ultracentrifugation (100,000xg, 4 degrees C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P <0.05, unpaired t test, n = 4) in the presence of DETA (75.31 +/- 3.59; 800 microM) and L-Arg (60.97 +/- 9.22; 1,000 microM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 microM; 99.19 +/- 0.58) and L-Gln (100 microM; 104.41 +/- 0.65). In addition, the presence of L-Lys (10 microM) significantly blocked the L-Arg (1,000 microM)-induced reduction in soluble ECE-1 levels (122.38 +/- 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.",
author = "Kuruppu, {D M Sanjaya} and Rajapakse, {Niwanthi W} and Dunstan, {Rhys A} and Smith, {Alexander Ian}",
year = "2014",
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language = "English",
volume = "396",
pages = "49 -- 54",
journal = "Molecular and Cellular Biochemistry",
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}

Nitric oxide inhibits the production of soluble endothelin converting enzyme-1. / Kuruppu, D M Sanjaya; Rajapakse, Niwanthi W; Dunstan, Rhys A; Smith, Alexander Ian.

In: Molecular and Cellular Biochemistry, Vol. 396, No. 1-2, 2014, p. 49 - 54.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Nitric oxide inhibits the production of soluble endothelin converting enzyme-1

AU - Kuruppu, D M Sanjaya

AU - Rajapakse, Niwanthi W

AU - Dunstan, Rhys A

AU - Smith, Alexander Ian

PY - 2014

Y1 - 2014

N2 - This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 microM), NOS substrate L-Arg (200-1,000 microM), a L-Arg transport inhibitor (L-Lys; 10 microM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 microM). The effect of L-Arg (1,000 microM) was also tested in the presence of L-Lys (10 microM), L-Gln (100 microM) and L-NAME (10-100 microM). Ultracentrifugation (100,000xg, 4 degrees C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P <0.05, unpaired t test, n = 4) in the presence of DETA (75.31 +/- 3.59; 800 microM) and L-Arg (60.97 +/- 9.22; 1,000 microM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 microM; 99.19 +/- 0.58) and L-Gln (100 microM; 104.41 +/- 0.65). In addition, the presence of L-Lys (10 microM) significantly blocked the L-Arg (1,000 microM)-induced reduction in soluble ECE-1 levels (122.38 +/- 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.

AB - This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 microM), NOS substrate L-Arg (200-1,000 microM), a L-Arg transport inhibitor (L-Lys; 10 microM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 microM). The effect of L-Arg (1,000 microM) was also tested in the presence of L-Lys (10 microM), L-Gln (100 microM) and L-NAME (10-100 microM). Ultracentrifugation (100,000xg, 4 degrees C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P <0.05, unpaired t test, n = 4) in the presence of DETA (75.31 +/- 3.59; 800 microM) and L-Arg (60.97 +/- 9.22; 1,000 microM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 microM; 99.19 +/- 0.58) and L-Gln (100 microM; 104.41 +/- 0.65). In addition, the presence of L-Lys (10 microM) significantly blocked the L-Arg (1,000 microM)-induced reduction in soluble ECE-1 levels (122.38 +/- 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.

UR - http://link.springer.com/article/10.1007%2Fs11010-014-2141-0

U2 - 10.1007/s11010-014-2141-0

DO - 10.1007/s11010-014-2141-0

M3 - Article

VL - 396

SP - 49

EP - 54

JO - Molecular and Cellular Biochemistry

JF - Molecular and Cellular Biochemistry

SN - 0300-8177

IS - 1-2

ER -