TY - JOUR
T1 - Identification of endothelin converting enzyme-1 in human non-pigmented ciliary epithelial cells
AU - Prasanna, Ganesh
AU - Dibas, Adnan
AU - Finkley, Alvin
AU - Yorio, Thomas
N1 - Funding Information:
This work was supported by Texas Advanced Research Program Grant from the Texas Higher Education Coordinating Board (Grant number: 09768-008}018) and NEI}NIH grant (Grant number: EY11979).
PY - 1999/8
Y1 - 1999/8
N2 - Endothelins (ETs), potent vasoactive peptides, are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin- converting enzyme (ECE). Although the role of ocular ET's are uncertain, they are elevated in the aqueous humor of normal as well as glaucomatous eyes and have been shown to lower the intraocular pressure for prolonged periods of time. In the current study, an endothelin-converting enzyme-1 (ECE-1) has been identified and its activity has been studied in SV-40 transformed human non-pigmented ciliary epithelial (HNPE) cells. Western blotting using polyclonal antibodies against ECE-1, detected a 124 kDa protein in the plasma membrane but not in the cytosol. Further characterization of the enzymatic activity of ECE-1 (conversion of Big ET-1 to ET-1) using the plasma membrane fraction of HNPE cells was performed by a novel assay involving 125I-Big ET-1 (substrate; 80 fmoles mg-1 protein) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as fmoles 125I-ET-1 produced mg protein-1 time-1) increased linearly with time and was similar to that observed in rat lung tissue. ECE-1 activity was enhanced with increasing concentrations of substrate (125I-Big ET-1: 30-200 fmoles mg protein-1 180 min-1) as well as with increasing concentrations of protein (5-20 μg proteins at the substrate concentration of 80 fmoles mg protein-1 180 min- 1). Treatments with CGS-26303 (100 μM), an inhibitor of ECE-1 and thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased ECE-1 activity. Furthermore, both, acidification of the reaction buffer (from pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mm) decreased ECE-1 activity by nearly 60%. These results suggest that the ECE-1 localized in HNPE cells, is a neutral pH-sensitive metalloprotease which is similar in its activity to that observed in lung tissue and is essential for the production of ET-1 in HNPE cells. The physiological importance of the unusual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure.
AB - Endothelins (ETs), potent vasoactive peptides, are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin- converting enzyme (ECE). Although the role of ocular ET's are uncertain, they are elevated in the aqueous humor of normal as well as glaucomatous eyes and have been shown to lower the intraocular pressure for prolonged periods of time. In the current study, an endothelin-converting enzyme-1 (ECE-1) has been identified and its activity has been studied in SV-40 transformed human non-pigmented ciliary epithelial (HNPE) cells. Western blotting using polyclonal antibodies against ECE-1, detected a 124 kDa protein in the plasma membrane but not in the cytosol. Further characterization of the enzymatic activity of ECE-1 (conversion of Big ET-1 to ET-1) using the plasma membrane fraction of HNPE cells was performed by a novel assay involving 125I-Big ET-1 (substrate; 80 fmoles mg-1 protein) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as fmoles 125I-ET-1 produced mg protein-1 time-1) increased linearly with time and was similar to that observed in rat lung tissue. ECE-1 activity was enhanced with increasing concentrations of substrate (125I-Big ET-1: 30-200 fmoles mg protein-1 180 min-1) as well as with increasing concentrations of protein (5-20 μg proteins at the substrate concentration of 80 fmoles mg protein-1 180 min- 1). Treatments with CGS-26303 (100 μM), an inhibitor of ECE-1 and thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased ECE-1 activity. Furthermore, both, acidification of the reaction buffer (from pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mm) decreased ECE-1 activity by nearly 60%. These results suggest that the ECE-1 localized in HNPE cells, is a neutral pH-sensitive metalloprotease which is similar in its activity to that observed in lung tissue and is essential for the production of ET-1 in HNPE cells. The physiological importance of the unusual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure.
KW - Big endothelin-1
KW - Ciliary epithelium
KW - Endothelin converting enzyme-1
KW - Endothelin converting enzyme-1 assay
KW - Endothelin-1
UR - http://www.scopus.com/inward/record.url?scp=0032806583&partnerID=8YFLogxK
U2 - 10.1006/exer.1999.0691
DO - 10.1006/exer.1999.0691
M3 - Article
C2 - 10433854
AN - SCOPUS:0032806583
SN - 0014-4835
VL - 69
SP - 175
EP - 183
JO - Experimental Eye Research
JF - Experimental Eye Research
IS - 2
ER -