title: Increased expression of endothelin-converting enzyme-1c isoform in response to high glucose levels in endothelial cells.
abstract: Endothelin-1 (ET-1) is both a potent vasoconstrictor and mitogenic factor that has been implicated as a cause of the micro- and macrovascular complications of diabetes mellitus. The pathway by which the high-glucose environment of diabetes mediates increased levels of endothelins has not been completely elucidated but appears to involve endothelin-converting enzyme (ECE-1), which converts inactive big ET-1 to active ET-1 peptide. To determine the effect of high glucose concentrations on the expression of ECE-1, hybrid endothelial cells (EA.hy926) and human umbilical vein endothelial cells (HUVEC) were both grown in various glucose concentrations. There was a 2-fold increase in ECE-1 immunoreactivity in the EA.hy926 cell line growing in medium containing 22.2 versus 5.5 mmol/l glucose after 24 h, which rose to greater than 20-fold after 5 days. Similar results were seen with HUVEC. Bradykinin or NG-nitro-L-arginine methyl ester did not change the effect of high glucose on ECE-1 protein expression. High glucose induced a 72 and 41% increase in total protein kinase C (PKC) activity in both EA.hy926 cells and HUVEC, respectively, and a 39, 49 and 109% elevation in PKC beta1, beta2 and delta expression, respectively, in EA.hy926 cells. The increase in ECE-1 expression was inhibited in both cell cultures by GF109203X (5 micromol/l), a general PKC inhibitor, while addition of 10 nmol/l phorbol myristic acid to EA.hy926 cells or HUVEC growing on medium containing 5.5 mmol/l glucose increased ECE-1 expression to a level similar to that of cells conditioned in high glucose. Human ECE-1 protein exists in four different isoforms, termed 1a, 1b, 1c and 1d. Northern blot analysis revealed that only ECE-1c isoform mRNA levels increased. Immunohistochemical staining of EA.hy926 cells grown in high glucose concentrations demonstrated an increase in the ECE-1c isoform, which occurred mainly in the plasma membrane. These results showed that the PKC pathway may play an important role in the glucose-mediated induction of ECE-1 expression. The main isoform to increase in response to high glucose was ECE-1c. This enzyme may be one of the factors contributing to the elevated ET-1 peptide levels observed in diabetes.
authors.affiliation: Diabetes Center and Department of Internal Medicine, Hadassah University Hospital, Jerusalem, Israel.
authors.forename: Shoshana
authors.initials: S
authors.lastname: Keynan
authors.forename: Mogher
authors.initials: M
authors.lastname: Khamaisi
authors.forename: Rachel
authors.initials: R
authors.lastname: Dahan
authors.forename: Kay
authors.initials: K
authors.lastname: Barnes
authors.forename: Carolyn D
authors.initials: CD
authors.lastname: Jackson
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Turner
authors.forename: Itamar
authors.initials: I
authors.lastname: Raz
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003928:Diabetic Nephropathies / Q000378:metabolism / Q000503:physiopathology; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D008666:Metalloendopeptidases; D009569:Nitric Oxide / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D009569:Nitric Oxide; D011493:Protein Kinase C; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D005947:Glucose