Endothelin Converting Enzyme Activity in Primary Rat Astrocytes Is Modulated by Endothelin B Receptors

• Published in: Biochemical and biophysical research communications Vol. 261; no. 1; pp. 149 - 155
• Main Authors: Ehrenreich, Hannelore, Löffler, Bernd-Michael, Hasselblatt, Martin, Langen, Hanno, Oldenburg, Jan, Subkowski, Thomas, Schilling, Lothar, Sirén, Anna-Leena
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.07.1999
• Subjects: Animals; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - metabolism; Astrocytes - cytology; Astrocytes - enzymology; Blotting, Western; Cell Membrane - enzymology; Cells, Cultured; Culture Media, Conditioned - chemistry; Endothelin Receptor Antagonists; Endothelin-1 - analysis; Endothelin-1 - metabolism; endothelin-converting enzyme; Endothelin-Converting Enzymes; Genotype; Glycosylation; Immunohistochemistry; Isoelectric Point; Kinetics; Metalloendopeptidases; Protein Processing, Post-Translational; Rats; Rats, Mutant Strains; Rats, Wistar; Receptor, Endothelin B; Receptors, Endothelin - genetics; Receptors, Endothelin - physiology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Deletion
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1999.0924

Astrocytes express endothelin-1 (ET-1), ET-3, and their receptors, ETA and ETB. We report here that activated astrocytes in vivo also express endothelin converting enzyme-1 (ECE-1). Higher basal ET-1 concentrations in astrocyte media from ETB-deficient (sl/sl) versus wildtype (+/+) rats suggested that altered ECE activity may be related to the absence of ETB receptors. Quantification of ECE activity in membranes from sl/sl astrocytes yielded a 50% higher conversion compared to +/+ astrocytes, with indistinguishable ECE-1 mRNA and protein levels. Kinetic analysis of ECE activity revealed similar Vmax values in sl/sl and +/+ astrocytes. Enzyme activity was competitively inhibited by phosphoramidon with Ki values of 0.6 and 0.3 μM, respectively. The Km value of ECE was 0.5 μM in +/+ and 0.2 μM in sl/sl astrocytes. Two-dimensional focussing of astrocytic ECE-1 uncovered heterogeneity of charge and molecular weight. ECE-1 from sl/sl revealed a glycosylation pattern different from +/+ astrocytes. In conclusion, the ETB receptor may, via ECE-1 glycosylation, exert a negative feedback on ECE activity in the astrocytic endothelin system.