Endothelin converting enzyme activity in primary rat astrocytes is modulated by endothelin B receptors.
Astrocytes express endothelin-1 (ET-1), ET-3, and their receptors, ET(A) and ET(B). 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 ET(B)-deficient (sl/sl) versus wildtype (+/+) rats suggested that altered ECE activity may be related to the absence of ET(B) 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 V(max) values in sl/sl and +/+ astrocytes. Enzyme activity was competitively inhibited by phosphoramidon with K(i) values of 0. 6 and 0.3 microM, respectively. The K(m) value of ECE was 0.5 microM in +/+ and 0.2 microM 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 ET(B) receptor may, via ECE-1 glycosylation, exert a negative feedback on ECE activity in the astrocytic endothelin system.
Departments of Neurology, Georg-August-University, Göttingen, Germany. ehrenreich@exmax1.mpiem.gwdg.de
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H
Ehrenreich
B M
BM
Löffler
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M
Hasselblatt
H
H
Langen
J
J
Oldenburg
T
T
Subkowski
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Schilling
A L
AL
Sirén
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000032:analysis / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D005838:Genotype; D006031:Glycosylation; D007150:Immunohistochemistry; D007526:Isoelectric Point; D007700:Kinetics; D008666:Metalloendopeptidases; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011922:Rats, Mutant Strains; D017208:Rats, Wistar; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion
D017077:Culture Media, Conditioned; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes