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, 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.