Abstract
Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET₁₈₋₃₄ (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5 nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET₁₈₋₃₄ were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.
Highlights
From the ‡Department of Biochemistry & Molecular Biology, Building 77, Monash University, Wellington Rd, Victoria 3800, Australia; §Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; ¶Department of Neurology, Brigham and Women’s Hospital, Boston, Massachusetts 02115; ʈDepartment of Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; **Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) [1]. It is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm
We have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of Endothelin Converting Enzyme-1 (ECE-1) in CSF of SAH subjects
Summary
From the ‡Department of Biochemistry & Molecular Biology, Building 77, Monash University, Wellington Rd, Victoria 3800, Australia; §Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; ¶Department of Neurology, Brigham and Women’s Hospital, Boston, Massachusetts 02115; ʈDepartment of Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; **Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) [1]. It is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. We have demonstrated that the catalytically active C terminus can be shed from the cell surface [2]. We have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects
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