Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

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 BigET 18 -34 (6 g/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ؎ 0.037 mols of substrate cleaved/ l of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET 18 -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. Endothelin-1 (ET-1) 1 is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, 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. EXPERIMENTAL PROCEDURESCSF Sample Collection-CSF samples were obtained from a prospective biorepository of SAH and non-SAH hydrocephalus human subjects in accordance with local institutional review board approved protocol. Detailed descriptions of biorepository methods have been previously pu...

Pharmacological hypothesis: Nitric oxide‐induced inhibition of ADAM‐17 activity as well as vesicle release can in turn prevent the production of soluble endothelin‐converting enzyme

“…We later confirmed the presence of this soluble form in the cerebrospinal fluid of patients who have suffered subarachnoid hemorrhage (Kuruppu et al. ,b). A circulating form of ECE‐1 with catalytic activity can result in the production of ET‐1 throughout the vasculature, thus having significant implications on vascular tone.…”

“…In subsequent studies, we examined the effect of exogenous NO on soluble ECE‐1 production in Ea.hy926 cells (Kuruppu et al. ,b). NO donor SNP inhibited the release of soluble ECE‐1.…”

“…This effect was mimicked by incubation of cells with L‐arginine, the substrate for NOS (Kuruppu et al. ,b). Furthermore, the presence of amino acids such as L‐lysine, which compete with L‐arginine for entry into cells, as well as the NOS inhibitor L‐NAME, prevented the L‐arginine‐induced inhibition of soluble ECE‐1 production (Kuruppu et al.…”

“…Furthermore, the presence of amino acids such as L‐lysine, which compete with L‐arginine for entry into cells, as well as the NOS inhibitor L‐NAME, prevented the L‐arginine‐induced inhibition of soluble ECE‐1 production (Kuruppu et al. ,b). Our results indicated that endogenous NO produced by these cells prevented the secretion of ECE‐1.…”

“…In contrast, the level of ECE‐1 expression in the cell membrane did not change in response to L‐arginine or SNP (Kuruppu et al. ,b). Therefore, according to studies conducted in our laboratory, both exogenous and endogenous NO can inhibit the release of soluble ECE‐1.…”