Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

Abstract

Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-D-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation. peptidase; bradykinin; inhibitor; endothelium THE VASODILATORY PEPTIDE bradykinin (BK) is readily degraded in the circulation by a number of metallopeptidases, including angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), aminopeptidase P, carboxypeptidases, and possibly endothelin-converting enzyme-1 (ECE-1) (12, 25). Recent evidence suggests that a portion of the hypotensive and particularly the cardioprotective effects of specific inhibitors of ACE and NEP are due to enhanced BK activity (5,17,22,23). BK is also efficiently cleaved in vitro by the closely related peptidases EP24.15 (also known as thimet oligopeptidase) and EP24.16 (also known as neurolysin) (21). We have recently shown that hypotensive responses to BK are markedly potentiated by the administration of a stable inhibitor of these enzymes N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate, termed JA2 (26). Unlike its predecessor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), JA2 is resistant to proteolytic cleavage by NEP and does not form an ACE inhibitor within the circulation. These results suggested that EP24.15 and/or EP24.16 contribute to BK metabolism in the vasculature.Although both EP24.15 and EP24.16 are primarily soluble, cytosolic enzymes, membrane-associated forms have been demonstrated in a number of cell types. For example, Crack et al. (4) have detected immunoreactive EP24.15 on the extracellular membrane of intact corticotrophic tumor cells, whereas EP24.16 has been localized to the plasma membrane in neurons, both in vivo (9, 31) and in vitro (28), as well as in human kidney cells transfected with the peptidase (29). Given that neither enzyme possesses a transmembrane domain, the exact mechanism of association with the membrane is not understood. Furthermore, the expression and subcellular distribution of EP24.15 or EP24.16 in cells of the cardiovascular system has not been explored.In the present study, we have used specific inhibitors that can distinguish EP24.15 and EP24.16 to characterize their presence in cultured vascular endothelial cells. The results show that both enzymes are present, although EP24.16 activity is higher, particularly in the membrane fraction. Furthermore, we present evidence that EP24.16 is present on the extrac...

Regulation of Endopeptidases EC3.4.24.15 and EC3.4.24.16 in Vascular Endothelial Cells by Cyclic Strain: Role of Gi Protein Signaling

“…[7][8][9][10]13 Several studies have detailed the effects of cyclic strain on secretory processes in vascular cells. Given the regulatory influence of cyclic strain on soluble levels of EP24.15 and EP24.16 activity described here, it was decided to examine its potential influence on endopeptidase release from BAECs.…”

“…6 Sitedirected inhibitors of both enzymes have been shown to potentiate bradykinin-induced vasodilation 11 and to decrease myocardial ischemia/reperfusion injury. 12 More recent studies by Norman et al 13,14 and Rioli et al 15 16,17 These cumulative observations clearly point to a physiological role for these related enzymes in regulating the balance between pressor and depressor peptides within the vasculature, thereby contributing significantly to the maintenance of vascular tone and homeostatic mechanisms.…”

Neurolysin Knockout Mice Generation and Initial Phenotype Characterization

“…It has been previously suggested that in vivo Nln contributes to the metabolism of several endogenous peptides including neurotensin, bradykinin, angiotensins, substance P, opioids (i.e. metorphamide, dynorphin A 1-8 ), somatostatin, and hemopressin (5,10,26,80). The presence of Nln activity in vascular endothelial cells suggested a possible physiological role for Nln in blood pressure control (79).…”

Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

“…Human umbilical vein endothelial cells (HUVECs) were maintained in Dulbecco's modified Eagle's medium supplemented with 8% (v/v) fetal bovine serum, 1% penicillin/streptomycin/glutamine and HAT supplement (1 vial/500 mL of media; Sigma), as mentioned previously [15].…”