A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells

Abstract

Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described

Met-Lys-bradykinin-Ser-Ser, a peptide produced by the neutrophil from kininogen, is metabolically activated by angiotensin converting enzyme in vascular tissue

“…The resulting peptide, possibly MetLys-BK, is antagonized by a low concentration of the specific B 2 receptor antagonist LF in the umbilical vein. We recently identified ACE in endothelial cells of the human umbilical vein (tissue sections, immunohistochemistry) [11]; in cultured endothelial cells derived from this vessel, the regulated expression of ACE has been described [11,24,25]. As previously reported [15], enalaprilat did not influence the apparent potency of BK in the contractility assay.…”

“…previously reported [10,11]. Both enalaprilat and BK have rather similar affinities for either catalytic site of somatic ACE [13,20], leading to apparently seamless radioligand saturation or competition curves.…”

“…HEK 293a cells were also exploited to transiently express human recombinant ACE and perform a [ 3 H]enalaprilat binding assay assay precisely as described (2 nM radioligand concentration) [11] (the peACE vector was a gift from Prof. P. Corvol, Paris, France). A 2 nM concentration of [ 3 H]enalaprilat was used to generate binding competition curves for kinins that are high affinity substrates of this peptidase and behave as bona fide competitors this radioligand in this binding assay [12].…”

Prolonged signalling and trafficking of the bradykinin B2 receptor stimulated with the amphibian peptide maximakinin: Insight into the endosomal inactivation of kinins

“…Complementary experiments on the mechanism of endosomal inactivation of BK were conducted. P. Corvol, Paris, France) was determined in transiently transfected HEK 293a cells as described [8]; this binding is displaced by BK-related substrates [9], which was verified for maximakinin.…”

Vasopeptidase-activated latent ligands of the histamine receptor-1

“…In addition to molecular studies based on recombinant B 2 R and H 1 R, we exploited the human umbilical vein contractility assay naturally expressing these two receptor types that mediate important contractile responses [13,14]. This vein also expresses ACE [2,15]. The ectopeptidase APN is present in the vascular smooth muscle cells of the umbilical artery [3,4] and at least in the endothelium of the umbilical vein [16,17].…”