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We reported previously a novel mode of action of angiotensin I-converting enzyme (kininase II; ACE) inhibitors mediated through the direct activation of bradykinin B 1 receptor, independent of endogenous kinins or ACE (J Biol Chem 277: [16847][16848][16849][16850][16851][16852] 2002). We aimed to further clarify the mechanism of activation of B 1 receptor, which leads to prolonged nitric oxide (NO) release. The ACE inhibitor enalaprilat and the peptide ligand desArg 10 -kallidin (in nanomolar concentrations) release NO by activating endothelial NO synthase (eNOS) in bovine and inducible NO synthase (iNOS) in stimulated human endothelial cells. The peptide and the ACE inhibitor ligands activate eNOS by facilitating different signaling pathways. DesArg 10 -kallidin enhances inositol-phosphate generation and elevates [Ca 2ϩ ] i by first augmenting intracellular release and then the influx of extracellular Ca 2ϩ . In contrast, enalaprilat stimulates only the influx of extracellular Ca 2ϩ through rare earth-sensitive channels, and its effect is blocked by cholera toxin or protein kinase C inhibitors. In addition, unlike desArg 10 -kallidin, enalaprilat can also release NO independent of Ca 2ϩ in bovine endothelial cells. The inflammatory cytokines interleukin-1␤ and interferon-␥ induce both B 1 receptor and iNOS in human endothelial cells. In contrast to eNOS, B 1 ligands activate iNOS similarly. Both desArg 10 -kallidin and ACE inhibitors enhance arginine uptake and release NO independent of [Ca 2ϩ ] i elevation. This is the first report on the direct activation of B 1 receptor by ACE inhibitors in human endothelial cells. This interaction leads to prolonged NO release and possibly contributes to the documented benefits of the use of ACE inhibitors.Bradykinin and related peptides (kinins) activate two types of G-protein coupled receptors: B 1 and B 2 . B 2 is ubiquitous, and B 1 is generally expressed under pathological conditions, in which it may protect or be detrimental (McLean et al., 2000). B 2 receptors are activated by bradykinin and kallidin, whereas the B 1 receptor agonists are desArg 9 -bradykinin and desArg 10 -kallidin (McLean et al., 2000) generated by membrane or plasma carboxypeptidases, M or N, which cleave the C-terminal arginine of the peptides (Erdös and Skidgel, 1997).In addition to these endogenous peptide ligands, inhibitors of angiotensin I-converting enzyme (kininase II; ACE) can also directly activate B 1 receptors in cultured cells (Ignjatovic et al., 2002). They activate through the HEAWH sequence in the second extracellular loop (residues 195-199 in human receptor). This sequence is conserved in B 1 receptors in several species, and it matches the consensus pentameric HEXXH sequence of the catalytic domains in ACE and other metalloenzymes required to couple the Zn 2ϩ cofactor. Activation of B 1 receptors by ACE inhibitor leads to prolonged nitric oxide (NO) release from cultured endothelial cells (Ignjatovic et al., 2002), indicating that B 1 receptors contribute to the widel...

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Angiotensin I-converting enzyme (ACE) inhibitors are widely used to treat patients with cardiovascular and kidney diseases, but inhibition of ACE alone does not fully explain the beneficial effects. We reported that ACE inhibitors directly activate bradykinin B 1 receptor at the canonical Zn 2ϩ binding site, leading to prolonged nitric oxide (NO) production in endothelial cells. Protein kinase C (PKC) ⑀, a novel PKC isoform, is up-regulated in myocardium after infarction, suggesting a role in the development of cardiac dysfunction. In cytokine-treated human lung microvascular endothelial cells, B 1 receptor activation by ACE inhibitors (enalaprilat, quinaprilat) or peptide ligands (desArg 10 -Lys 1 -bradykinin, des-Arg 9 -bradykinin) inhibited PKC⑀ with an IC 50 ϭ 7 ϫ 10 Ϫ9 M. Despite the reported differences in binding affinity to receptor, the two peptide ligands were equally active, even when inhibitor blocked the cleavage of Lys 1 , thus the conversion by aminopeptidase. The synthetic undecapeptide (LLPHEAWHFAR) representing the binding site for ACE inhibitors on human B 1 receptors reduced PKC⑀ inhibition by enalaprilat but not by peptide agonist. A combination of inducible and endothelial NO synthase inhibitors, 1400W [N-(3(aminomethyl) benzyl) acetamidine dihydrochloride] and N-nitro-L-arginine (2 M), significantly reduced inhibition by enalaprilat (100 nM), whereas the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (100 M) inhibited PKC⑀ activity just as the B 1 ligands did. In conclusion, NO generated by B 1 receptor activation inhibits PKC⑀.