Angiotensin 1-9 and 1-7 release in human heart: role of cathepsin A.

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A's presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B(2) receptor in Chinese hamster ovary cells transfected to express human ACE and B(2) (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B(2) receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC(50)s) with ACE. They probably induce conformational changes in the ACE/B(2) receptor complex via interaction with ACE.

Neprilysin inhibitors potentiate effects of bradykinin on b2 receptor.

Some beneficial effects of angiotensin-I--converting enzyme (ACE, kininase II) inhibitor therapy are attributed to enhancing the activity of bradykinin on its B(2) receptor. Independent of inhibition of bradykinin hydrolysis, ACE inhibitors enhance the action of bradykinin on its B(2) receptor by inducing crosstalk between ACE and the receptor. We investigated whether inhibitors of another kininase II-type enzyme, neprilysin (neutral endopeptidase 24.11; NEP), could augment bradykinin effects unrelated to blocking its breakdown using a NEP-resistant bradykinin analog as ligand. We used transfected Chinese hamster ovary (CHO) cells stably expressing human B(2) receptor and NEP (CHO/NEP-B(2)) or only B(2) (CHO/B(2)) as control and human pulmonary fibroblasts (IMR90), expressing B(2), but more NEP than ACE. NEP inhibitor phosphoramidon (100 nmol/L), or omapatrilat, which inhibits both NEP and ACE, did not potentiate bradykinin in CHO/B(2) cells. In IMR90 cells, 10 nmol/L bradykinin elevated [Ca(2+)](i) and desensitized the receptor. Adding either 100 nmol/L omapatrilat or phosphoramidon resensitized the receptor to the ligand, which was abolished by receptor blocker HOE 140. Arachidonic acid release by bradykinin from CHO/NEP-B(2) cells was also augmented by 100 nmol/L phosphoramidon or omapatrilat about 3-fold, and again, the inhibitors resensitized the desensitized B(2) receptor. The inhibitors did not potentiate bradykinin when soluble rNEP was added to the medium of CHO/B(2) cells. Similar to ACE, NEP inhibitors potentiated bradykinin independent of inhibiting inactivation. Consequently, omapatrilat could augment bradykinin effects on B(2), when either ACE or NEP is expressed close to receptor on cell membrane.