Hydrolysis of angiotensin peptides by human angiotensin I-converting enzyme and the resensitization of B2 kinin receptors

Abstract

We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I-converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B2 receptors coupled to green fluorescent protein (B2GFP) or to express only coupled B2GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B2 receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18x slower than Ang I and &30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although micromol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B2GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-alpha, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B2 activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B2 receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Figure 1

Peptides and enzymes that hydrolyze them. Arrows indicate the bonds cleaved by enzymes and the relative rate of hydorlysis (→) indicates rapid hydrolysi; (→); slow hydrolysis.

Figure 2

Resensitization of bradykinin B₂GFP receptors to ACE-resistant BKans. A, CHO/ABG cells were stimulated with 1 μmol/L BKan for 30 minutes to desensitize, then treated with medium alone, 1 μmol/L BKan, or 1 to 10 nmol/L Ang 1-7 or Ang 1-9 for 5 minutes; 1 μmol/L HOE 140 blocked resensitization. Ordinate: [³H]AA released by the peptides (5 minutes) relative to amount released by medium alone. Values are mean±SEM (n=8). *P<0.001; †P<0.01; experiments done in triplicate. B, CHO/ABG were desensitized with 10 μmol/L DidnsKBK, then resensitized to DidnsKBK in medium with 1 μmol/L Ang 1-9 or EPT (n=3). ‡P<0.001. HOE 140 blocked the effect (data not shown).

Figure 3

Effect of signal transduction inhibitors. CHO/ABG cells were incubated with 1 of the following inhibitors for 30 minutes: 1.0 μmol/L staurosporine, 0.5 μmol/L calphostin C, 1.0 μmol/L okadaic acid, 0.1 μmol/L calyculin A, or 0.1 μmol/L Gö976, washed, desensitized with BKan (1 μmol/L), then exposed to medium alone or 1 μmol/L BKan or 10 nmol/L Ang 1-9. Values are mean±SEM (n=3). *P<0.01 vs medium control. Kinase or phosphatase inhibitors blocked resensitization also by Ang 1-7 and enalaprilat (data not shown).

Figure 4

Effect of tyrosine kinase inhibitor. CHO/ABG cells were incubated with or without 20 μmol/L genistein, desensitized with BKan (1 μmol/L) followed by medium, 1 μmol/L BKan, 10 nmol/L Ang 1-7 or Ang 1-9, or enalaprilat for 5 minutes. Values are mean±SEM (n=4); *P<0.005 vs medium control. Tyrosine kinase inhibitor abolished resensitization by peptide or enalaprilat. EPT indicates enalaprilat.