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

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 B-2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B-2 receptors coupled to green fluorescent protein (B(2)GFP) or to express only coupled B(2)GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B-2 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 approximate to 30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although mu mol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1- 9 inhibit ACE, they resensitize the desensitized B(2)GFP 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 B-2 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 B-2 activation by bradykinin. Ang 1- 9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B-2 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.