D-Arg(0)-Bradykinin-Arg-Arg, a Latent Vasoactive Bradykinin B(2) Receptor Agonist Metabolically Activated by Carboxypeptidases

We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B(2) receptor (B(2)R) agonists activated by vascular or plasma peptidases. D-Arg(0)-BK-Arg-Arg (r-BK-RR) is a novel prodrug peptide hypothetically activated by two catalytic cycles of Arg-carboxypeptidases (CPs) to release the direct agonist D-Arg(0)-BK. N-terminally extending the BK sequence with D-Arg(0) in the latter peptide was meant to block the second kinin inactivation pathway in importance, aminopeptidase P. The affinity of r-BK and r-BK-RR for recombinant B(2)R was assessed using a [(3)H]BK binding displacement assay. Their pharmacology was evaluated in human isolated umbilical vein, a contractile bioassay for the B(2)R, in a morphological assay involving the endocytosis of B(2)R-green fusion protein (GFP) and in anesthetized rats instrumented to record hemodynamic responses to bolus intravenous injection of both peptides. r-BK exhibited an affinity equal to that of BK for the rat B(2)R, while r-BK-RR was 61-fold less potent. In the vein and the B(2)R-GFP internalization assay, r-BK was a direct agonist unaffected by the blockade of angiotensin converting enzyme (ACE) with enalaprilat, or Arg-CPs with Plummer’s inhibitor. However, the in vitro effects of r-BK-RR were reduced by these inhibitors, more so by enalaprilat. In anesthetized rats, r-BK and r-BK-RR were equipotent hypotensive agents and their effects were inhibited by icatibant (a B(2)R antagonist). The hypotensive effects of r-BK were potentiated by enalaprilat, but not influenced by the Arg-CPs inhibitor, which is consistent with a minor role of Arg-CPs in the metabolism of r-BK. However, in rats pretreated with both enalaprilat and Plummer’s inhibitor, the hypotensive responses and the duration of the hypotensive episode to r-BK were significantly potentiated. The hypotensive responses to r-BK-RR were not affected by enalaprilat, but were reduced by pre-treatment with the Arg-CPs inhibitor alone or combined with enalaprilat. Therefore, in vivo, Arg-CPs activity is dominant over ACE to regenerate the B(2)R agonist r-BK from r-BK-RR, a prodrug activator of the B(2)R. A B(2)R agonist activated only at the level of the microcirculation by resident peptidases could be developed as an intravenously infused drug for ischemic diseases.