AUTHOR=Bachelard Hélène , Charest-Morin Xavier , Marceau François 

TITLE=D-Arg0-Bradykinin-Arg-Arg, a Latent Vasoactive Bradykinin B2 Receptor Agonist Metabolically Activated by Carboxypeptidases

JOURNAL=Frontiers in Pharmacology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2018.00273

DOI=10.3389/fphar.2018.00273

ISSN=1663-9812

ABSTRACT=<p>We previously reported hypotensive and vasodilator effects from C-terminally extended bradykinin (BK) sequences that behave as B<sub>2</sub> receptor (B<sub>2</sub>R) agonists activated by vascular or plasma peptidases. D-Arg<sup>0</sup>-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<sup>0</sup>-BK. N-terminally extending the BK sequence with D-Arg<sup>0</sup> 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<sub>2</sub>R was assessed using a [<sup>3</sup>H]BK binding displacement assay. Their pharmacology was evaluated in human isolated umbilical vein, a contractile bioassay for the B<sub>2</sub>R, in a morphological assay involving the endocytosis of B<sub>2</sub>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<sub>2</sub>R, while r-BK-RR was 61-fold less potent. In the vein and the B<sub>2</sub>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 <italic>in vitro</italic> 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<sub>2</sub>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, <italic>in vivo</italic>, Arg-CPs activity is dominant over ACE to regenerate the B<sub>2</sub>R agonist r-BK from r-BK-RR, a prodrug activator of the B<sub>2</sub>R. A B<sub>2</sub>R agonist activated only at the level of the microcirculation by resident peptidases could be developed as an intravenously infused drug for ischemic diseases.</p>