1. INTRODUCTION
Bradykinin [BK-(1-9)] is a nonapeptide of the kallikrein-kinin system (KKS) and was first characterized over 70 years ago (Rochaet al. , 1949), as a potent hypotensive agent in vivo, and to induce a slow contraction of guinea-pig uterus (hencebrady kinin). This biological activity was generated by incubating ox plasma with snake venom (from Bothrops jararaca ) or trypsin. A decade later, the nonapeptide BK-(1-9) was purified by (Elliott et al. , 1960) and shown to induce hypotension, slow contraction of smooth muscle and pro-inflammatory effects. Since then, our knowledge of the biochemistry, physiology, and pharmacology of the KKS and BK-(1-9) has greatly increased. In the present context the most important features include (i) the inactivation of BK-(1-9) by plasma kininases I and II (Yang et al. , 1970), (ii) the rapid and extensive inactivation of BK-(1-9) in the pulmonary circulation (Ferreira et al. , 1967), later associated with the angiotensin-converting enzyme (ACE) (Bakhle, 1968; Ferreira et al. , 1970); ii) elucidation of two proteoforms of kininogens (Habal et al. , 1976); iii) identification of des-Arg9-BK [BK-(1-8)] as a biologically active component of KKS (Regoli et al. , 1977); iv) cDNA cloning of the constitutive B2 receptor, a selective target for BK-(1-9) (McEachern et al. , 1991); v) cDNA cloning of B1 receptor, a selective target for BK-(1-8) (Menkeet al. , 1994) and (vi) FDA approval of the selective B2 receptor antagonist icatibant (Cicardi et al. , 2010) as a treatment for hereditary angiodema.
The bioactivities of the peptide fragments of BK-(1-9) have been mostly overlooked for the past 40 years. In the late 1960s, BK-(1-9) was shown to have a biological half-life of about 17 seconds in blood, being extensively metabolized in a single pass on the pulmonary circulation (clearance of about 80%) (Ferreira et al. , 1967; Ryan et al. , 1968). Given this short half-life, several studies (Suzukiet al. , 1969; Regoli et al. , 1977; Park et al. , 1978; Redman et al. , 1979; Roch-Arveiller et al. , 1983) aimed at characterizing the potential activity of the fragments of BK-(1-9) in different species and using different protocols. However, the overall conclusion was that the fragments derived from BK-(1-9) were devoid of biological activity, except for the BK-(1-8) (des-Arg9-BK).
Over the last 20 years, several new biological activities for BK-(1-9) have been uncovered (Regoli et al. , 2015; Regoli et al. , 2016), particularly its ability to generate NO, which have considerably increased the range of effects that could be mediated by the nonapeptide. However, the fragments of BK-(1-9) have not been assessed for these new, potential, activities. One problem inherent in such re-assessment of the fragments is the choice of the peptides to study. BK-(1-9) is a substrate for carboxypeptidases (e.g., ACE and ACE2), aminopeptidases (e.g., aminopeptidase P) and endopeptidases (e.g., neutral endopeptidase, now neprilysin), thus generating many different cleavage products (Campbell, 2013; Verano-Braga et al. , 2020). A general conclusion of BK-(1-9) metabolism is that its major proteolytic fragment is the BK-(1-8), which is acknowledged as the only biologically active BK peptide known to date, together with BK-(1-7) and BK-(1-5) that are regarded as biologically inert fragments (Kopylov et al. , 2016; Semis et al. , 2019).
Thus, we present here a re-evaluation of the biological activities exhibited by BK-(1-7) and BK-(1-5), compared with those of the parent nonapeptide BK-(1-9), in a range of in vitro, ex vivo and in vivo systems, using human and rodent tissues. Our results show that, contrary to previous results, these BK-(1-9) fragments are indeed biologically active, stimulating NO production in vitro, and inducing vasorelaxation ex vivo. Such actions were not mediated by either of the kinin B1 or B2 receptors. We also reportin vivo actions of the BK-(1-9) peptide fragments in cardiovascular parameters (arterial blood pressure and heart rate), and under inflammatory conditions.