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.