4. DISCUSSION
One of the most significant features of the KKS is that its major
agonist component, the nonapeptide BK-(1-9) is rapidly and extensively
inactivated in vitro or in vivo, in the presence of biological tissues.
Such rapid loss of biological activity is shared by many other mediators
and neurotransmitters, including NO, prostaglandins, angiotensin II and
acetylcholine, and provides an immediate target for pharmacological
intervention. Indeed, the isolation of BK-(1-9) (Elliott et al. ,
1960) was soon followed by the discovery and characterization of the
bradykinin potentiating factor (BPF) as a group of peptides that blocked
inactivation of the nonapeptide and potentiated its activities
(Ferreira, 1965; Ferreira et al. , 1970).
Our re-assessment of the biological activities of two peptide fragments
of BK has clearly shown that, in contrast to earlier results (Suzukiet al. , 1969; Park et al. , 1978; Redman et al. ,
1979; Roch-Arveiller et al. , 1983), BK-(1-7) and BK-(1-5) do
exhibit a range of biological activities, some of which are similar to
those of BK-(1-9) and others which are significantly different from
those of the parent nonapeptide. Although, as did BK-(1-9), the two
peptide fragments increased NO production in vitro, exhibited
vasorelaxant effects ex vivo and induced hypotension in vivo, these
biological activities were, unlike those of BK-(1-9), resistant to
antagonists of B1 or B2 receptors.
Moreover, whereas BK-(1-9) showed, as expected, increased nociception
and increased microvascular permeability, the two peptide fragments were
clearly less potent nociceptive agents and did not affect microvascular
permeability.
4.1. Which peptide fragments of BK-(1-9) are relevant in vivo?
Because BK-(1-9) is a substrate for a variety of peptidases,
aminopeptidases, carboxypeptidases and endopeptidases, an equal variety
of peptide fragments could be generated in vivo. This situation
immediately raises the question of which fragments are, in fact, the
most likely endogenous peptides formed from BK-(1-9) in vivo. In several
earlier studies, BK-(1-8), BK-(1-7) and BK-(1-5) were identified as the
major peptide fragments of BK-(1-9) (Murphey et al. , 2000;
Marshall et al. , 2002; Ahmad et al. , 2006; Ramirez-Molinaet al. , 2006; Kopylov et al. , 2016; Semis et al. ,
2019). In the present work, by monitoring the stable isotope-labelled
[Pro3(13C5;15N)]-BK-(1-9), we were able to detect in vivo
production of BK-(1-7) and BK-(1-5), after an infusion of BK-(1-9), thus
confirming that these fragments are endogenously produced BK-(1-9)
metabolites. Taking all the data together, we could demonstrate that
BK-(1-7) and BK-(1-5) were endogenously formed stable metabolites of
BK-(1-9) in plasma. As such, it is relevant in the context of the
overall response to BK-(1-9) in vivo, to assess the biological
activities of these two peptide fragments.
4.2. Earlier work on biological activities of BK-(1-9) fragments
The first evidence for important biological activity of a BK-(1-9)
metabolite was presented by (Regoli et al. , 1977) who found
activity in the octapeptide fragment, BK-(1-8) or
des-Arg9-BK, and who postulated the existence of two
kinin receptors, which was later confirmed by the cloning of the B2
receptor (McEachern et al. , 1991) and the B1receptor (Menke et al. , 1994). Whereas BK-(1-9) is an agonist at
both B1 and B2 receptors, its metabolite BK-(1-8) is a
selective agonist of B1 receptors. In our work, the
activity of either BK-(1-7) or BK-(1-5) on stimulating NO production and
inducing vasorelaxation was not affected by either B1 or
B2 receptor antagonists. Almost 15 years ago, BK-(1-5)
and BK-(1-9) were shown to inhibit α-thrombin-induced platelet
aggregation and secretion (Hasan et al. , 1996) but BK-(1-5)
appeared less potent than BK-(1-9) and both peptides were active at
higher concentrations than those we have used (0.1 – 1 mM). No further
experiments to identify the kinin receptors involved were reported.
Later work with BK-(1-5) showed this peptide to increase the survival of
rats in a sepsis model and to antagonize the effects of LPS on the
contractile response of aortic rings (Morinelli et al. , 2001).
The latter effect was observed at 1 nM, a concentration similar to those
we have used, and these authors discredited the involvement of either
B1 or B2 receptors although no data with
either receptor antagonist was provided. Moreover, these effects of
BK-(1-5) were not endothelium-dependent, excluding a possible mediation
by NO. In neither of these earlier reports was the heptapeptide BK-(1-7)
studied.
4.3. Effects on NO production in cells
We found significant stimulation by BK-(1-7) and BK-(1-5) of NO
production in vitro, using neonatal rat and adult mouse cardiomyocytes,
as well as human glioblastoma cells. We chose to study cardiomyocytes
because the parent peptide BK-(1-9) is known to induce NO production in
cardiac myocytes (Oldenburg et al. , 2004) and may contribute to
cardiac pre-conditioning (Schoemaker et al. , 2000; Heuschet al. , 2015). We decided to use an immortalized
glioblastoma-like cell line derived from humans (U-87 MG) as this cell
line expresses mRNA for both B1 and B2receptors (Uhlen et al. , 2017), which most likely translates to a
high density of these receptors in the cell membrane. Although the
concentrations of the peptides used were not physiological, they were
comparable to those used in other similar pharmacological studies, for
example, in reports of Ang-(1-7) (Gomes et al. , 2010), alamandine
(Jesus et al. , 2018) and BK-(1-9) actions in vitro (Oldenburget al. , 2004). We acknowledge that a concentration-response curve
was needed to confirm that BK-(1-9) fragments action on inducing NO
production is not a non-selective event and we were able to show that in
rat neonatal cardiomyocytes that BK-(1-9) and BK-(1-5) displayed
activity at the lowest concentration used (1 nM), whereas BK-(1-7) was
active at a higher concentration (10 nM). Taken together, these in vitro
results suggest that the two fragments of BK-(1-9) tested were
biologically active in stimulating NO production and that these
activities were most likely driven by receptor activation and not by a
non-specific interaction, given the nanomolar activity of BK-(1-7) and
BK-(1-5). Further studies are necessary to identify which target(s)
these molecules act upon.
4.4. Vascular effects ex vivo and in vivo
Blood pressure is controlled by complex mechanisms that modulate cardiac
output and peripheral vascular resistance (see (Guyenet, 2006). The
characteristic effect of BK-(1-9) in vivo is a marked hypotension, which
was first observed by (Rocha et al. , 1949) and later attributed
to vasodilation of systemic vessels, leading to a consequent reduction
of peripheral vascular resistance (Leeb-Lundberg et al. , 2005).
In our ex vivo experiments with aortic rings, the two fragments were as
effective as the parent nonapeptide, although the contribution of the
endothelium, NO and vasodilator prostanoids to this relaxation differed
between the three peptides, implying differences in the mechanisms of
vasorelaxation. Another important difference was that the
B1 or B2 receptor antagonists were
ineffective in blocking the vasorelaxation induced by the fragments, as
observed for the stimulation of NO production in cultured cells.
When BK-(1-9) was administered in conscious Wistar rats, we observed the
expected transient dose-dependent hypotensive response, while the two
fragments induced a transient but dose-independent hypotensive response.
The acute hypotensive response observed for BK-(1-7) and BK-(1-5),
although less prominent than that for BK-(1-9), was similar to that
observed for alamandine (Santos et al. , 2019), which is a known
cardiovascular modulator of the renin-angiotensin system. However, the
acute hypotensive effect mediated by BK-(1-7) and BK-(1-5) was not
altered by either ACE inhibition or by bypassing the pulmonary
circulation (as per i.a. administration), implying a resistance
of the two fragment peptides to ACE. This result may seem
counter-intuitive as ACE is the main enzyme responsible for the
metabolism, in vivo, of BK-(1-9) and some of its fragments (Kopylovet al. , 2016), but it is also known that BK-(1-9) is a substrate
for several other peptidases and it is highly likely in our in vivo
model, that both BK-(1-7) and BK-(1-5) are metabolized by peptidases
other than ACE. It is important that inhibitors of bradykininases other
than ACE are tested to evaluate precisely which enzymes contribute
significantly to the cleavage of these metabolites to even smaller
products.
4.5. Effects on inflammation
When BK-(1-9) or its fragments were administered to conscious rats, we
observed augmented locomotion, a sign of nociception. Nociception is
associated with inflammation and the KKS is known to play a major role
in this pathophysiological process (Leeb-Lundberg et al. , 2005;
Marceau et al. , 2020). To assess whether BK-(1-7) and BK-(1-5)
played any part in inflammatory events similar to BK-(1-9), we evaluated
their activities in two known inflammatory effects of the nonapeptide,
nociception (Cayla et al. , 2012) and increased microvascular
permeability (Kempe et al. , 2020). We observed that BK-(1-7) and
BK-(1-5) increased nociceptive reflexes in C57Bl/6 mice, but the two
fragments were significantly less effective than BK-(1-9). On the other
hand, we did not observe increased microvascular permeability mediated
by the two BK-(1-9) fragments. Our data suggest that BK-(1-7) and
BK-(1-5) would be less potent pro-inflammatory agents than BK-(1-9) and
that, in the context of inflammation, cleavage of the nonapeptide to
BK-(1-7) and BK-(1-5) would present as a reduction in pro-inflammatory
activity. Also, the lack of effects on microvascular permeability could
imply that hypotension induced by the fragments in vivo would not be
accompanied by oedema, as it is for the parent BK-(1-9). Overall, our
data suggest that these peptide fragments may have important outcomes
beyond the cardiovascular system, and further and more detailed
experiments are needed to evaluate their potential roles in nociception
and inflammation. On this regard, since the KSS seems to play an
important role in the current SARS-CoV-2 pandemic and its associated
disease, COVID-19 (van de Veerdonk et al. , 2020; Verano-Bragaet al. , 2020), and some COVID-19 symptoms like thrombosis, lung
inflammation and pulmonary edema may be a direct consequence of the
so-called “bradykinin storm” (Garvin et al. , 2020), the
relevance of BK-(1-7) and BK-(1-5) in COVID-19 should also be studied.