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.