Caracterização da atividade biológica de fragmentos N-terminais da bradicinina

Abstract

The Kallikrein-Kinin System (KKS) is a complex peptide-based hormonal system acting on various tissues throughout the body, but its most relevant actions are on cardiovascular system and inflammation. Bradykinin [BK-(1-9)] and its biologically active metabolite des-Arg9-bradykinin [BK-(1-8)] are the known effectors of KKS and endogenous ligands of bradykinin receptors type 2 (B2) and type 1 (B1), respectively. Since the late 1970’s, all other metabolites of BK-(1-9) have been considered inactive. Thus, the main objective of this work is to evaluate whether some N-terminal fragments of BK-(1-9), namely BK-(1-7), BK-(1-5) and BK-(1-3) are, indeed, devoid of biological activity. In vitro NO quantification on DAF-FM diacetate loaded mouse, rat and human cells was employed to assess biological activity of BK-(1-9) metabolites. BK-(1-9), BK-(1-7), BK-(1-5) and BK-(1-3) were able to induce NO production in all cell types tested. The observed effects of BK-(1-7), BK-(1-5) and BK-(1-3) were not affected by kinin receptor antagonists nor by kinin receptor knockout mice. NO is one of the main mediators in vasodilation. Thus, it was very reasonable to presume that BK-(1-9) metabolites could also induce vasodilation. By vascular reactivity assays of rat thoracic aorta rings, it was observed that BK-(1-7), BK-(1-5) and BK-(1-3) were able to produce a dose-dependent vasodilation in rings pre-contracted by phenylephrine, while BK-(1-9) induced vasodilation in low concentrations and vasoconstriction in high concentrations. The mechanism by which vascular responses were mediated by BK-(1-9) and its metabolites are slightly different among them, but it was observed that these responses were not mediated by kinin receptors. Vasodilation is often translated in vivo in hypotension; thus, blood pressure of non-anesthetized rats was measured after administration of BK-(1-9) and its metabolites to assess whether theses peptides could reduce blood pressure. BK-(1-9) induced a transient dose-dependent hypotension while, and surprisingly, BK-(1-7), BK-(1-5) and BK-(1-3) were also able to promote transient hypotension, however, the observed effect was not dose-dependent. Biological activity of BK-(1-9) was also assessed by two important inflammatory components: vascular permeability and hypernociception. When administered in the footpad of mice, BK-(1-9) provoked an increase vascular permeability, while its metabolites did not induce this effect. Pain stimulus was observed in footpad of mice injected with BK-(1-9), BK-(1-7), BK-(1-5) or BK-(1-3), but the response of BK-(1-9) was almost 100% higher than of its metabolites. Taken together, these data indicate that BK-(1-7), BK-(1-5) and BK-(1-3) are biologically active in vitro in the most relevant species employed in pharmacological studies, by which were independent from kinin receptor activation. These peptides were also shown to have biological activity in vivo using cardiovascular and inflammatory models. Further studies are necessary to uncover the molecular target(s) and the mechanism of action of BK-(1-9) metabolites.