2.6 Vascular reactivity
Vascular reactivity of BK-(1-9) and its fragments were assessed as
previously described (Rocha-Resende et al. , 2017). Briefly, 8-12
weeks old male Wistar rats were killed by decapitation. The thoracic
aorta was quickly dissected, connective and adipose tissues were removed
and then the vessel was cut into 3-5mm wide rings. The rings were
carefully placed between two stainless-steel stirrups and coupled to an
isometric force transducer. The preparations were kept at 37°C, bathed
in Krebs-Henseleit salt solution (118 mM NaCl; 4.7 mM KCl; 1.17 mM
KH2PO4; 1.12 mM
MgSO4.7H2O; 11.2 mM glucose; 25 mM
NaHCO3; 2.5 mM
CaCl2.2H2O; pH = 7.3), lightly bubbled
with 95% O2 / 5% CO2. Endothelial
cells of some aortic rings were removed by gently rubbing the rings with
a thin wire, to evaluate the contribution of endothelial cells to the
vasorelaxation. Aortic rings were stabilized for at least 60 minutes
under a passive tension of 1.6 g. During the stabilization period,
Krebs-Henseleit solution was replaced every 15 minutes and passive
tension was corrected to 1.6 g if needed. Endothelium integrity was
assessed by pre-contracting the rings with phenylephrine (100 nM),
dissolved in saline. After stabilization of contraction, acetylcholine
(ACh, 1 µM) was added to induce endothelium-dependent vasorelaxation.
Endothelium was considered intact when vasorelaxation mediated by this
concentration of ACh was greater than 80%. For experiments using rings
without functional endothelium [E (-)], only preparations with no
response to ACh were used.
Vasorelaxation mediated by BK-(1-9), BK-(1-7) and BK-(1-5) was assessed
in endothelium-intact rings by constructing a concentration-response
curve for each peptide, after pre-contraction of rings with
phenylephrine (100 nM). Peptide concentrations tested ranged from 1 pM
(10-12 M) to 1µM (10-6 M). A vehicle
(saline) curve was also generated by adding the same volume of saline to
the rings, as used with the peptides, as a negative control. Endothelium
dependency to those effects was assessed in denuded rings (E (-)). The
contribution of NO and prostanoids to the vasorelaxation response was
assessed by blocking nitric oxide synthases (NOS) and cyclooxygenases
(COX) with Nω-nitro-L-arginine methyl ester (L-NAME) and
indomethacin, respectively. Both inhibitors were used at a final
concentration of 1 µM. L-NAME was dissolved in saline and indomethacin
was dissolved in DMSO, with further dilutions in saline. The involvement
of kinin receptors in vasorelaxation induced by BK-(1-9), BK-(1-7) or
BK-(1-5), was assessed by selectively blocking B1 or
B2 receptors with
Lys-(des-Arg9-Leu8)-BK-(1-9), or
HOE-140, respectively (final concentration 100 nM). Inhibitors and
antagonists were incubated with preparations for 20 minutes before
contraction by phenylephrine.