Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R−/−), B2- (B2R−/−) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R−/−). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R−/− presented similar level of vascular dysfunction as found in B1R−/− or B2R−/− mice. In accordance, aortic rings from B1R−/− or B2R−/− mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R−/− and B2R−/− mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R−/− or B2R−/− mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

B 1 -and B 2 -kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B 1 -(B 1 R −/− ), B 2 -(B 2 R −/− ) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B 1 B 2 R −/− ). For pharmacological studies, selective B 1 -and B 2 -kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B 1 -and B 2 -kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B 1 B 2 R −/− presented similar level of vascular dysfunction as found in B 1 R −/− or B 2 R −/− mice. In accordance, aortic rings from B 1 R −/− or B 2 R −/− mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B 1 R −/− and B 2 R −/− mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B 1 R −/− or B 2 R −/− mice was rescued by the SOD mimetic compound. Taken together, our findings show that B 1 -and B 2 -kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B 1 -and B 2 -kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.
Kallikrein mRNA and protein expression have been identified in blood vessels, which indicate the presence of KKS components in vascular tissue (Saed et al., 1990). KKS biologically active peptides act through two different types of G protein-coupled receptors, B 1 -and B 2 -kinin receptors (B 1 R and B 2 R), that are expressed at different molecular levels. In the vasculature, both receptors have been implicated in the activation of a wide spectrum of vasoactive pathways (Tsutsui et al., 2000;Felipe et al., 2007;Regoli et al., 2012). Indeed, B 2 R is ubiquitously expressed whereas B 1 R is virtually absent in healthy tissues, but its expression is rapidly increased under certain pathological conditions (McLean et al., 2000). However, in spite of the degree of expression, there are compelling evidence supporting that even barely expressed, B 1 R plays a physiological role and its activation induces vasoconstriction (Felipe et al., 2007) or vasorelaxation (Tsutsui et al., 2000) under unstressed conditions.
It is well-known that the endothelium-dependent vasodilatation in mouse aorta can be primarily attributed to NO· release as the result of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) activity (Pohl et al., 2000;Capettini et al., 2010), which are highly regulated by changes in intracellular Ca 2+ concentration, phosphorylation levels and, less known, by protein-protein interactions (Kone et al., 2003). Although previous studies have demonstrated molecular interactions between B 2 R/eNOS and B 2 R/nNOS (Ju et al., 1998;Golser et al., 2000), the functionality of such interactions in vascular tissue have not yet been described. Moreover, it has been shown that mice lacking B 1 -or B 2 -kinin receptors (B 1 R −/− or B 2 R −/− ) exhibit impairments in acetylcholine (ACh)-induced vasodilation (Loiola et al., 2011). Therefore, based on the above considerations, the present study was designed to evaluate the participation of kinin receptors in ACh-induced vascular relaxation, focusing on protein-protein interactions involving kinin receptors with eNOS and nNOS.

Animals
All experimental protocols were performed in accordance with the guidelines for the humane use of laboratory animals and were previously approved by the Animal Ethics Committee of the Federal University of Minas Gerais. Male B 1 R (B 1 R −/− ), B 2 R (B 2 R −/− ), double kinin receptors (B 1 B 2 R −/− ) knockout mice with their wild-type (WT) background controls (C57BL/6J, 10-to 14-wk-old, 25-30 g) were maintained in a temperature-controlled room (22-26 • C) on a 12 h light/dark cycle and free access to standard mouse chow and tap water.

Measurements of NO· Production and Superoxide Generation
NO· production in the aorta was determined using a fluorescent cell-permeable dye, 4,-amino-5 methylamino-2 ′ ,7 ′ -diaminofluorescein diacetate (DAF-FM, 10 µM; Molecular Probes) as previously described (Mota et al., 2015). Intracellular superoxide levels were assessed using dihydroethidium (DHE, 10 µM; Calbiochem). Freshly isolated aortas were incubated with dyes and loaded for 30 min at 37 • C in K-H solution and then washed for 30 min. Thoracic aorta segments were frozen and cut into sections that were 10 µm thick. For some experiments, fresh aortic rings from WT mice were incubated in the presence or absence of Des-Arg 10 HOE 140, HOE 140 (1 µM) or MnTMPyP (10 µM) for 30 min prior to ACh stimulation (10 µM, 30 min). Images were recorded using a fluorescence microscope (IX2-ICB, Olympus R , USA), and image analyses were performed in ImageJ software (NIH).

Statistical Analysis
All data are expressed as mean ± SEM. Vascular responses are expressed as % relaxation of the tone induced by phenylephrine. Significant differences between groups were determined using Two-way ANOVA followed by Bonferroni's post-hoc tests to compare the concentration-response curves obtained in aortic rings. Fluorescence microscopy images were analyzed according to the intensity of the fluorescence per area, both represented in arbitrary units (a.u.). The delta of the area under the curve was calculated as the difference between the concentration-response curves in the presence and the absence of MnTMPyP. One-way ANOVA followed by Bonferroni's post-hoc tests were used for all other analyses. All statistical comparisons were made using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) and values of P < 0.05 were considered to be statistically significant.

Protein-Protein Interactions between Constitutive NOS Isoforms and Kinin Receptors
In order to identify the existence of protein-protein interactions involving kinin receptors and constitutive NOS in native vascular tissue, thoracic aortas from WT mice were lysed and proteins were immunoprecipitated with anti-B 1 R, anti-B 2 R, anti-eNOS, and anti-nNOS antibodies. As shown in Figures 1A,B, the positive control, non-precipitated aortic lysate (input), show a strong signal at proper molecular weight, whereas IgG signal was barely detected ( Figure 1A) or absent ( Figure 1B) in samples immunoprecipitated with normal rabbit serum. Moreover, we show that eNOS ( Figure 1A) and nNOS ( Figure 1B) physically interact with B 1 -and B 2 -kinin receptors. We further validate our findings by performing opposite protein immunoprecipitation experiments (Figures 1C,D).

Vascular Reactivity
Based on our findings that both B 1 -and B 2 -kinin receptors are expressed and physically interact with nNOS and eNOS, we FIGURE 1 | Protein-protein interactions between constitutive NOS and kinin receptors. Thoracic aorta proteins of wild type mice were used for immunoprecipitation experiments (IP). (A,B) Non-precipitated aortic lysates was used as a positive control (input, 50 µg of protein), whereas immunoprecipitation with normal rabbit serum was used as an IgG control. Proteins were immunoprecipitated using anti-B 1 R or anti-B 2 R antibody followed by WB with anti-eNOS (A) or anti-nNOS (B). (C,D) Proteins were immunoprecipitated using anti-eNOS or anti-nNOS antibody followed by WB with anti-B 1 R (C) or anti-B 2 R (D). Data shown are representative of four separate experiments, each of which provided nearly identical results. next sought to investigate the functionality of these interactions. To address this question, we evaluated whether kinin receptors are involved in the endothelial vasodilator response to ACh, in which leads to vasorelaxation via NOS activation. As shown in the Figure 2, aortic rings exhibited concentrationdependent vasodilation in response to ACh, which was partially reduced by pre-incubation with the selective inhibitor of nNOS (TRIM ; Figures 2A,C) and markedly decreased by the non-selective NOS inhibitor (L-NNA; Figures 2B,D). To assess the contribution of B 1 -and B 2 -kinin receptors in the endothelium-dependent vasodilation response elicited by ACh, aortas were pre-incubated with either a selective B 1 R or B 2 R antagonist. Interestingly, blockage of B 1 R (Figures 2A,B) or B 2 R (Figures 2C, D) led to a significant reduction in ACh-induced vasorelaxation.
To better understand the individual contribution of eNOS and nNOS in the reduced vasorelaxation response to ACh upon B 1 -and B 2 -kinin receptor blockage, we performed experiments combining kinin receptor antagonists and NOS inhibitors. Our results show that pre-incubation with Des-Arg 10 HOE 140 in combination with L-NNA ( Figure 2B) or HOE 140 plus L-NNA ( Figure 2D) fully abolished the vasorelaxation induced by ACh. However, pre-incubation with TRIM in combination with kinin receptors antagonists did not potentiate the inhibitory effect of Des-Arg 10 HOE 140 (Figure 2A) or HOE 140 ( Figure 2C) on vasodilator responses.
To assess whether B 1 -and B 2 -kinin receptors affect endothelium-independent vasorelaxation, the NO· donor FIGURE 3 | Endothelium-independent vascular relaxation mediated by NO· donor (sodium nitroprusside, SNP) was not modified in aortic rings treated with B 1 -and B 2 -kinin receptor antagonists. Cumulative concentration-response curves for SNP were determined using an antagonist of the B 1 -kinin receptor (Des-Arg 10 HOE 140; 1 µM), B 2 -kinin receptor (HOE 140; 1 µM) or NOS inhibitor (L-NNA; 1 µM). The results are expressed as mean ± SEM for 4-6 experiments in each group.
SNP was used to induce relaxation in endothelium-denuded aortic rings. As shown in the Figure 3, pre-incubation of B 1 -and B 2 -kinin receptors antagonists did not change the SNP-induced relaxation. As expected, non-selective NOS inhibition did not affect the endothelium-independent relaxation induced by the NO· donor. Collectively, these findings suggest that blockade of kinin receptors affects ACh-induced vascular relaxation via impairment of nNOS activation.

Genetic Deletion of B 1 -or B 2 -Kinin Receptors Impairs ACh-Induced Vasorelaxation
To unequivocally demonstrate that kinin receptors contribute to the endothelium-dependent vasodilation elicited by ACh, we made use of knock-out mice that lack either B 1 -or B 2 -kinin receptors. As shown in Figure 4A, impaired AChinduced vasorelaxation was found in B 1 R −/− and B 2 R −/− mice compared with WT. Furthermore, substance P, another endothelium-dependent vasodilator via NOS activation, induced a concentration-dependent vasodilation in WT mice, which was significantly decreased in B 1 R −/− and B 2 R −/− (Figure 4B). Consistent with pharmacological approach, the selective inhibition of nNOS did not potentiate the impaired ACh-induced vasodilation in B 1 R −/− or B 2 R −/− mice whereas pre-incubation with L-NNA fully abolished the residual AChinduced vasodilation (Figures 4C,D). Moreover, impaired ACh-induced vasorelaxation was not amplified in B 1 B 2 R −/− (E max : 52.1 ± 6.5%, Figure 5) when compared with B 1 R −/− or B 2 R −/− (Table 1). Overall, these data validate our findings with pharmacological approach and indicate that uncoupled nNOS activity is the main cause of endothelial dysfunction in aorta of B 1 R −/− and B 2 R −/− mice. Cumulative concentration-response curves for ACh in WT (n = 15) and B 1 B 2 R −/− (n = 11) mice. ***P < 0.001.

Contribution of B 1 -or B 2 -Kinin Receptors on NO· Production and Superoxide Generation
Taking into account that oxidative stress is a major contributor and marker of endothelial dysfunction, we next investigated the superoxide generation in thoracic aortic rings. As shown in the Figure 6A, ACh was able to raise superoxide levels, in which were fully restored by the SOD-like membrane permeable compound, MnTMPyP, a superoxide scavenger. In line with reduced vasodilation, acute pharmacological blockage of B 1 -or B 2 -kinin receptors markedly potentiates DHE fluorescence when compared to ACh-stimulated aortic rings in the absence of kinin receptors antagonists ( Figure 6A). Moreover, we also show that NO· levels were significantly decreased in rings from B 1 R −/− and B 2 R −/− mice compared to WT ( Figure 6B). Accordingly, B 1 R −/− and B 2 R −/− aortas exhibited an increased superoxide levels when compared to WT ( Figure 6C). Altogether, our data show that reduced NO· bioavailability and greater ROS generation are linked with the impaired endothelium-dependent vasorelaxation of B 1 R −/− and B 2 R −/− mice.

SOD Mimetic MnTMPyP Rescues Impaired Endothelium-Dependent Vasorelaxation in Mice Lacking B 1 -or B 2 -Kinin Receptors
In order to confirm whether oxidative stress is involved in the endothelial dysfunction of B 1 R −/− and B 2 R −/− mice,  The results are expressed as mean ± SEM.
ACh-induced vasorelaxation curves were performed in the presence of MnTMPyP. As shown in the Figures 7A,B, AChinduced vasorelaxation was leftward shifted in MnTMPyPtreated aortic rings when compared to non-treated vessels of WT mice. Interestingly, in aortic rings from B 1 R −/− and B 2 R −/− mice, MnTMPyP restored the ACh-induced vasorelaxation (Figures 7A,B). The difference of the area under curve shows that the SOD mimetic enhanced ACh-induced vasorelaxation in all tested groups; however, B 1 R −/− and B 2 R −/− mice reached a significant enhancement when compared to WT mice ( Figure 7C). Taken together, these data support the oxidative pathogenesis hypothesis of the endothelial dysfunction found in B 1 R −/− and B 2 R −/− mice.

DISCUSSION
The main findings of this study are as follows: (1)  Kinin receptors have been reported to play a pivotal role in numerous vascular responses (Berthiaume et al., 1997;Felipe et al., 2007). Although it remains controversial whether B 1 R −/− and B 2 R −/− mice are normotensive (Milia et al., 2001;Trabold et al., 2002;Abadir et al., 2003) or hypertensive (Madeddu et al., 1997(Madeddu et al., , 1998Emanueli et al., 1998Emanueli et al., , 1999, these mice exhibit increased susceptibility to develop hypertension in response to different stressor stimuli (Alfie et al., 1997;Madeddu et al., 1997;Duka et al., 2001). Furthermore, it was previously demonstrated that mice lacking B 1 -or B 2 -kinin receptors display a decreased NO· bioavailability, despite the increased enzymatic  activity of the constitutive NOS isoform (Loiola et al., 2011). Thus, these findings indicate that the vascular NOS activity might be uncoupled in B 1 R −/− and B 2 R −/− mice. Moreover, whether ROS generation is involved, or not, in this vascular dysfunction remains unknown. Besides of these important issues, identification of the uncoupled NOS isoform represents another relevant question that could explain the underlying mechanisms and molecular basis of NOS uncoupling in B 1 R −/− and B 2 R −/− mice.
Ca 2+ -dependent NOS, which include eNOS and nNOS, are tightly regulated by several regulatory sites. Furthermore, new molecular regulators have been identified affecting their activities and, consequently, the amount of critical end-products for vascular function. Notably, protein-protein interactions have been proposed as an important mechanism that regulates the activity of NOS (Kone et al., 2003). Indeed, several studies have already described the functional relevance of physical interactions between NOS with a wide variety of structural and regulatory proteins such as, calmodulin (Rizzo et al., 1998), caveolin-1 (Ghosh et al., 1998), heat shock protein 90 (Russell et al., 2000), α1A adrenergic receptors (Pupo and Minneman, 2002), and angiotensin II receptor type 1 (AbdAlla et al., 2000). Although the heteromerization of B 2 -kinin receptor with nNOS and eNOS was previously reported (Ju et al., 1998;Golser et al., 2000), this study is the first to demonstrate B 1 R-eNOS/nNOS heteromerization. Moreover, although we are the first group to actually demonstrate such macromolecular protein complexes in native vascular tissue, if these interactions are functionally coupled to each other and, more importantly, whether this affects vascular function, it has remained unknown until now.
Here, we showed that eNOS and nNOS inhibition strongly decreases the ACh-induced vasorelaxation, confirming the large contribution of both constitutive NOS as the main source of NO· production. Intriguingly, pharmacologic antagonism or genetic ablation of B 1 -or B 2 -kinin receptors decreased the endothelium-dependent vasorelaxation response to ACh. Therefore, our results show that kinin receptors act as regulators of NOS activity in endothelial cells. To identify whether kinin receptors differently modulate eNOS and nNOS activities, the vasorelaxation response to ACh was assessed combining kinin receptors antagonists and NOS inhibitors. Our results demonstrated that B 1 -and B 2 -kinin receptors blockage fully abolished the relaxation upon non-selective NOS inhibition whereas nNOS inhibition did not potentiate the reduction of vasorelaxation induced by kinin receptors antagonism. In addition, we found similar pattern of vascular response in aorta from B 1 R −/− and B 2 R −/− mice. It is worth noting that non-selective NOS inhibition and B 1 -or B 2 -kinin receptors antagonists led to vascular contraction. These vascular responses further support that inhibition of the main vasodilation pathway favors contractile mechanisms, in which the oxidative stress is well-known to play a dysfunctional role in several vascular disorders (Johansson et al., 2005;Loria et al., 2014). Moreover, a reasonable explanation for selecting ACh as the agonist to trigger the vasorelaxation in the present study is the well-known NOS activation by muscarinic pathway whereas, the vascular response of agonists of kinin receptors is still controversial in aorta of different species, where it produces vasoconstriction (Levesque et al., 1993;Felipe et al., 2007) or vasodilation (Tsutsui et al., 2000). Furthermore, previous evidence has already demonstrated the absence of protein-protein interactions between kinin and muscarinic receptors (Willars et al., 1999). Importantly, we also validated the vascular dysfunction in B 1 R −/− and B 2 R −/− mice assessing the vasodilation induced by another well-known NOS activator, substance P. Taken together, our results show that B 1and B 2 -kinin receptors represent a functional regulatory domain of NOS activity. Moreover, our findings indicate that eNOS activity remained unaffected whereas the loss of nNOS function may represent a potential uncoupled activity.
Taking into account that B 1 R and B 2 R receptors are expressed in endothelial and smooth muscle layers (Leeb-Lundberg et al., 2005), kinin receptors may also affect the vascular relaxation by changing the responsiveness to NO· or, by acting on other targets resident in the vascular smooth muscle. To test this hypothesis, the vasodilator response to an exogenous NO· donor was used. Our results showed that kinin receptors did not affect the vasodilator response to NO· donor, which indicate a preserved smooth muscle function. Therefore, our findings strongly indicate that the endothelial dysfunction is the main cause of impaired vascular relaxation in aorta of B 1 R −/− and B 2 R −/− mice. Importantly, a cross-talk between B 1 R and B 2 R has been reported ( Barki-Harrington et al., 2003), indicating an interdependence with each other for the proliferative response. Moreover, it has been also shown that B 1 B 2 R −/− mice display protection against endotoxin-induced hypotension (Cayla et al., 2007) and obesity induced by high fat diet (Morais et al., 2015), while there are evidence showing amplified renal injury after ischemia/reperfusion (Kakoki et al., 2007) and increased nephropathy, neuropathy and bone mineral loss in Akita diabetic mice (Kakoki et al., 2010). Thus, in the present study we show that B 1 B 2 R −/− mice present similar level of vascular dysfunction as found in mice lacking B 1 R or B 2 R, and therefore ruling out a possible synergistic response between these receptors in this mechanism. Although still under debate whether endothelial dysfunction is a primary cause or consequence of vascular diseases, such vascular dysfunction is generally associated with decreased NO· bioavailability and increased superoxide generation which ultimately lead to an impaired endotheliumdependent vasorelaxation (Vanhoutte et al., 2017).
Under physiological conditions, the activity of eNOS is able to produce NO·, whereas nNOS activity produces NO· and hydrogen peroxide (H 2 O 2 ; Rosen et al., 2002;Gao et al., 2007). However, during certain pathological stimuli, the activity of eNOS and nNOS is drastically modified and becomes primarily uncoupled, producing a large amount of ROS, mainly, superoxide (Vasquez-Vivar et al., 1998;Weaver et al., 2005). Consistent with impaired vasodilation, acute blockage of kinin receptors markedly potentiate ACh-induced superoxide generation in aorta of WT mice. Additionally, we also demonstrated that B 1 R −/− and B 2 R −/− mice show a reduced NO· bioavailability associated with marked increase in intracellular levels of superoxide. Therefore, although the enhanced constitutive NOS activity in vascular tissue of B 1 R −/− and B 2 R −/− mice, our findings indicate that the uncoupled activity of nNOS represents a remarkable source of superoxide and, may be the major cause of the endothelial dysfunction in B 1 R −/− and B 2 R −/− mice.
Indeed, the oxidative stress is the major cause of endothelial dysfunction in several forms of vascular diseases, mainly via disruption of NO· signaling pathway (Drummond et al., 2011;Silva et al., 2016). Therefore, enhanced superoxide anion dismutation through the SOD mimetic can prevents the inactivation of NO· and formation of the highly reactive intermediate peroxynitrite (Weaver et al., 2005), and thus yielding augmented H 2 O 2 concentration, an endotheliumdependent relaxing factor in aorta (Capettini et al., 2008(Capettini et al., , 2010. Accordingly, the left-shifted ACh-induced vasodilation in WT may be explained by the above factors. Moreover, the stronger enhancement of vascular relaxation induced by the SOD mimetic in B 1 R −/− and B 2 R −/− mice than in WT mice further support our hypothesis of oxidative stress involvement in the endothelial dysfunction. These findings are consistent with previous studies, in which the exogenous SOD mimetic restores endothelium-dependent vasodilation due increased NO· bioavailability (Fontana et al., 1999;Braga et al., 2015). nNOS uncoupling has been implicated in several endothelium-dependent vascular disorders such as, in mesenteric artery of DOCA-salt hypertensive mice , thoracic aorta subjected to experimental atherosclerosis model (Capettini et al., 2011) and penile arteries of obese Zucker rat (Sánchez et al., 2012). Accordingly, deficient mice of B 1 -kinin receptor has been described to aggravate atherosclerosis and the development of abdominal aorta aneurysms in ApoE −/− mice under cholesterol rich-diet (Merino et al., 2009). B 2 -kinin receptor deficient mice also demonstrate higher blood pressure in response to chronic excess of angiotensin II or dietary salt (Madeddu et al., 1997) and chronic mineralocorticoid excess (Emanueli et al., 1998). Moreover, pharmacologic blockade or genetic disruption of the B 2 -kinin receptor accelerates the development of renovascular hypertension induced by clipping of the left renal artery (Madeddu et al., 1998). On the other hand, a recently study demonstrated that B 2 -kinin receptor antagonist inhibited AngII-induced neutrophil activation and inflammatory phenotype in ApoE −/− mice, thus suggesting B 2 -kinin receptor antagonism as potential therapy for abdominal aortic aneurysm (Moran et al., 2016). Therefore, further studies are necessary in disease models to test the hypothesis whether the conformational rearrangement of protein-protein interactions, involving kinin receptors and NOS isoforms, is relevant to vascular dysfunction.
In summary, we provide data supporting a novel implication of kinin receptors in the endothelial dysfunction. We report here that B 1 -and B 2 -kinin receptors regulate the endotheliumdependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interactions between B 1 -and B 2 -kinin receptors with nNOS is involved in the oxidative pathogenesis of endothelial dysfunction.

AUTHOR CONTRIBUTIONS
TM participated in all steps of this study. GC and IJ performed experiments. SL, LC, VL, JC, EC, JLP, and JBP contributed to the experimental design, data analyses, data interpretation, and the preparation and revision of the manuscript.