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Background: The blood–brain barrier (BBB) describes the brain's highly specialized capillaries, which form a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, by preventing the entry of potentially harmful circulating molecules into the brain. However, this specialized function is challenging for the development of CNS therapeutics. Several strategies to facilitate drug delivery into the brain parenchyma via disruption of the BBB have been proposed. Bradykinin has proven effective in disrupting mechanisms across the blood–tumor barrier. Unfortunately, bradykinin has limited therapeutic value because of its short half-life and the undesirable biological activity elicited by its active metabolites. Objective: To evaluate NG291, a stable bradykinin analog, with selective agonist activity on the bradykinin-B2 receptor and its ability to disrupt the BBB transiently. Methods: Sprague Dawley rats and CD-1 mice were subjected to NG291 treatment (either 50 or 100 μg/kg, intravenously). Time and dose-dependent BBB disruption were evaluated by histological analysis of Evans blue (EB) extravasation. Transcellular and paracellular BBB leakage were assessed by infiltration of 99mTc-albumin (66.5 KDa) and 14C-sucrose (340 Da) radiolabeled probes into the brains of CD-1 mice treated with NG291. NG291 influence on P-glycoprotein (P-gp) efflux pump activity was evaluated by quantifying the brain accumulation of 3H-verapamil, a known P-gp substrate, in CD-1 mice. Results: NG291-mediated BBB disruption was localized, dose-dependent, and reversible as measured by EB extravasation. 99mTc-albumin leakage was significantly increased by 50 μg/kg of NG291, whereas 100 μg/kg of NG291 significantly augmented both 14C-sucrose and 99mTc-albumin leakage. NG291 enhanced P-gp efflux transporter activity and was unable to increase brain uptake of the P-gp substrate pralidoxime. NG291 did not evoke significant short-term neurotoxicity, as it did not increase brain water content, the number of Fluoro-Jade C positive cells, or astrocyte activation. Conclusion: Our findings strongly suggest that NG291 increases BBB permeability by two different mechanisms in a dose-dependent manner and increases P-gp efflux transport. This increased permeability may facilitate the penetration into the brain of therapeutic candidates that are not P-gp substrates. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are essential mediators of G protein‐coupled receptors (GPCRs) signalling to intracellular effectors. There is a considerable diversity of G protein subunits that channel signals initiated by GPCRs into specific outcome. In particular, mammalian genomes contain 16 conserved genes encoding G protein α subunits with unique properties. Of four Gα subfamilies (Gi/o, Gq, Gs and G12/13), members of the G12/13 group have received considerable attention for their roles in carcinogenesis. However, our ability to study activation of G12/13 by GPCRs with the power to distinguish between the two subunits is limited. Here, we present an adaptation of the bioluminescence resonance energy transfer (BRET)‐based assay to specifically monitor activity of Gα12 in living cells. In this kinetic assay, agonist‐induced release of Venus‐tagged Gβγ subunits from Gα12 is followed in real time using nano‐luciferase (Nluc)–tagged BRET donor. Using this assay, we characterized bradykinin B2 receptor (BDKRB2) and found that the receptor couples to Gα12 in addition to Gαo, and Gαq, but not to Gαs. We demonstrated the utility of this assay to quantify rates of G protein activation and inactivation as well as performing dose‐response studies while rank ordering signalling via individual Gα subunits. We further showed the utility of this assay to other GPCRs by demonstrating Gα12 coupling of cholecystokinin A receptor (CCKAR). Introduction of the Gα12‐coupling BRET assay is expected to accelerate characterization of GPCR actions on this understudied G protein. [ABSTRACT FROM AUTHOR]

Muscle damage is one of the most important factors that affect muscle fatigue during endurance exercise. Recent evidence suggests that the renin–angiotensin system impacts on skeletal muscle wasting. The aim of this study was to determine association between the AGT Met235Thr, ACE I/D and BDKRB2 −9/+9 polymorphisms with inflammation, myocardial and muscle injury induced by endurance exercise. Eighty-one Brazilian male runners participated in this study and completed the International Marathon of Sao Paulo. Muscle and myocardial damage markers (alanine transaminase, ALT, aspartate transaminase, AST, lactic dehydrogenase, LDH, creatine kinase, CK, Troponin, pro BNP, myoglobin, and CK-MB) and inflammatory mediators (IL-6, IL-8, IL-10, IL12p70, IL1β, and TNF-α) were determined one day before, immediately after, one day after, and three days after the event. Muscle damage was also determined fifteen days after race and angiotensinogen (AGT) Met235Thr, angiotensin-converting enzyme (ACE) I/D, and Bradykinin B2 receptor (BDKRB2) −9/+9 polymorphisms were determined. Marathon race participation induced an increase in all muscle damage and inflammatory markers evaluated (p < 0.0001). The muscle damage markers, troponin and pro BNP, CK and LDH and inflammatory markers, IL-6, IL-8, IL-1β and IL-10 were also higher in ACE II genotype immediately after race, compared to DD genotype. The percentage of runners higher responders (>500U/I) to CK levels was higher for II genotypes (69%) compared to DD and ID genotypes (38% and 40%, respectively) immediately after. Troponin, pro BNP and IL-1β, IL-8 levels were also elevated in AGT MM genotype compared to TT genotype athletes after and/or one day after race. BDKRB2 −9/−9 had pronounced response to LDH, CK, CK-MB and ALT and AST activities, myoglobin, troponin, IL-6, IL-8 levels immediately, one day and/or three days after race. The percentage of runners higher responders (>500U/I) to CK levels was greater for −9−9 and −9+9 genotypes (46 and 48%, respectively) compared to +9+9 genotypes (31%) immediately after. ACE II, AGT MM, and BDKRB2 −9−9 genotypes may increase the susceptibility to inflammation, muscle injury after endurance exercise and could be used to predict the development of clinical conditions associated with muscle damage and myocardial injury. [ABSTRACT FROM AUTHOR]

Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. [ABSTRACT FROM AUTHOR]

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that tissue kallikrein ( TK) protects against cerebral ischemia injury mainly through inhibition of apoptosis via bradykinin B2 receptor (B2R). In this study, we proposed that autophagy induction contributed to the neuroprotective mechanism of TK. To validate this hypothesis, we investigated TK-induced autophagy and its signaling mechanisms in human SH- SY5Y cells exposed to oxygen and glucose deprivation ( OGD). We found that TK treatment enhanced autophagy induction, reflected by augmented LC3 conversion and Beclin1 expression, decreased p62 levels and increased monomeric red fluorescent protein- LC3 puncta formation. Green fluorescent protein-monomeric red fluorescent protein- LC3 adenovirus assay indicated that TK maintained autophagic flux. Moreover, bafilomycin A1 (Baf.A1) caused obvious LC3- II accumulation either in the presence or absence of TK. Autophagy inhibition by Beclin1 knockdown or Baf.A1 treatment abrogated the neuroprotective effects of TK. Mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase ( ERK)1/2 and AMP-activated protein kinase ( AMPK)/tuberous sclerosis complex 2 ( TSC2)/mammalian target of rapamycin ( mTOR) signaling were induced by OGD stress and enhanced by TK. MEK/ ERK inhibitor U0126 alone elevated autophagy in OGD conditions, but impaired TK-induced autophagy. Blockade of AMPK/ TSC2/ mTOR signaling by AMPK inhibitor compound C and sh RNA mediated the knockdown of AMPK α1 and TSC2 but abolished autophagy in SH- SY5Y cells exposed to OGD treated either with or without TK. Moreover, B2R expression was up-regulated by OGD exposure. B2R knockdown attenuated autophagy and suppressed MEK1/2/ ERK1/2 and AMPK/ TSC2/ mTOR signaling in OGD conditions in either the presence or absence of TK. In sum, we revealed the significance of B2R-mediated MEK/ ERK and AMPK signaling in autophagy induction under OGD stress, and proposed novel mechanisms involved in the neuropotective function of TK through B2R-dependent regulation of autophagy. [ABSTRACT FROM AUTHOR]

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, and pain. This receptor attracts special attention as one of the factors that have cardioprotective and infarct-limiting effects. Certain genetic variants of the coding and noncoding regions of the bradykinin B2 receptor gene ( BDKRB2) may play a role in modulating its expression. The 3′-untranslated region of BDKRB2 exon 3 harbors a minisatellite repeat (B2-VNTR), which affects the mRNA stability. Hence, it is of interest to study a possible association of B2-VNRT alleles with various forms of coronary heart disease (CHD). In our work the allele and genotype frequency distributions of B2-VNTR were compared between healthy individuals and patients with CHD (angina pectoris or myocardial infarction (MI)) of the Russian ethnic group. Based on its length polymorphism, B2-VNTR was classed with low-polymorphic non-hypervariable minisatellites. Three B2-VNTR alleles, which consisted of 43, 38, and 33 repeats, were observed in all investigated cohorts. The alleles with 43 and 33 repeats were the most prevalent. The allele and genotype frequencies of B2-VNTR did not significantly differ between males and females in control group, and also between healthy males and males with angina pectoris or MI. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of CHD in males. However, we do not exclude the possibility of an association of the short B2-VNTR alleles (38 and 33 repeats) with a cardioprotective effect in females with CHD. This hypothesis requires further investigation. [ABSTRACT FROM AUTHOR]

Abstract: Bradykinin is a vasoactive peptide that participates in numerous inflammatory processes, vasodilation, and cell growth/survival; it mainly acts through two receptor subtypes, bradykinin B1 and bradykinin B2 receptors, which are G protein-coupled receptor (GPCR) family members. Details on ubiquitin-dependent degradation via the lysosome and/or proteasome, and the recycling process that directs bradykinin B2 receptor to the cell surface after agonist-induced endocytosis remain unclear; nevertheless, intracellular localization and internalization of GPCRs following stimulation by ligands are well known. Evidence concerning the nuclear localization and functions of GPCRs has been accumulating. The bradykinin B2 receptor has been shown to localize in the nucleus and suggested to function as a transcriptional regulator of specific genes. The transfer of membrane GPCRs (regardless of liganding), including the bradykinin B2 receptor to the nucleus can be attributed to the presence of a peptide sequence referred to as the nuclear localization signal (NLS). More recently, we found that nuclear bradykinin B2 receptors form heterodimers with the nuclear lamina protein, lamin C. The function of heterodimerization of the bradykinin B2 receptor with lamin C is still unclear. However, nuclear proteins lamin A/C are involved in a variety of diseases. Although further studies are required to elucidate the precise functions and mechanisms of intracellular and nuclear bradykinin B2 receptors, here we discuss the role of lamin A/C in laminopathies and examine the clinical significance of the bradykinin B2 receptor heterodimer. [Copyright &y& Elsevier]

Abstract: The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303–320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function. [Copyright &y& Elsevier]

Abstract: Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [3H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10−11 M) and α-SMA expression/polymerization at higher concentration (10−6 M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients. [Copyright &y& Elsevier]

Abstract: Bradykinin-induced interleukin (IL)-8 release should potentially activate neutrophils releasing myeloperoxidase (MPO) and subsequently generating “nitrosative stress”. We studied bradykinin-induced expression of bradykinin B2 receptor and bradykinin- and lipopolysaccharide (LPS)-induced IL-8 release, MPO (marker of neutrophil activation) and 3-nitrotyrosine (3-NT; marker of “nitrosative stress”) production in human bronchial epithelial cells BEAS-2B alone or in co-culture with human neutrophils. We evaluated B2 receptor protein expression in BEAS-2B cells by immunostainings and Western blot analysis, and measured respectively bradykinin- or LPS-induced IL-8 release in BEAS-2B cells and bradykinin- and/or LPS-induced MPO and 3-NT production in BEAS-2B cells co-cultured with human neutrophils by ELISA. In addition, we evaluated bradykinin- and/or LPS-induced 3-NT formation in BEAS-2B cells co-cultured with human neutrophils by immunocytochemistry. Bradykinin up-regulates B2 receptor expression (P<0.05) and stimulate IL-8 release (P<0.001) in BEAS-2B cells. Either the selective bradykinin B2 receptor antagonist HOE 140 or the selective bradykinin B1 receptor antagonist Lys-(des-Arg9, Leu8)-bradykinin alone halved IL-8 release and the combination of both drugs suppressed this effect. In BEAS-2B cells co-cultured with human neutrophils bradykinin increased MPO release and 3-NT production compared to BEAS-2B cells with human neutrophils (P<0.001), and the addition of LPS in BEAS-2B cells with human neutrophils and bradykinin induced a further dramatically increase of MPO release and 3-NT formation (P<0.001). Bradykinin and LPS provoked “nitrosative stress”, potentially mediated by IL-8, in bronchial epithelium co-cultured with neutrophils suggesting a role for bradykinin in the amplification of chronic airway inflammation via production of “nitrosative stress”. [Copyright &y& Elsevier]

BACKGROUND AND PURPOSE A 3H-labelled derivative of the novel small-molecule bradykinin (BK) B2 receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B2 receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [3H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. KEY RESULTS [3H]-JSM10292 bound to B2 receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B2 receptor agonists or antagonists were added. [3H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B2 receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [3H]-JSM10292 and competition experiments with unlabelled JSM10292 and [3H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. CONCLUSIONS AND IMPLICATIONS [3H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors. [ABSTRACT FROM AUTHOR]

Introduction. Bradykinin, a vasodilator by nature has been documented to have a protective role against hypertension and cardiovascular complications. Polymorphisms of bradykinin B2 receptor ( BDKRB2) gene are reported to be predisposing factors for hypertension. Evaluation of the association between −58C>T and ±9 bp polymorphisms of BDKRB2 with essential hypertension (EHT) was attempted. Methods. Two hundred and fourteen primary hypertensives and 249 controls were genotyped for the selected markers by polymerase chain reaction, gel electrophoresis (±9 bp), and SSCP (−58C>T). Results. While −58C>T polymorphism did not reveal any association with EHT, ±9 bp polymorphism showed a significant association with high risk for heterozygotes (++9/−9) when tested against the pooled frequencies of homozygotes (OR [odds ratio] == 1.63, 95%% confidence interval [CI] == 1.12-2.38, P == .02), and this risk was 1.7 folds high in males (OR == 1.74, 95%% CI == 1.05-2.86, P == .06) and 1.9 folds high in familial cases (OR == 1.96, 95%% CI == 1.09-3.53, P == .04). In contrast, significant protective effect was observed for −9/-9 genotype against EHT when tested under dominant model in general (OR == 0.59, 95%% CI == 0.41-0.86, P == .01), in males (OR == 0.49, 95%% CI == 0.30-0.82, P == .01), and in familial cases (OR == 0.50, 95%% CI == 0.28-0.89, P == .04). Significant risk for ++9 bp allele was observed in general (OR == 1.39, 95%% CI == 1.05-1.86, P == .04) and in males (OR == 1.65, 95%% CI == 1.13-2.41, P == .02). The interaction information analysis revealed a synergistic effect between the two polymorphisms contributing to EHT. ++9/++9 genotype of ±9 bp polymorphism when present in combination with CC genotype of −58C>T polymorphism showed 2.2-fold higher risk for developing EHT. Conclusions. The results suggest that allele ++9 bp might be a risk factor for EHT in general and specially in males. Markers −58C>T and ±9 bp may act synergistically causing susceptibility to EHT. [ABSTRACT FROM AUTHOR]

Abstract: The plasma contact activation (CAS) and kallikrein/kinin (KKS) systems consist of 4 proteins: factor XII, prekallikrein, high molecular weight kininogen, and the bradykinin B2 receptor. Murine genetic deletion of factor XII (F12-/- ), prekallikrein (Klkb1-/- ), high molecular weight kininogen (Kgn1-/- ) and the bradykinin B2 receptor (Bdkrb2-/- ) yield animals protected from thrombosis. With possible exception of F12-/- and Kgn1-/- mice, the mechanism(s) for thrombosis protection is not reduced contact activation. Bdkrb2-/- mice are best characterized and they are protected from thrombosis through over expression of components of the renin angiotensin system (RAS) leading to elevated prostacyclin with vascular and platelet inhibition. Alternatively, prolylcarboxypeptidase, a PK activator and degrader of angiotensin II, when deficient in the mouse leads to a prothrombotic state. Its mechanism for increased thrombosis also is mediated in part by components of the RAS. These observations suggest that thrombosis in mice of the CAS and KKS are mediated in part through the RAS and independent of reduced contact activation. [Copyright &y& Elsevier]