Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Discovery of dihydroquinoxalinone acetamides containing bicyclic amines as potent Bradykinin B1 receptor antagonists

Replacement of the core beta-amino acid in our previously reported piperidine acetic acid and beta-phenylalanine-based Bradykinin B1 antagonists by dihydroquinoxalinone acetic acid increases the in vitro potency and metabolic stability. The most potent compounds from this series have IC(50)s<0.2 nM in a human B1 receptor functional assay. A molecular modeling study of the binding modes of key compounds, based on a B1 homology model, explains the structure-activity relationship (SAR) for these analogs.     

Differential role of kinin B1 and B2 receptors in ischemia-induced apoptosis and ventricular remodeling

We investigated the role of kinin receptors in cardiac remodeling after ischemia/reperfusion (I/R). Bradykinin injection improved cardiac contractility, diastolic function, reduced infarct size and prevented left ventricular thinning after I/R, whereas des-Arg(9)-BK injection had no protective effects. Bradykinin, but not des-Arg(9)-BK, reduced cardiomyocyte apoptosis and increased Akt and GSK-3beta phosphorylation. Furthermore, myocardial infarct size was similar between wild type and B2 knockout mice after I/R, but significantly reduced in kinin B1 receptor knockout mice. These results indicate that the kinin B2 receptor, but not the B1 receptor, protects against I/R-induced cardiac dysfunction by inhibiting apoptosis and limiting ventricular remodeling.     

Balance between the two kinin receptors in the progression of experimental focal and segmental glomerulosclerosis in mice

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). In order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. The blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease.KEY WORDS: Focal and segmental glomerulosclerosis, Bradykinin receptors, Inflammation, Podocyte, Fibrosis     

Expression of bradykinin B2 receptor in the mouse ovary

The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B(2) receptor mRNA was constitutively expressed. Bradykinin B(2) receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B(2) receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B(2) receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B(2) receptor mRNA level. The present data indicate that the level of B(2) receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.     

Regulation of kinin B(2) receptors by bradykinin in human lung cells

Bradykinin is a potent mediator of inflammation that has been shown to participate in allergic airway inflammation. The biologic effects of bradykinin are mediated by binding and activation of its cognate receptor, the B(2) receptor (B(2)R). In the lung fibroblast cell line IMR-90, binding of bradykinin to B(2)R triggers down-regulation of receptor surface expression, suggesting that bradykinin-induced inflammation is transient and self-limited. Notably, subjects with chronic airway inflammation continue to respond to BK following a first challenge. B(2)Rs are expressed on many different lung cell types, including airway epithelial cells. We therefore compared IMR-90 cells with the human lung epithelial cell line BEAS2B and found that B(2)R expression in the two cell types is differently regulated by BK. Whereas BK induces down-regulation of B(2)R in IMR-90 cells, the same treatment leads to up-regulation of the receptor in BEAS2B cells. These results provide a possible explanation for the potency of bradykinin in inducing ongoing airway inflammation.     

Bradykinin receptor antagonists attenuate neointimal proliferation postangioplasty

Bradykinin has been linked to the development of restenosis in response to vascular injury. We therefore examined the effect of bradykinin on vascular smooth muscle cell growth and neointimal formation in organ culture. Bradykinin stimulated both RNA and DNA synthesis (by 175%) in smooth muscle cells from either porcine or human coronary arteries and increased cell number in a concentration-dependent manner. Both p42/44 mitogen-activated protein kinase (MAPK) and p38 kinase were also activated. Treatment with [Hyp(3),Tyr(Me)(8)]bradykinin, a B(2) receptor agonist, stimulated thymidine incorporation by 146%, whereas B(1)-selective Lys-des-Arg(9)-bradykinin had no effect. Addition of the B(2) antagonist HOE-140 reduced the stimulation by 56%, whereas B(1)-selective des-Arg-HOE-140 had no significant effect. Similarly, HOE-140 attenuated angioplasty-induced neointimal formation in organ culture with an efficacy approaching 100% inhibition. These experiments suggest that bradykinin promotes smooth muscle proliferation after vascular injury, presumably via B(2) receptor-dependent activation of MAPK family pathways, and may explain the negative outcome of angiotensin converting enzyme inhibitor therapy on restenosis in nonrodent models.     

研究报告：缓激肽上调豚鼠肠道黏膜下神经丛环氧合酶2的表达

目的缓激肽和缓激肽B2受体在肠神经系统中起重要作用。缓激肽通常参与肠道的炎症反应和神经保护,这种作用取决于缓激肽诱导前列腺素的形成。环氧合酶1 (COX1)和环氧合酶2 (COX2)催化花生四烯酸转化为前列腺素。本研究旨在探讨缓激肽刺激对豚鼠肠神经前列腺素E2 (p GE2)释放和COX2表达的影响及信号机制。方法本文通过免疫荧光检测肠神经细胞中COX2与神经细胞标志物Anti-Hu和ch AT的表达;采用PCR及蛋白质印迹(Western blot)检测不同条件下缓激肽刺激对COX2表达的影响;使用缓激肽B1受体的选择性拮抗剂Leu-8和B2受体的选择性拮抗剂HOE-140,研究缓激肽影响COX2表达的信号机制;利用COX2选择性拮抗剂NS398和COX1拮抗剂FR12207,观察COX2在缓激肽诱导p EG2释放的作用。结果 COX2与神经细胞标志物Anti-Hu和ch AT在肠神经细胞上共同表达,缓激肽可通过B2受体诱导肠神经细胞COX2的表达。缓激肽刺激引起的肠神经细胞p GE2的释放与COX2表达升高密切相关。结论缓激肽通过B2R影响肠道黏膜下神经丛COX2的表达,肠道缓激肽...     

Response to: The BRAIN TRIAL: a randomized, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury - authors' reply

A response to the letter regarding The BRAIN TRIAL: a randomized, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury, by Mr Vincent Simmon President and CEO of Xytis Inc.     

Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system

Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 receptor (B2R). B2R, a member of the G protein-coupled receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The recombinant receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the recombinant receptor, which indicated the activation of endogenous G alpha(q) protein by the recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the recombinant receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged recombinant B2R been expressed at high levels in BHK cells and extensively characterized.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Decreased renal NO excretion and reduced glomerular tuft area in mice lacking the bradykinin B2 receptor

Bradykinin B(2) receptor knockout mice (B(2)-/-) have been useful to study the role of bradykinin under pathological conditions. With the use of these mice, it was shown that bradykinin plays an important role in angiogenesis, heart failure, salt-induced hypertension, and kidney fibrosis. Data on the role of the bradykinin B(2) receptor under physiological conditions using these mice are controversial and scarce, because these mice have no typical phenotype. For this reason, we have studied, under physiological conditions, renal hemodynamics as well as a number of morphometric glomerular parameters of B(2)-/- mice on a homogenized genetic background and on mice bred in a pathogen-free environment. Backcrossed B(2)-/- mice had normal blood pressure and normal apparent renal hemodynamics and morphology. However, reduced renal nitrite excretion and glomerular cGMP content were found, which was associated with a reduced glomerular capillary surface area. These differences had, however, no detectable effects on renal hemodynamics. These differences between B(2)-/- and wild-type mice might become important under pathological conditions as shown by a number of studies using these bradykinin B(2) receptor knockout mice.     

Is the expression of kinin B(1) receptor related to intrahepatic vascular response?

Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B(2) receptor (B(2)R). The biological effects of kinins may also be mediated by the inducible B(1) receptor (B(1)R). AIM: To verify if the hepatic B(1)R expression modulates IHVR to kinins. METHOD: We evaluated the ability of bradykinin and B(1)R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. RESULTS: Bradykinin-induced IHVR was similar in all groups. B(1)R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B(1) receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. CONCLUSION: B(1)R hepatic expression did not modulate IHVR to kinins.     

Bradykinin antagonists: new opportunities

The pro-inflammatory, pain producing, and cardiovascular effects of bradykinin B2 receptor activation are well characterized. Bradykinin B1 receptors also produce inflammation and pain. Therefore, antagonists are expected to be anti-inflammatory/analgesic drugs. Other exploitable clinical opportunities may exist. The newly discovered non-peptide B2 receptor antagonists and the equivalent B1 receptor pharmacological agents, which are in the pipeline, are suitable preclinical tools to properly evaluate potential utilities.     

Bradykinin augments fibroblast-mediated contraction of released collagen gels

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.     

Bradykinin protects cardiac c-kit positive cells from high-glucose-induced senescence through B2 receptor signaling pathway

Cardiac c-kit positive cells are cardiac-derived cells that exist within the heart and have a great many protective effects. The senescence of cardiac c-kit positive cells probably leads to cell dysfunction. Bradykinin plays a key role in cell protection. However, whether bradykinin prevents cardiac c-kit positive cells from high-glucose-induced senescence is unknown. Here, we found that glucose treatment causes the premature senescence of cardiac c-kit positive cells. Bradykinin B2 receptor (B2R) expression was declined by glucose-induced senescence. Bradykinin treatment inhibited senescence and reduced intracellular oxygen radicals according to senescence-associated β-galactosidase staining and 2′,7′-dichlorodihydrofluorescein diacetate staining. Moreover, the mitochondrial membrane potential was damaged, as measured by JC-1 staining. The mitochondrial membrane potential was preserved under bradykinin treatment. The concentration of superoxide was decreased, and the concentration of intracellular adenosine triphosphate was increased after bradykinin treatment. Western blot showed that bradykinin leads to AKT and mammalian target of rapamycin (mTOR) phosphorylation and decreased levels of P53 and P16 when compared with glucose treatment alone. Antagonists of B2R, phosphoinositide 3-kinase (PI3K), mTOR, and B2R small interfering RNA prevented the protective effect of bradykinin. P53 antagonist also inhibited the glucose-induced senescence of cardiac c-kit positive cells. In conclusion, bradykinin prevents the glucose-induced premature senescence of cardiac c-kit positive cells through the B2R/PI3K/AKT/mTOR/P53 signal pathways.     

B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.     

Synthesis and analysis of potent, more lipophilic derivatives of the bradykinin B2 receptor antagonist peptide Hoe 140.

Bradykinin (BK) is an endogenous peptide that has been implicated in several pathological conditions, hence antagonists of its activity have therapeutic potential. The decapeptide Hoe 140 is currently one of the best BK antagonists, but interest remains in finding even more potent compounds. A library of Hoe 140 derivatives was synthesized that incorporated non-natural analogs of the cationic, naturally occurring amino acids arginine (Arg) and lysine (Lys). The modified amino acids were designed to form enhanced ionic interactions due to an increase in local hydrophobicity, which promotes desolvation of the cation in water. The potencies of the resulting peptides were determined by competitive binding assays in human A431 cells expressing the BK B2 receptor. Two of the peptides synthesized were equipotent to Hoe 140 (IC(50s) 2.99 and 3.36 nM) and the most potent was demonstrated as a functional antagonist in vitro by blocking BK-mediated phosphorylation of mitogen-activated protein (MAP) kinases. The new derivatives are more hydrophobic than Hoe 140 and thus may exhibit changes in pharmacokinetic properties when evaluated in vivo.