Pharmacological evaluation of a selective bradykinin B1 antagonist in a canine model of arthritis.

The effects of a selective bradykinin 1 receptor antagonist, compound A, were evaluated in a canine model of acute inflammatory model of arthritis. Despite detection of the B1 receptor in canine type B synoviocytes using a fluorescent ligand, oral administration of compound A (9 and 27 mg/kg) did not improve weight bearing of dogs injected intra-articularly with IL-1ß in a force plate analysis. Analysis of the synovial fluid of IL-1ß-treated dogs indicated high levels of bradykinin postchallenge. Excellent exposure, coupled with evidence of the presence of the B1 receptor during an acute inflammatory model of pain, indicates an inability of the receptor to mediate inflammatory pain in canines.

Acute hypothalamo-pituitary-adrenal axis response to LPS-induced endotoxemia: expression pattern of kinin type B1 and B2 receptors.

Bradykinin (BK) and des-Arg9-BK are pro-inflammatory mediators acting via B2 (B2R) and B1 (B1R) receptors, respectively. We investigated the role of B2R and B1R in lipopolysaccharide (LPS)-induced hypothalamo-pituitary-adrenal (HPA) axis activation in SD rats. LPS given intraperitoneally (ip) up-regulated B1R mRNA in the hypothalamus, both B1R and B2R were up-regulated in pituitary and adrenal glands. Receptor localization was performed using immunofluorescence staining. B1R was localized in the endothelial cells, nucleus supraopticus (SON), adenohypophysis and adrenal cortex. B2R was localized nucleus paraventricularis (PVN) and SON, pituitary and adrenal medulla. Blockade of B1R prior to LPS further increased ACTH release and blockade of B1R 1 h after LPS decreased its release. In addition, we evaluated if blockade of central kinin receptors influence the LPS-induced stimulation of hypothalamic neurons. Blockade of both B1R and B2R reduced the LPS-induced c-Fos immunoreactivity in the hypothalamus. Our data demonstrate that a single injection of LPS induced a differential expression pattern of kinin B1R and B2R in the HPA axis. The tissue specific cellular localization of these receptors indicates that they may play a crucial role in the maintenance of body homeostasis during endotoxemia.

Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An In Vitro Biochemical and Neuroproteomics Assessment.

Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a "survival" capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.

In vivo radioimaging of bradykinin receptor b1, a widely overexpressed molecule in human cancer.

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids ((68)Ga-SH01078 and (68)Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers.

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus.

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1ß and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus.

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1β and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus. .

Effect of interferon-γ on inflammatory cytokine-induced bradykinin B1 receptor expression in human vascular cells.

The expression of the bradykinin B(1) receptor is strongly regulated in vascular tissue following injury, with little or no expression in healthy tissues. The present work aimed to verify whether primary human vascular cells (umbilical vein endothelial cells, umbilical artery smooth muscle cells) respond to tumor necrosis factor (TNF)-α and interferon (IFN)-γ by an upregulation of B(1) receptors and whether these pathways interact. B(1) receptor expression was quantified using a [(3)H]Lys-des-Arg(9)-bradykinin binding assay (cell surface protein) and RT-PCR (mRNA). A pharmacological approach exploiting several inhibitory drugs related to cytokine signaling was applied. The combined treatment with TNF-α and IFN-γ had a synergistic effect on B(1) receptor expression in both cell types, increasing primarily receptor abundance in both cell types (16 h) and mRNA concentration (4h) in endothelial cells. The synergistic effect of the IFN-γ-TNF-α combination was abated by drugs targeted at the signaling of either cytokine (for TNF-α: etanercept or the IκB kinase 2 inhibitor TPCA-1; for IFN-γ: neutralizing antibodies to IFN-γ, a pan-Jak inhibitor but not the Jak2 inhibitor AG490). Thus, Jak2 signaling may not be recruited by the IFN-γ receptors in vascular cells; however, Stat1 phosphorylation was correlated as expected to the effect of IFN-γ on B(1) receptor expression. Random migration was inhibited by the B(1) receptor agonist Lys-des-Arg(9)-bradykinin only in smooth muscle cells pretreated with the cytokine combination. The amplificatory effect of IFN-γ on TNF-α-induced bradykinin B(1) receptor expression is relevant to vasculopathies associated with T helper 1 cytokines.

Expression of kinin receptors on eosinophils: comparison of asthmatic patients and healthy subjects.

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B(1) and B(2) receptors (B(1)R and B(2)R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca(2+) was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg(9)]-BK were assessed. Eosinophils expressed kinin B(1)R and B(2)R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B(1)R and B(2)R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B(1)R and B(2)R mRNA expression did not differ significantly between these groups. Expression of kinin B(1)R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B(1) and B(2) agonists up-regulated expression of their respective receptors but did not increase intracellular Ca(2+) or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B(1)R.

Role of nuclear factor-kappaB and protein kinase C signaling in the expression of the kinin B1 receptor in human vascular smooth muscle cells.

Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-kappaB (NF-kappaB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-kappaB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-kappaB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1beta, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-kappaB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1beta, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1beta-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-kappaB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-kappaB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.

Expression and localisation of kinin receptors in colorectal polyps.

Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n=18), villous (n=15) and hyperplasic polyps (n=15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/microg total RNA+/-312 SE) which is significantly higher as compared with controls (683+/-197 SE, p<0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852+/-936 SE) as compared with controls (843+/-263 SE, p<0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.

Coronary vasomotor response to the selective B1-kinin-receptor agonist Des-Arg9-bradykinin in humans.

OBJECTIVES: The aim of the present study was to assess the effects of selective B1-receptor stimulation with des-Arg9-bradykinin on coronary vasomotion in transplanted and non-transplanted patients. BACKGROUND: Bradykinin B1-receptors have been identified on endothelial and smooth muscle cells in human coronary arteries in vitro; however, their physiologic role in the coronary circulation is unknown. METHODS: Twelve heart transplant patients were compared with 10 control subjects at 3.2 +/- 2.2 months after surgery. Coronary flow velocity was measured using guide-wire Doppler. The diameter of 3 epicardial segments of the left coronary artery and coronary blood flow were assessed at baseline, immediately after infusions of increasing doses of des-arginine(Arg9)-bradykinin at estimated coronary blood concentrations of 5.4 x 10(-9), 5.4 x 10(-8), 5.4 x 10(-7) and 1.6 x 10(-6) mol/liter, and of acetylcholine at 10(-8), 10(-7) and 10(-6) mol/liter). RESULTS: Des-Arg9-bradykinin induced a similar decrease in all measured epicardial diameters in both groups and no change in coronary blood flow. Vasoconstriction was significant only at the 2 highest concentrations: -6 +/- 9% (p < 0.01) and -7 +/- 11% (p < 0.01) in control subjects, and -8 +/- 8% (p < 0.001) and -9 +/- 11% (p < 0.001) in heart transplant patients. Acetylcholine induced significant epicardial vasodilation in control subjects and vasoconstriction in transplant patients. The presence of allograft rejection did not modify the responses to des-Arg9-bradykinin with regard to both conductance and resistance vessels. CONCLUSIONS: Kinin B1-receptors exist and can be stimulated in humans. The vasoconstrictive action on epicardial coronary arteries of des-Arg(9)-bradykinin in humans argues for a predominant action of B1-receptor stimulation at the level of smooth muscle cells.

The bradykinin B1 receptor contributes to the cardioprotective effects of AT1 blockade after experimental myocardial infarction.

OBJECTIVE: To investigate the role of the bradykinin B1 receptor (B1R) on the angiotensin receptor AT1 blockade-dependent cardioprotective effects, we studied the B1R regulation in wild-type rats treated with the AT1 antagonist, irbesartan (IRB), and also in transgenic rats with cardiac overexpression of the human AT1 (TGR-alphaMHCAT1) after induction of myocardial infarction (MI). In addition, we treated wild-type rats with IRB and/or the B1R antagonist, B9958, and determined the left ventricular (LV) function. METHODS: Untreated, IRB (50 mg/kg/day/p.o.), B9958 (0.1 mg/kg/48 h/s.c.), and IRB/B9958-treated Sprague-Dawley rats were submitted to a permanent occlusion of the left descending coronary artery. Six days and three weeks after induction of MI, the LV function was characterized by using a Millar-tip catheter. Myocardial AT1- and B1-mRNA expression were analyzed by RNase-protection assays, B1R protein density by immunohistochemistry. RESULTS: At both time points, LV function had improved by almost 50% after treatment with IRB but remained unchanged in TGR-alphaMHCAT1 after induction of MI compared to their untreated controls. The beneficial effect of IRB was reversed by co-treatment with B9958. The B1R antagonist treatment alone had no effect. A cross-talk between AT1 and B1R was also indicated by an up-regulation of B1R after treatment with IRB on protein and RNA level, while AT1 overexpression reduced B1R expression after induction of MI. CONCLUSION: These results indicate that the mechanisms of B1R regulation are influenced by the AT1 receptor. Thus, we are able to demonstrate for the first time that the B1R contributes to the cardio-beneficial effects of AT1 blockade.