Kinin B1 receptor: A target for neuroinflammation in hypertension.

Kinins are a family of oligopeptides of the kallikrein-kinin system that act as potent vasoactive hormones and inflammatory mediators. The bioactive kinins mainly consist of bradykinin and kallidin, and their metabolites des-Arg9-bradykinin and des-Arg10-kallidin. Physiological effects of kinins are mediated by activation of highly selective G-protein coupled kinin B1 and B2 receptors. Growing evidence suggests that B1 receptor activation mediates diverse physiological and pathological features of cardiovascular diseases. However, studies are limited regarding the impact of B1 receptor mediated neuroinflammation on the development of hypertension and other cardiovascular diseases. Given the potential role for B1 receptor activation in immune cell infiltration, microglia activation, and cytokine production within the central nervous system, B1 receptor mediated signaling cascades might result in elevated neuroinflammation. In this review, we will discuss the potential pro-inflammatory role of B1 receptor activation in hypertension. A better understanding of B1 receptor inflammatory signaling may lead to the development of therapeutics that target B1 receptors to treat neurogenic hypertension.

The role of kinin B1 and B2 receptors in the mouse model of oxazolone-induced atopic dermatitis.

This study evaluated the role of kinin B1 and B2 receptors in the pre-clinical mouse model of oxazolone-induced atopic dermatitis. The B1 R715 or B2 HOE140 receptor antagonists were dosed at different schemes of treatment. After assessment of clinical lesion scores and pruritus, lesional skin samples were collected for histopathological analysis. The plasma extravasation and the expression of the metalloproteinase ADAMTS5 were also assessed. The immunopositivity for kinin receptors was evaluated in the skin, dorsal root ganglion (DRG), thoracic spinal cord and brain cortex sections. Marked upregulation of B1 and B2 receptors was observed in the skin of oxazolone-treated mice. The induction of atopic dermatitis led to a downregulation of both receptors in the DRG, without any alteration in the spinal cord and brain cortex. The repeated administration of HOE140 (50 nmol/kg; i.p.) partially inhibited the oxazolone-related pruritus, associated with a reduction of ADAMTS5 immunolabelling in the skin. Alternatively, R715 (438 nmol/kg; i.p.) produced a mild inhibition of plasma extravasation in oxazolone-challenged mice. Noteworthy, the repeated i.d. injection of R715 (30 nmol/site) or HOE140 (3 nmol/site) significantly reduced the histiocyte numbers, according to the histopathological analysis. Either B1 or B2 kinin antagonists, irrespective of the protocol of treatment, did not alter any other evaluated clinical or histological parameters. Data brings novel evidence about the role of kinin receptors in allergy-related conditions, such as atopic dermatitis. Further studies to test different protocols of treatment with kinin antagonists on in-depth cellular alterations underlying oxazolone-induced atopic dermatitis remain to be performed.

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.

MAPK/NF-κB-dependent upregulation of kinin receptors mediates airway hyperreactivity: a new perspective for the treatment.

Airway hyperreactivity (AHR) is a major feature of asthmatic and inflammatory airways. Cigarette smoke exposure, and bacterial and viral infections are well-known environmental risk factors for AHR, but knowledge about the underlying molecular mechanisms on how these risk factors lead to the development of AHR is limited. Activation of intracellular mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) and their related signal pathways including protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and protein kinase A (PKA) signaling pathways may result in airway kinin receptor upregulation, which is suggested to play an important role in the development of AHR. Environmental risk factors trigger the production of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukins (ILs) that activate intracellular MAPK- and NF-κB-dependent inflammatory pathways, which subsequently lead to AHR via kinin receptor upregulation. Blockage of intracellular MAPK/NF-κB signaling prevents kinin B1 and B2 receptor expression in the airways, resulting in a decrease in the response to bradykinin (kinin B2 receptor agonist) and des-Arg9-bradykinin (kinin B1 receptor agonist). This suggests that MAPK- and NF-κB-dependent kinin receptor upregulation can provide a novel option for treatment of AHR in asthmatic as well as in other inflammatory airway diseases.

Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma.

In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis.

Comparison of kinin B(1) and B(2) receptor expression in neutrophils of asthmatic and non-asthmatic subjects.

Kinins have been implicated in the pathophysiology of asthma and activation of kinin receptors stimulates neutrophil chemotaxis. However, the expression of kinin receptors on neutrophils of asthmatic subjects has not been assessed. The aim of this study was to compare the expression of kinin B(1) and B(2) receptor mRNA and proteins in neutrophils of asthmatic and non-asthmatic subjects, and to assess whether inhaled corticosteroid treatment may influence expression of the kinin receptors. Neutrophils were isolated from peripheral blood of asthmatic (n=27) and non-asthmatic subjects (n=14). The presence of kinin B(1) and B(2) receptor protein on neutrophils was confirmed by immunolabeling with specific antibodies followed by immunoperoxidase, immunofluorescence and FACS detection. Kinin B(1) and B(2) receptor mRNA expression was assessed by RT-PCR. Quantitative image analysis of fluorescence immunolabeled neutrophils showed no differences in kinin B(1) or B(2) receptor protein expression between asthmatic and non-asthmatic subjects. Similarly, quantitative real time RT-PCR analysis demonstrated no differences in expression of mRNA for the kinin B(1) or B(2) receptors between asthmatic and non-asthmatic subjects. However, B(1) receptor mRNA expression was significantly lower in asthmatic subjects using > or =2000 microg of inhaled corticosteroid per day (p<0.05) and B(1) receptor protein levels also tended to be lower in these subjects. Corticosteroids may have a beneficial anti-inflammatory effect in asthma by down-regulating B(1) receptor expression on neutrophils, thereby decreasing the migration of these inflammatory cells into the airways.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

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.

Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium.

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium (EC(50) = 8.4 nM) in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca(2+)](i) in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg(9)-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg(10)-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.