Hypothalamic kinin B1 receptor mediates orexin system hyperactivity in neurogenic hypertension.

Brain orexin system hyperactivity contributes to neurogenic hypertension. We previously reported upregulated neuronal kinin B1 receptor (B1R) expression in hypertension. However, the role of central B1R activation on the orexin system in neurogenic hypertension has not been examined. We hypothesized that kinin B1R contributes to hypertension via upregulation of brain orexin-arginine vasopressin signaling. We utilized deoxycorticosterone acetate (DOCA)-salt hypertension model in wild-type (WT) and B1R knockout (B1RKO) mice. In WT mice, DOCA-salt-treatment increased gene and protein expression of orexin A, orexin receptor 1, and orexin receptor 2 in the hypothalamic paraventricular nucleus and these effects were attenuated in B1RKO mice. Furthermore, DOCA-salt- treatment increased plasma arginine vasopressin levels in WT mice, but not in B1RKO mice. Cultured primary hypothalamic neurons expressed orexin A and orexin receptor 1. B1R specific agonist (LDABK) stimulation of primary neurons increased B1R protein expression, which was abrogated by B1R selective antagonist R715 but not by the dual orexin receptor antagonist, ACT 462206, suggesting that B1R is upstream of the orexin system. These data provide novel evidence that B1R blockade blunts orexin hyperactivity and constitutes a potential therapeutic target for the treatment of salt-sensitive hypertension.

Bradykinin 1 receptor blockade subdues systemic autoimmunity, renal inflammation, and blood pressure in murine lupus nephritis.

OBJECTIVE: The goal of this study was to explore the role of bradykinins and bradykinin 1 receptor (B1R) in murine lupus nephritis. METHODS: C57BL/6 and MRL/lpr mice were compared for renal expression of B1R and B2R by western blot and immunohistochemistry. MRL/lpr lupus-prone mice were administered the B1R antagonist, SSR240612 for 12 weeks, and monitored for blood pressure, proteinuria, renal function, and serum autoantibodies. RESULTS: Renal B1R:B2R ratios were significantly upregulated in MRL/lpr mice compared with B6 controls. B1R blockade ameliorated renal pathology lesions, proteinuria, and blood pressure, accompanied by lower serum IgG and anti-dsDNA autoantibody levels, reduced splenic marginal zone B cells and CD4+ T cells, and renal infiltrating CD4+ T cells, macrophages, and neutrophils. Both urine and renal CCL2 and CCL5 chemokines were also decreased in the B1R blocked MRL/lpr mice. CONCLUSION: Bradykinin receptor B1R blockade ameliorates both systemic immunity and renal inflammation possibly by inhibiting multiple chemokines and renal immune cell infiltration. B1R blockade may be particularly attractive in subjects with concomitant lupus nephritis and hypertension.

Bradykinin B2 receptor is essential to running-induced cell proliferation in the adult mouse hippocampus.

Physical exercise is a strong external effector that induces precursor cell proliferation in the adult mouse hippocampus. Research into mechanisms has focused on central changes within the hippocampus and we have established that serotonin is the signaling factor that transduces physical activity into adult neurogenesis. Less focus has been given on potential peripheral signals that may cause pro-mitotic running effects. Vasoactive kinin peptides are important for blood pressure regulation and inflammatory processes to maintain cardiovascular homeostasis. Acting via the two receptors termed B1 (B1R) and B2R, the peptides also function in the brain. In particular, studies attribute B2R a role in cell proliferation and differentiation into neurons in vitro. Here, we determined B1R and B2R mRNA expression levels in the adult mouse hippocampus and prefrontal cortex in vivo, and in response to running exercise. Using mice depleted in either or both receptors, B1-knockout (KO), B2KO and B1/2KO we observed changes in running performance overnight and in running distances. However, voluntary exercise led to the known pro-mitotic effect in the dentate gyrus of B1KO mice while it was attenuated in B2KO accompanied by an increase in microglia cells. Our data identify B2R as an important factor in running-induced precursor cell proliferation.

Inhibition on angiotensin-converting enzyme exerts beneficial effects on trabecular bone in orchidectomized mice.

BACKGROUND: This study aimed to study the osteo-preservative effects of captopril, an inhibitor on angiotensin-converting enzyme (ACE), on bone mass, micro-architecture and histomorphology as well as the modulation of captopril on skeletal renin-angiotensin system (RAS) and regulators for bone metabolism in mice with bilateral orchidectomy. METHODS: The orchidectomized (ORX) mice were orally administered with vehicle or captopril at low dose (10mg/kg) and high dose (50mg/kg) for six weeks. The distal femoral end, the proximal tibial head and the lumbar vertebra (LV) were stained by hematoxylin and eosin, Safranin O/Fast Green and masson-trichrome. Micro-computed tomography was performed to measure bone mineral density (BMD). RESULTS: Treatment with captopril increased trabecular bone area at distal metaphysis of femur, proximal metaphysis of tibia and LV-4, moreover, high dose of captopril significantly elevated trabecular BMD of LV-2 and LV-5. The mRNA expressions of renin receptor, angiotensinogen, carbonic anhydrase II, matrix metalloproteinase-9, and tumor necrosis factor-alpha were significantly decreased in tibia of ORX mice following treatment with captopril. The administration with captopril enhanced the ratio of OPG/RANKL mRNA expression, the mRNA expression of transforming growth factor-beta and the protein expression of bradykinin receptor-1. CONCLUSIONS: The inhibition on ACE by captopril exerts beneficial effects on trabecular bone of ORX mice. The therapeutic efficacy may be attributed to the regulation of captopril on local RAS and cytokines in bone.

Expression and function of TLR4- induced B1R bradykinin receptor on cardiac fibroblasts.

Cardiac fibroblasts (CF) are key cells for maintaining extracellular matrix (ECM) protein homeostasis in the heart, and for cardiac repair through CF-to-cardiac myofibroblast (CMF) differentiation. Additionally, CF play an important role in the inflammatory process after cardiac injury, and they express Toll like receptor 4 (TLR4), B1 and B2 bradykinin receptors (B1R and B2R) which are important in the inflammatory response. B1R and B2R are induced by proinflammatory cytokines and their activation by bradykinin (BK: B2R agonist) or des-arg-kallidin (DAKD: B1R agonist), induces NO and PGI2 production which is key for reducing collagen I levels. However, whether TLR4 activation regulates bradykinin receptor expression remains unknown. CF were isolated from human, neonatal rat and adult mouse heart. B1R mRNA expression was evaluated by qRT-PCR, whereas B1R, collagen, COX-2 and iNOS protein levels were evaluated by Western Blot. NO and PGI2 were evaluated by commercial kits. We report here that in CF, TLR4 activation increased B1R mRNA and protein levels, as well as COX-2 and iNOS levels. B1R mRNA levels were also induced by interleukin-1α via its cognate receptor IL-1R1. In LPS-pretreated CF the DAKD treatment induced higher responses with respect to those observed in non LPS-pretreated CF, increasing PGI2 secretion and NO production; and reducing collagen I protein levels in CF. In conclusion, no significant response to DAKD was observed (due to very low expression of B1R in CF) - but pre-activation of TLR4 in CF, conditions that significantly enhanced B1R expression, led to an additional response of DAKD.

Kinin B1 Receptor Promotes Neurogenic Hypertension Through Activation of Centrally Mediated Mechanisms.

Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension.

Blockade of the kinin B1 receptor affects the cytokine/chemokine profile in rat brain subjected to autoimmune encephalomyelitis.

Kinins are bioactive peptides which provide multiple functions, including critical regulation of the inflammatory response. Released during tissue injury, kinins potentiate the inflammation which represents a hallmark of numerous neurological disorders, including those of autoimmune origin such as multiple sclerosis (MS). In the present work, we assess the expression of B1 receptor (B1R) in rat brain during the course of experimental autoimmune encephalomyelitis (EAE) which is an animal model of MS. We apply pharmacological inhibition to investigate the role of this receptor in the development of neurological deficits and in shaping the cytokine/chemokine profile during the course of the disease. Overexpression of B1R is observed in brain tissue of rats subjected to EAE, beginning at the very early asymptomatic phase of the disease. This overexpression is suppressed by a specific antagonist known as DALBK. The involvement of B1R in the progression of neurological symptoms in immunized rats is confirmed. Analysis of an array of cytokines/chemokines identified a sub-group as being B1R-dependent. Increase of the protein levels for the proinflammatory cytokines (Il-6, TNF-α but not IL-1ß), chemokines attracting immune cells into nervous tissue (MCP-1, MIP-3α, LIX), and protein levels of fractalkine and vascular endothelial growth factor observed in EAE rats, were significantly diminished after DALBK administration. This may indicate the protective potential of pharmacological inhibition of B1R. However, simultaneously reduced protein levels of anti-inflammatory and neuroprotective factors (IL-10, IL-4, and CNTF) was noticed. The results show that B1R-mediated signaling regulates the cellular response profile following neuroinflammation in EAE.

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.

Brain kinin B1 receptor is upregulated by the oxidative stress and its activation leads to stereotypic nociceptive behavior in insulin-resistant rats.

Kinin B1 receptor (B1R) is virtually absent under physiological condition, yet it is highly expressed in models of diabetes mellitus. This study aims at determining: (1) whether B1R is induced in the brain of insulin-resistant rat through the oxidative stress; (2) the consequence of B1R activation on stereotypic nocifensive behavior; (3) the role of downstream putative mediators in B1R-induced behavioral activity. Sprague-Dawley rats were fed with 10% D-glucose in their drinking water or tap water (controls) for 4 or 12 weeks, combined either with a standard chow diet or a diet enriched with α-lipoic acid (1 g/kg feed) for 4 weeks. The distribution and density of brain B1R binding sites were assessed by autoradiography. Behavioral activity evoked by i.c.v. injection of the B1R agonist Sar-[D-Phe(8)]-des-Arg(9)-BK (10 µg) was measured before and after i.c.v. treatments with selective antagonists (10 µg) for kinin B1 (R-715, SSR240612), tachykinin NK1 (RP-67580) and glutamate NMDA (DL-AP5) receptors or with the inhibitor of NOS (L-NNA). Results showed significant increases of B1R binding sites in various brain areas of glucose-fed rats that could be prevented by the diet containing α-lipoic acid. The B1R agonist elicited head scratching, grooming, sniffing, rearing, digging, licking, face washing, wet dog shake, teeth chattering and biting in glucose-fed rats, which were absent after treatment with α-lipoic acid or antagonists/inhibitors. Data suggest that kinin B1R is upregulated by the oxidative stress in the brain of insulin-resistant rats and its activation causes stereotypic nocifensive behavior through the release of substance P, glutamate and NO.

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.

Heterodimerization of human apelin and bradykinin 1 receptors: novel signal transduction characteristics.

Apelin receptor (APJ) and bradykinin 1 receptor (B1R) are involved in a variety of important physiological processes, which share many similar characteristics in distribution and functions in the cardiovascular system. This study explored the possibility of heterodimerization between APJ and B1R, and investigated the impact of heterodimer on the signal transduction characteristics and the physiological functions in human endothelial cells after stimulation with their agonists. We first identified the endogenous expression of APJ and B1R in HUVECs and their co-localization on HEK293 membrane. The constitutive heterodimerization between the APJ and B1R was then demonstrated by BRET and FRET assays. Stimulation with Apelin-13 and des -Arg(9)-BK enhanced the phosphorylation of eNOS in HUVECs, which could be dampened by the knockdown of APJ or B1R, indicating the co-existence of APJ and B1R is critical for eNOS phosphorylation in HUVECs. Furthermore, APJ/B1R heterodimers were found to enhance the activity of PKC signaling pathway and increase intracellular Ca(2+) concentration in HEK293 cells, which might be the mechanism of APJ/B1R heterodimers promoting the phosphorylation of eNOS and leads to increased Gαq, PKC signal pathway activities and a significant increase in cell proliferation. The results provide a new theoretical and experimental base for revealed intracellular molecular mechanisms of physiological function involved in the APJ and B1R and provide potential new targets for the development of drugs and treating cardiovascular disease.

Bradykinin modulates spontaneous nerve growth factor production and stretch-induced ATP release in human urothelium.

The urothelium plays a crucial role in integrating urinary bladder sensory outputs, responding to mechanical stress and chemical stimulation by producing several diffusible mediators, including ATP and, possibly, neurotrophin nerve growth factor (NGF). Such urothelial mediators activate underlying afferents and thus may contribute to normal bladder sensation and possibly to the development of bladder overactivity. The muscle-contracting and pain-inducing peptide bradykinin is produced in various inflammatory and non-inflammatory pathologies associated with bladder overactivity, but the effect of bradykinin on human urothelial function has not yet been characterized. The human urothelial cell line UROtsa expresses mRNA for both B1 and B2 subtypes of bradykinin receptors, as determined by real-time PCR. Bradykinin concentration-dependently (pEC50=8.3, Emax 4434±277nM) increased urothelial intracellular calcium levels and induced phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2. Activation of both bradykinin-induced signaling pathways was completely abolished by the B2 antagonist icatibant (1µM), but not the B1 antagonist R715 (1µM). Bradykinin-induced (100nM) B2 receptor activation markedly increased (192±13% of control levels) stretch-induced ATP release from UROtsa in hypotonic medium, the effect being dependent on intracellular calcium elevations. UROtsa cells also expressed mRNA and protein for NGF and spontaneously released NGF to the medium in the course of hours (11.5±1.4pgNGF/mgprotein/h). Bradykinin increased NGF mRNA expression and accelerated urothelial NGF release to 127±5% in a protein kinase C- and ERK1/2-dependent manner. Finally, bradykinin up-regulated mRNA for transient-receptor potential vanilloid (TRPV1) sensory ion channel in UROtsa. In conclusion, we show that bradykinin represents a versatile modulator of human urothelial phenotype, accelerating stretch-induced ATP release, spontaneous release of NGF, as well as expression of sensory ion channel TRPV1. Bradykinin-induced changes in urothelial sensory function might contribute to the development of bladder dysfunction.

Endothelium dependent expression and underlying mechanisms of des-Arg9-bradykinin-induced B1R-mediated vasoconstriction in rat portal vein.

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B(1) receptor (B(1)R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B(1)R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B(1)R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B(1)R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B(1)R increased phospholipase A(2)/COX-2-derived thromboxane A(2) (TXA(2)) levels, which in turn mediated portal vein contraction through binding to TXA(2) receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B(1)R expression and identify a critical role for the endothelial B(1)R in the modulation of portal vein vascular tone. Our study suggests a potential role for B(1)R antagonists as therapeutic tools for diseases where portal hypertension may be involved.

Blocking of bradykinin receptor B1 protects from focal closed head injury in mice by reducing axonal damage and astroglia activation.

The two bradykinin receptors B1R and B2R are central components of the kallikrein-kinin system with different expression kinetics and binding characteristics. Activation of these receptors by kinins triggers inflammatory responses in the target organ and in most situations enhances tissue damage. We could recently show that blocking of B1R, but not B2R, protects from cortical cryolesion by reducing inflammation and edema formation. In the present study, we investigated the role of B1R and B2R in a closed head model of focal traumatic brain injury (TBI; weight drop). Increased expression of B1R in the injured hemispheres of wild-type mice was restricted to the later stages after brain trauma, i.e. day 7 (P<0.05), whereas no significant induction could be observed for the B2R (P>0.05). Mice lacking the B1R, but not the B2R, showed less functional deficits on day 3 (P<0.001) and day 7 (P<0.001) compared with controls. Pharmacological blocking of B1R in wild-type mice had similar effects. Reduced axonal injury and astroglia activation could be identified as underlying mechanisms, while inhibition of B1R had only little influence on the local inflammatory response in this model. Inhibition of B1R may become a novel strategy to counteract trauma-induced neurodegeneration.

An in vitro reconstitution system to address the mechanism of the vascular expression of the bradykinin B1 receptor in response to angiotensin converting enzyme inhibition.

The expression of the bradykinin (BK) B1 receptor (B1R), lacking in normal vascular tissues, is induced following innate immune system activation and chronic blockade of angiotensin converting enzyme (ACE). To identify cytokine-dependent or -independent mechanisms for the latter phenomenon, the ACE inhibitor enalaprilat and several peptides potentiated in vivo by ACE blockade were applied either directly to human umbilical artery smooth muscle cells (hUA-SMCs) or to differentiated monoblastoid U937 cells to produce a conditioned medium (CM) that was later transferred to hUA-SMCs. A phagocyte stimulant, lipopolysaccharide, did not upregulate B1R, measured using [³H]Lys-des-Arg9-BK binding, or translocate NF-κB to the nuclei if applied directly to the hUA-SMCs. However, the CM of lipopolysaccharide-stimulated U937 cells was active in these respects (effects inhibited by etanercept and correlated to TNF-α presence in the CM). A peptidase-resistant B1R agonist had no significant direct or indirect acute effect (4h) on B1R expression, but repeated hUA-SMC stimulations over 40 h were stimulatory in the absence of NF-κB activation. Other peptides regulated by ACE or enalaprilat did not directly or indirectly stimulate B1R expression. The reconstitution system supports the rapid cytokine-dependent vascular induction of B1Rs and a slow "autoregulatory" one potentially relevant for the ACE blockade effect.

Up-regulation of human bradykinin B1 receptor by secreted components of Pseudomonas aeruginosa via a NF-κB pathway in epithelial cells.

Pulmonary epithelial cells produce neutrophil chemotactic activity in response to pathogenic bacterial infections, resulting in neutrophil migration to infection sites. Elicited neutrophils in the inflamed tissues were found to be dependent on bradykinin B1 receptor (B1R), which shows high affinity for the active metabolites derived from bradykinin. Thus, the up-regulation of bradykinin and B1R expression represents an important host defense response against invading microbes such as Pseudomonas aeruginosa. However, the effect of P. aeruginosa on the expression of B1R remains unclear, while P. aeruginosa infection is known to stimulate the production of bradykinin. Here, we report that human B1R (hB1R) transcription is up-regulated in host cells co-cultured with P. aeruginosa. Components secreted from P. aeruginosa play a major role in the up-regulation, and the secretion of the components is not controlled by either type III secretion system or quorum sensing. Moreover, the B1R induction is mediated by a NF-κB signaling pathway in human lung epithelial cells. Taken together, this study demonstrates that P. aeruginosa is capable of up-regulating hB1R expression via the NF-κB signaling pathway.

Enalaprilat-mediated activation of kinin b(1) receptors and vasodilation in the rat isolated perfused kidney.

The present study evaluated whether enalaprilat (the active form of enalapril, an angiotensin-converting enzyme inhibitor) activates B(1) receptors. We observed that the levels of B(1) receptor mRNA and protein expression were upregulated in the kidneys of diabetic rats. Bradykinin (BK)-induced renal vasodilation decreased in isolated perfused kidneys of diabetic rats, but des-Arg(9)-BK-induced renal vasodilation increased. Enalaprilat also produced vasodilation in the isolated perfused kidneys of control and diabetic rats. The response to des-Arg(9)-BK or enalaprilat was blocked by Lys-(des-Arg(9), Leu(8))-BK (a B(1) receptor antagonist) and N-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). These results suggest that enalaprilat activates B(1) receptors and stimulates the production of nitric oxide in the kidneys of both control and diabetic rats.

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice.

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B1 receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57Bl/6 mice, the bradykinin B1 receptor expression was up-regulated 24h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B1 receptor antagonist, R-954 (Ac-Orn-[Oic2, alpha-MePhe5, D-betaNal7, Ile8]desArg9-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B1 receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Blockade of bradykinin B2 receptor more effectively reduces postischemic blood-brain barrier disruption and cytokines release than B1 receptor inhibition.

Blood-brain barrier disruption and brain edema are detrimental in ischemic stroke. The kallikrein-kinin system appears to play an important role in the regulation of vascular permeability and is invoked in edema formation. The effects of kinins are mediated by bradykinin receptors B1R and B2R. However, little is known about the exact roles of bradykinin receptors in the early stage of cerebral ischemia. In this study, we demonstrated that ischemia upregulated the level of B1R and B2R at 24h after reperfusion by immunofluorescence assays, mainly expressed in astrocytes and neurons, respectively, in the ischemic penumbra. Moreover, B2R inhibition more effectively reduced neurological severity scores, blood-brain barrier permeability and cytokines release than B1R inhibition did. Additionally, B2R inhibition also significantly suppressed B1R protein level. Therefore, blockade of B2R may be a more effective strategy for the treatment of ischemic brain injury than B1R inhibition within 24h after reperfusion.