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.

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.

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.

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.

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.

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.

Fluorescent ligands of the bradykinin B1 receptors: pharmacologic characterization and application to the study of agonist-induced receptor translocation and cell surface receptor expression.

Unlike the widely distributed and preformed B(2) receptors, the bradykinin B(1) receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B(1) receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-epsilon-aminocaproyl-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-bradykinin] and the agonist B-10378 [5(6)CF-epsilon-aminocaproyl-Lys-des-Arg(9)-bradykinin]. B-10376 has a K(i) of 10 to 20 nM to displace [(3)H]Lys-des-Arg(9)-bradykinin from rabbit or human recombinant B(1) receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA(2), 7.49). B-10378 was a full agonist at the naturally expressed B(1) receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K(i) of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B(1) receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B(1) receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B(1) receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B(1) receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).

In vivo up-regulation of kinin B1 receptors after treatment with Porphyromonas gingivalis lipopolysaccharide in rat paw.

It has been demonstrated that kinin B(1) receptors are highly up-regulated under several stressful stimuli, such as infection. However, there is no evidence indicating whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) might lead to B(1) receptor up-regulation. In this study, we demonstrate that Pg-LPS injection into the rat paw resulted in a marked functional up-regulation of B(1) receptors (as measured by an increase of B(1) receptor-induced edema), which was preceded by a rapid rise in B(1) receptor mRNA expression. The local administration of Pg-LPS also resulted in a prominent production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), followed by an increase of neutrophil influx; both events were observed at periods before B(1) receptor induction. The functional and molecular Pg-LPS-elicited B(1) receptor up-regulation was significantly reduced by the glucocorticoid dexamethasone (0.5 mg/kg s.c.), and to a lesser extent by the chimeric anti-TNF-alpha antibody infliximab (1 mg/kg s.c.). Of high relevance, we show for the first time that a single administration of the proresolution lipid mediator (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (resolvin E1; 300 ng/rat i.p.) was able to markedly down-regulate Pg-LPS-driven B(1) receptor expression, probably by inhibiting TNF-alpha production and neutrophil migration. Collectively, the present findings clearly suggest that Pg-LPS is able to induce the up-regulation of B(1) receptors through mechanisms involving TNF-alpha release and neutrophil influx, which are largely sensitive to resolvin E1. It is tempting to suggest that kinin B(1) receptors might well represent a pivotal pathway for the inflammatory responses evoked by P. gingivalis and its virulence factors.

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.

Interleukin-1beta enhances the action of bradykinin in rat myenteric neurons through up-regulation of glial B1 receptor expression.

Interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) are released under pathological conditions in the gastrointestinal tract such as inflammatory bowel diseases (IBD). We examined the effects of IL-1beta and TNFalpha on bradykinin (BK) -induced increases in the intracellular Ca(2+) concentration ([Ca(2+)]i) and prostaglandin (PG) E(2) release in neonatal rat myenteric plexus cells. BK evoked a [Ca(2+)]i increase in myenteric neurons and glial cells, both of which were potentiated by treatment with IL-1beta but not TNFalpha. In both cell types, the [Ca(2+)]i responses to BK were abolished by D-Arg(0)[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-BK (HOE140), a B2R antagonist, but not affected by des-Arg(9)-HOE140, a B1R antagonist. After culture with IL-1beta, however, the B1R antagonist suppressed the BK-induced [Ca(2+)]i increase. Only in glial cells did the B1R agonists des-Arg(9)-BK and BK fragment 1-8 evoke a [Ca(2+)]i rise in a dose-dependent manner. Real time RT-PCR and immunocytochemical analyses showed that IL-1beta treatment increased expression of B1R mRNA in myenteric plexus cells and B1R protein in glial cells, respectively. Either indomethacin or an EP1 receptor antagonist suppressed the increased [Ca(2+)]i response to BK invoked by treatment with IL-1beta. IL-1beta treatment increased BK-induced PGE(2) release from cultured myenteric plexus cells. These results suggest that IL-1beta promotes up-regulation of B1R expression in glial cells, resulting in the potentiation of neural responses to BK through the elevation of PGE(2) released from glial cells. The alteration of phenotypes of glial cells may be the cause of the changes in neural function in the enteric nervous system in IBD.

The relevance of kinin B1 receptor upregulation in a mouse model of colitis.

BACKGROUND AND PURPOSE: Kinins are implicated in many pathophysiological conditions, and recent evidence has suggested their involvement in colitis. This study assessed the role of the kinin B1 receptors in a mouse model of colitis. EXPERIMENTAL APPROACH: Colitis was induced in mice by 2,4,6-trinitrobenzene sulphonic acid (TNBS), and tissue damage and myeloperoxidase activity were assessed. B1 receptor induction was analysed by organ bath studies, binding assay and reverse transcription PCR. KEY RESULTS: TNBS-induced colitis was associated with tissue damage, neutrophil infiltration and time-dependent increase of colon B1 receptor-mediated contraction, with the maximal response observed at 72 h. The upregulation of the B1 receptor at this time point was also confirmed by means of binding studies. B1 receptor mRNA levels were elevated as early as 6 h after colitis induction and remained high for up to 48 h. TNBS-evoked tissue damage and neutrophil influx were reduced by the selective B1 receptor antagonist SSR240612, and in B1 receptor knockout mice. In vivo treatment with inhibitors of protein synthesis, nuclear factor-kappaB activation, inducible nitric oxide synthase (iNOS) or tumour necrosis factor alpha (TNFalpha) significantly reduced B1 receptor agonist-induced contraction. Similar results were observed in iNOS and TNF receptor 1-knockout mice. CONCLUSIONS AND IMPLICATIONS: These results provide convincing evidence on the role of B1 receptors in the pathogenesis of colitis. Therefore, the blockade of kinin B1 receptors might represent a new therapeutic option for treating inflammatory bowel diseases.

Bradykinin stimulates glutamate uptake via both B1R and B2R activation in a human retinal pigment epithelial cells.

AIMS: We were to examine the effect of bradykinin (BK) in the regulation of glutamate transporter and its related signaling molecules in a human retinal pigment epithelial (ARPE) cells, which are important cells to support retina. MAIN METHODS: d-[2,3-(3)H]-aspartate uptake, western immunoblotting, reverse transcription polymerase chain reaction, [(3)H]-arachidonic acid release, and siRNA transfection techniques were used. KEY FINDINGS: BK stimulated glutamate uptake as well as the mRNA expression of excitatory amino acid transporter 4 (EAAT4) and excitatory amino acid carrier 1 (EAAC1), which was blocked by treatment with bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R) siRNA, suggesting the role of B1R and B2R in this process. The BK-induced stimulation of glutamate uptake was also blocked by [des-Arg(10)]-HOE 140, a B1R antagonist, and HOE 140, a B2R antagonist, as well as by the tyrosine kinase inhibitors genistein and herbimycin A. In addition, the BK-induced stimulation of glutamate uptake was blocked by treatment with the phospholipase A(2) inhibitors mepacrine and AACOCF(3), the cyclooxygenase (COX) inhibitor indomethacin, and the COX-2 inhibitor Dup 697. Furthermore, the BK-induced increase in COX-2 expression was blocked by the PI-3 kinase inhibitors wortmannin and LY294002, Akt inhibitor, and the protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide I, suggesting the role of PI-3 kinase and PKC in this process. BK stimulated Akt activation and the translocation of PKC activation via the activation of B1R and B2R. SIGNIFICANCE: BK stimulates glutamate uptake through a PKC-Akt-COX-2 signaling cascade in ARPE cells.

Expression of kinin B1 and B2 receptors in immature, monocyte-derived dendritic cells and bradykinin-mediated increase in intracellular Ca2+ and cell migration.

The kinins, bradykinin (BK) and Lys-des[Arg(9)]-BK, are important inflammatory mediators that act via two specific G protein-coupled kinins, B(1) and B(2) receptors (B(2)R). Kinins influence the activity of immune cells by stimulating the synthesis of cytokines, eicosanoids, and chemotactic factors. Whether human dendritic cells (DC) express kinin receptors and whether kinins influence DC function are unknown. Fluorescence immunocytochemistry and RT-PCR were used to demonstrate that immature human monocyte-derived DC (hMo-DC) constitutively expressed kinins B(1)R and B(2)R. Kinin receptor expression was induced on the 3rd and 4th days of culture during differentiation of hMo-DC from monocytes and was not dependent on the presence of IL-4 or GM-CSF. Although monocytes also expressed B(2)R mRNA, the protein was not detected. The kinin agonists BK and Lys-des[Arg(9)]-BK up-regulated the expression of their respective receptors. BK, acting via the B(2)R, increased intracellular Ca(2+), as visualized by confocal microscopy using the fluorescent Ca(2+) dye, Fluor-4 AM. Evaluation of migration in Trans-well chambers demonstrated significant enhancement by BK of migration of immature hMo-DC, which was B(2)R-dependent. However, kinins did not induce maturation of hMo-DC. The novel finding that kinin receptors are constitutively expressed in immature hMo-DC suggests that these receptors may be expressed in the absence of proinflammatory stimuli. BK, which increases the migration of immature hMo-DC in vitro, may play an important role in the migration of immature DC in noninflammatory conditions and may also be involved in the recruitment of immature DC to sites of inflammation.

Expression of genes encoding kinin receptors in peripheral blood mononuclear cells from patients with acute coronary syndromes.

BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis, including plaque destabilization leading to the rupture and local thrombosis, clinically manifested as unstable angina (UA) or myocardial infarction (MI). Recent data report enhanced expression of numerous pro-inflammatory genes in patients with acute coronary syndrome (ACS) both in plaque and in inflammatory cells. Kinins are peptides involved in vasodilation, vascular permeability, pain and inflammation. Their effects are mediated by two receptors, B1 and B2. As the role of kinins in ACS is not clear, the aim of the study was to assess the expression of the genes encoding kinin receptors in patients with ACS. METHODS: The study was carried out on 40 patients with ACS and 10 age-matched healthy subjects (control (C)). To evaluate gene expression of B1 and B2 kinin receptors, total mRNA was extracted from peripheral blood mononuclear cells and the number of mRNA copies was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: In patients with MI and UA, the B1 receptor (B1R)/B2 receptor (B2R) ratio was inversed compared with healthy subjects (C group) (MI vs C: 1.54 +/- 0.39 vs 0.36 +/- 0.04; P < 0.01; UA vs C: 2.13 +/- 0.98 vs 0.36 +/- 0.04; P < 0.05 respectively). B2R gene mRNA level was markedly lower in MI group versus C group (24 216 +/- 5409 copies/microg vs 39 908 +/- 5309 copies/microg; P < 0.05). The difference in B1R gene expression between MI and C group was negligible. We have not observed differences in studied genes expression between UA and C groups. CONCLUSION: Patients with ACS show inverted B1R/B2R ratio. Such disturbance in kinin signalling may reflect increased activation of circulating mononuclears, which are important participants of atherosclerotic plaque development and eventually rupture.

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.

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.

Bradykinin B1 receptor expression induced by tissue damage in the rat portal vein: a critical role for mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways.

The bradykinin B1 receptor (B1R) is normally absent under physiological conditions, but is highly inducible during inflammatory conditions or following tissue damage. The present study attempted to determine some of the mechanisms underlying B1R upregulation following tissue injury in rat portal vein. Damage induced by tissue isolation and in vitro incubation caused a significant and time-dependent increase in des-Arg9-bradykinin (des-Arg9-BK) responsiveness that paralleled the B1R mRNA expression, as confirmed by real-time quantitative PCR. In vitro incubation of rat portal vein also induced the activation of some members of the mitogen activated protein kinase (MAPK) family, namely, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK, an effect accompanied by degradation of the inhibitory protein IkappaBalpha and translocation of nuclear transcription factor-kappaB (NF-kappaB) to the nucleus. The blockade of p38 MAPK, JNK or NF-kappaB, but not ERK pathways with selective inhibitors, resulted in a significant reduction of the upregulated contractile response caused by the selective B1R agonist des-Arg9-BK, and largely prevented the induction of B1R mRNA expression in the rat portal vein. Together, these results demonstrate that in vitro tissue damage induces activation of several intracellular signaling pathways that have a key role in the control of B1R expression. B1R could exert a pivotal role in the development of the cardiovascular response associated with vascular damage.

Cytokines and neutrophils as important mediators of platelet-activating factor-induced kinin B1 receptor expression.

PAF injection into the rat paw is accompanied by the concomitant activation of NF-kappaB and neutrophil influx, which appears to be relevant to the up-regulation of kinin B1 receptors. Herein, we analyse the role of TNF-alpha and IL-1beta production for PAF-induced B1 receptor upregulation in the rat paw. Additionally, we evaluate how cytokine production and neutrophil migration fit into the temporal sequence of events leading to PAF-induced B1 receptor upregulation. In our experiments, treatment with PAF resulted in a marked increase of B1 receptor-mediated paw oedema and in situ production of TNF-alpha at 1 h and IL-1beta at 3 and 6 h later. B1 receptor-mediated paw oedema was significantly inhibited by anti-TNF-alpha antibody and by interleukin-1 receptor antagonist (IRA). TNF-alpha was necessary for the local PAF-induced IL-1beta production. NF-kappaB blocker PDTC prevented the production of both TNF-alpha and IL-1beta, indicating that cytokine production is NF-kappaB dependent. Depletion of neutrophils with an anti-PMN antibody prevented IL-1beta, but not TNF-alpha, production. Although both TNF-alpha and IL-1beta are relevant to functional B1 receptor upregulation, PAF-induced increase in B1 receptor mRNA was markedly suppressed by anti-TNF-alpha and, to a lesser extent, by IRA. B1 receptor mRNA expression was also prevented by the anti-PMN antibody. In conclusion, the activation of the TNF-alpha/neutrophil axis by PAF seems to be sufficient for B1 receptor mRNA production. However, the TNF-alpha/neutrophil axis is also necessary for IL-1beta production. These two processes might lead to the appearance of functional kinin B1 upregulation receptors in vivo after PAF treatment.