Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor.

The angiotensin type 2 (AT2) receptor and the bradykinin type 2 (B2) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT2 receptor and the B2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gαq, GRK2 and ß-arrestin2 recruitment proximal to AT2 receptors only occurred upon B2 receptor coexpression and activation, all of which is indicative of AT2-B2 receptor heteromerization. Additionally, we also observed specific coupling of the B2 receptor with the Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, GRK2 and ß-arrestin2 was inhibited by B2 receptor but not AT2 receptor antagonism, indicating the importance of B2 receptor activation within AT2-B2 heteromers. The close proximity between the AT2 receptor and B2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT2 receptor and B2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gαq/GRK2/ß-arrestin2 recruitment (AT2 receptor) and Gαz protein coupling (B2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.

Penicillin causes non-allergic anaphylaxis by activating the contact system.

Immediate hypersensitivity reaction (IHR) can be divided into allergic- and non-allergic-mediated, while "anaphylaxis" is reserved for severe IHR. Clinically, true penicillin allergy is rare and most reported penicillin allergy is "spurious". Penicillin-initiated anaphylaxis is possible to occur in skin test- and specific IgE-negative patients. The contact system is a plasma protease cascade initiated by activation of factor XII (FXII). Many agents with negative ion surface can activate FXII to drive contact system. Our data showed that penicillin significantly induced hypothermia in propranolol- or pertussis toxin-pretreated mice. It also caused a rapid and reversible drop in rat blood pressure, which did not overlap with IgE-mediated hypotension. These effects could be countered by a bradykinin-B2 receptor antagonist icatibant, and consistently, penicillin indeed increased rat plasma bradykinin. Moreover, penicillin not only directly activated contact system FXII-dependently, but also promoted bradykinin release in plasma incubated-human umbilical vein endothelial cells. In fact, besides penicillin, other beta-lactams also activated the contact system in vitro. Since the autoactivation of FXII can be affected by multiple-factors, plasma from different healthy individuals showed vastly different amidolytic activity in response to penicillin, suggesting the necessity of determining the potency of penicillin to induce individual plasma FXII activation. These results clarify that penicillin-initiated non-allergic anaphylaxis is attributed to contact system activation, which might bring more effective diagnosis options for predicting penicillin-induced fatal risk and avoiding costly and inappropriate treatment clinically.

Tibial post fracture pain is reduced in kinin receptors deficient mice and blunted by kinin receptor antagonists.

BACKGROUND: Tibial fracture is associated with inflammatory reaction leading to severe pain syndrome. Bradykinin receptor activation is involved in inflammatory reactions, but has never been investigated in fracture pain. METHODS: This study aims at defining the role of B1 and B2-kinin receptors (B1R and B2R) in a closed tibial fracture pain model by using knockout mice for B1R (B1KO) or B2R (B2KO) and wild-type (WT) mice treated with antagonists for B1R (SSR 240612 and R954) and B2R (HOE140) or vehicle. A cyclooxygenase (COX) inhibitor (ketoprofen) and an antagonist (SB366791) of Transient Receptor Potential Vaniloid1 (TRPV1) were also investigated since these pathways are associated with BK-induced pain in other models. The impact on mechanical and thermal hyperalgesia and locomotion was assessed by behavior tests. Gene expression of B1R and B2R and spinal cord expression of c-Fos were measured by RT-PCR and immunohistochemistry, respectively. RESULTS: B1KO and B2KO mice demonstrated a reduction in post-fracture pain sensitivity compared to WT mice that was associated with decreased c-Fos expression in the ipsilateral spinal dorsal horn in B2KO. B1R and B2R mRNA and protein levels were markedly enhanced at the fracture site. B1R and B2R antagonists and inhibition of COX and TRPV1 pathways reduced pain in WT. However, the analgesic effect of the COX-1/COX-2 inhibitor disappeared in B1KO and B2KO. In contrast, the analgesic effect of the TRPV1 antagonist persisted after gene deletion of either receptor. CONCLUSIONS: It is suggested that B1R and B2R activation contributes significantly to tibial fracture pain through COX. Hence, B1R and B2R antagonists appear potential therapeutic agents to manage post fracture pain.

Cigarette Smoke Particles-Induced Airway Hyperreactivity in Vivo and in Vitro.

Cigarette smoke is a well-known strong risk factor for inducing airway hyperreactivity (AHR), but the underlying molecular mechanisms are not fully understood. In the present study, mouse in-vivo and in-vitro models were used to study effects of dimethyl sulfoxide (DMSO)-extracted cigarette smoke particles (DSP) on the airway, and to explore the underlying molecular mechanisms that are involved in DSP-induced AHR. In mouse in-vivo model, DSP (0.75, 1.5 or 3 µL/mL) was administered intranasally daily for 7 d. At the end of this period, lung functions were measured with flexiVent™. The results showed that the mice exhibited AHR in a dose-dependent manner following methacholine inhalation in vivo. In mouse in-vitro organ culture model, exposure of mouse tracheal segments to DSP (0.1 µL/mL) with or without the following pharmacological inhibitors: specific c-Jun-N-terminal kinase (JNK) inhibitor SP600125 (10 µM) or the anti-inflammatory drug dexamethasone (1 µM). DSP-induced bradykinin receptor-mediated airway contraction with increased mRNA and protein expressions for bradykinin B1 and B2 receptors could be significantly reduced by SP600125 or dexamethasone. In conclusion, the present study demonstrates that DSP could induce AHR in vivo and in vitro. In addition to this, the upregulation of bradykinin receptors in airway is most likely one of the underlying molecular mechanisms involved.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells.

Leukocyte adhesion to the vascular endothelium contributes to many immunological and inflammatory disorders. These processes have been shown to be mediated by bradykinin receptor type 2 (B2R) and dopamine receptor type 2 (D2R). In a previous study, we reported the formation of a B2R-D2R heterodimer, possibly altering cellular functions. Hence, in the present study, we examined the effect of co-activation of endothelial cells with B2R and D2R agonists on the interaction of these cells with neutrophils. Bradykinin, the main B2R agonist, significantly increased cell adhesion, and this effect was reversed when the endothelial cells were additionally co-treated with a selective D2R agonist, sumanirole. These results were dependent on the incubation time, showing an opposite tendency after prolonged stimulation. Significant changes in the expression of adhesion proteins, such as E-selectin and intercellular adhesion molecule 1 in endothelial cells were observed. Additionally, the cells preincubated with tumor necrosis factor-α showed decreased cell adhesion and IL-8 release after long incubation with both agonists. The modulation of cell adhesion by D2R and B2R seem to be mediated via STAT3 phosphorylation. In summary, this study demonstrated a protective role of D2R in neutrophil-endothelial cell adhesion induced by bradykinin, especially in cytokine-stimulated endothelial cells.

The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats.

PURPOSE: This study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B1 and B2 receptors on LPS- induced fever and the POA cells involved in this response. MATERIAL AND METHODS: Male Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca++ signaling in POA cells was performed in rat pup brain tissue microcultures. RESULTS: Icatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE2concentration. Effect abolished by indomethacin (i.p.). CONCLUSIONS: LPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B2-receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B2-receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever.

Resveratrol inhibits BK-induced COX-2 transcription by suppressing acetylation of AP-1 and NF-κB in human rheumatoid arthritis synovial fibroblasts.

Bradykinin (BK) induces inflammation in rheumatoid arthritis (RA). Resveratrol is a potent activator of Sirt1 which could modulate inflammation through deacetylating histones of transcription factors. Here, we investigated the mechanisms underlying BK-induced COX-2 expression which is modulated by resveratrol/Sirt1 in human rheumatoid arthritis synovial fibroblasts (RASFs). We found that BK-induced COX-2 protein and mRNA expression associated with PGE2 synthesis, and promoter activity was mediated through B2R receptors, which were attenuated by selective B2R antagonist Hoe140 or transfection with B2R siRNA. BK-induced responses were mediated through PKCµ, MAPKs, AP-1 and NF-κB which were inhibited by their respective inhibitors or siRNAs. Up-regulation of Sirt1 by resveratrol suppressed the BK-induced COX-2/PGE2 production through inhibiting the interaction of AP-1 and NF-κB with COX-2 promoter in RASFs. BK-induced COX-2/PGE2 expression is mediated through a B2R-PKCµ-dependent MAPKs, AP-1, and NF-κB cascade. Resveratrol inhibited the phosphorylation and acetylation of p65, c-Jun, and Fos and reduced the binding to the COX-2 promoter, thereby attenuated the COX-2 expression. Therefore, resveratrol may be a promising therapeutic intervention for treatment of inflammatory arthritis.

Physiological Roles of Bradykinin and Involvement of Bradykinin B2 Receptor in Urethral Function in Humans and Animals.

OBJECTIVE: We investigated the role of bradykinin in urethral function by examining contractile responses in urethral smooth muscle strips isolated from humans and the intraurethral pressure in rats and dogs. METHODS: The contractile responses of human urethral tissue for bradykinin (0.01-10 µmol/L) were examined, and changes in intraurethral pressure induced by bradykinin (0.003-10 µg/kg) in anesthetized rats or dogs were measured. In addition, the effects of pretreatment with the bradykinin B2 receptor antagonist FK3657 were also examined. RESULTS: In smooth muscle strips obtained from human urethra, bradykinin induced contraction, which was inhibited by FK3657 in a concentration-dependent manner. In anesthetized rats and dogs, intravenously administered bradykinin dose-dependently increased intraurethral pressure. FK3657 shifted the intraurethral pressure dose-response curve for bradykinin to the right in rats. The bradykinin-induced elevation of intraurethral pressure was also dose-dependently inhibited by FK3657 in dogs. CONCLUSIONS: The present study provides evidence that bradykinin elicits urethral smooth muscle contraction via the bradykinin B2 receptor, suggesting the potential utility of this receptor as a novel target for the treatment of voiding dysfunction.

Kinin receptors in skin wound healing.

BACKGROUND: Wound healing is a complex and dynamic process that includes 3 different phases: inflammation, proliferation, and remodeling. Kinins are vasoactive peptides released after tissue injury, and are directly involved in the development and maintenance of inflammatory processes, and their actions are mediated by the activation of receptors called B1 and B2. OBJECTIVE: We aimed to evaluate the involvement of kinin receptors in the skin healing process. METHODS: Knockout mice for kinin receptors (KOB1, KOB2 and KOB1B2) and wild type controls (WT) were subjected to a skin excision model, and tissue repair process was evaluated during different phases of wound healing. RESULTS: In knockout animals for kinin receptors differences were observed in the resolution period of injury exceeding 17 days for the total closure of wounds. The absence of kinin receptors promotes a significant reduction in infiltration of polymorphonuclear cells on day 2 of the inflammatory phase. Already at the late stage of this phase (3 days) there was a negative influence on the infiltration of polymorphonuclear and mononuclear cells at the site of injury in comparison to WT. Collagen was significantly diminished in tissue of KOB1, KOB2 and KOB1B2 from day two to the end of the healing process. Moreover, wound tissue from KOB2 and KOB1B2, but not KOB1, presented impaired parameters of re-epitheliazation, reduced proliferation of cells (PCNA immunostaining), and a lower number of myofibroblasts (α-SMA immunostaining). CONCLUSION: These data reveal the involvement of kinin receptors in processes of skin repair. Both kinin receptors participate especially during the inflammatory phase, while B2 receptors seem to be more relevant in the quality of the wound scar. Thus, a better understanding of the contribution of kinins to skin wound healing may reveal novel options for therapy.

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance.

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.

Involvement of B2 receptor in bradykinin-induced proliferation and proinflammatory effects in human nasal mucosa-derived fibroblasts isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa either accompanied by polyp formation (CRSwNP) or without polyps (CRSsNP). CRSsNP accounts for the majority of CRS cases and is characterized by fibrosis and neutrophilic inflammation. However, the pathogenesis of CRS, especially CRSsNP, remains unclear. Immunohistochemistry of CRSsNP specimens in the present study showed that the submucosa, perivascular areas, and the mucous glands were abundant in fibroblasts. Therefore, we investigated the effects bradykinin (BK), an autacoid known to participate in inflammation, on human CRSsNP nasal mucosa-derived fibroblasts (NMDFs). BK increased CXCL1 and -8 secretion and mRNA expression with EC50 ranging from 0.15~0.35 µM. Moreover, BK enhanced cell proliferation and upregulated the expressions of proinflammatory molecules, including cell adhesion molecules (CAMs) and cyclooxygenase (COX)-1 and -2. These functionally caused an increase in monocyte adhesion to fibroblast monolayer. Using pharmacological intervention and BKR siRNA knockdown, we demonstrated that the BK-induced CXCL chemokine release, cell proliferation and COX and CAM expressions were mainly through the B2 receptor (B2R). Accordingly, the B2R was preferentially expressed in the NMDFs than B1R. The B2R was highly expressed in the CRSsNP than the control specimens, while the B1R and kininogen (KNG)/BK expression slightly increased in the CRSsNP mucosa. Collectively, we report here for the first time that fibroblasts, KNG/BK, and BKRs are overexpressed in CRSsNP mucosa and BK upregulates chemokine expression, proliferation, and proinflammatory molecule expression in NMDFs via B2R activation, which lead to a functional increase in monocyte-fibroblast interaction. Our findings reveal a critical role of fibroblast, KNG/BK, and BKRs in the development of CRSsNP.

ß-blockade abolishes the augmented cardiac tPA release induced by transactivation of heterodimerised bradykinin receptor-2 and ß2-adrenergic receptor in vivo.

Bradykinin (BK) receptor-2 (B2R) and ß2-adrenergic receptor (ß2AR) have been shown to form heterodimers in vitro. However, in vivo proofs of the functional effects of B2R-ß2AR heterodimerisation are missing. Both BK and adrenergic stimulation are known inducers of tPA release. Our goal was to demonstrate the existence of B2R-ß2AR heterodimerisation in myocardium and to define its functional effect on cardiac release of tPA in vivo. We further investigated the effects of a non-selective ß-blocker on this receptor interplay. To investigate functional effects of B2R-ß2AR heterodimerisation (i. e. BK transactivation of ß2AR) in vivo, we induced serial electrical stimulation of cardiac sympathetic nerves (SS) in normal pigs that underwent concomitant BK infusion. Both SS and BK alone induced increases in cardiac tPA release. Importantly, despite B2R desensitisation, simultaneous BK infusion and SS (BK+SS) was characterised by 2.3 ± 0.3-fold enhanced tPA release compared to SS alone. When ß-blockade (propranolol) was introduced prior to BK+SS, tPA release was inhibited. A persistent B2R-ß2AR heterodimer was confirmed in BK-stimulated and non-stimulated left ventricular myocardium by immunoprecipitation studies and under non-reducing gel conditions. All together, these results strongly suggest BK transactivation of ß2AR leading to enhanced ß2AR-mediated release of tPA. Importantly, non-selective ß-blockade inhibits both SS-induced release of tPA and the functional effects of B2R-ß2AR heterodimerisation in vivo, which may have important clinical implications.

Intracellular and nuclear bradykinin B2 receptors.

Bradykinin is a vasoactive peptide that participates in numerous inflammatory processes, vasodilation, and cell growth/survival; it mainly acts through two receptor subtypes, bradykinin B1 and bradykinin B2 receptors, which are G protein-coupled receptor (GPCR) family members. Details on ubiquitin-dependent degradation via the lysosome and/or proteasome, and the recycling process that directs bradykinin B2 receptor to the cell surface after agonist-induced endocytosis remain unclear; nevertheless, intracellular localization and internalization of GPCRs following stimulation by ligands are well known. Evidence concerning the nuclear localization and functions of GPCRs has been accumulating. The bradykinin B2 receptor has been shown to localize in the nucleus and suggested to function as a transcriptional regulator of specific genes. The transfer of membrane GPCRs (regardless of liganding), including the bradykinin B2 receptor to the nucleus can be attributed to the presence of a peptide sequence referred to as the nuclear localization signal (NLS). More recently, we found that nuclear bradykinin B2 receptors form heterodimers with the nuclear lamina protein, lamin C. The function of heterodimerization of the bradykinin B2 receptor with lamin C is still unclear. However, nuclear proteins lamin A/C are involved in a variety of diseases. Although further studies are required to elucidate the precise functions and mechanisms of intracellular and nuclear bradykinin B2 receptors, here we discuss the role of lamin A/C in laminopathies and examine the clinical significance of the bradykinin B2 receptor heterodimer.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control.

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Aliskiren reduces myocardial ischemia-reperfusion injury by a bradykinin B2 receptor- and angiotensin AT2 receptor-mediated mechanism.

Angiotensin-converting enzyme inhibitors and angiotensin AT1 receptor blockers reduce myocardial ischemia-reperfusion injury via bradykinin B2 receptor- and angiotensin AT2 receptor-mediated mechanisms. The renin inhibitor aliskiren increases cardiac tissue kallikrein and bradykinin levels. In the present study, we investigated the effect of aliskiren on myocardial ischemia-reperfusion injury and the roles of B2 and AT2 receptors in this effect. Female Sprague-Dawley rats were treated with aliskiren (10 mg/kg per day) and valsartan (30 mg/kg per day), alone or in combination, together with the B2 receptor antagonist icatibant (0.5 mg/kg per day) or the AT2 receptor antagonist PD123319 (30 mg/kg per day), for 4 weeks before myocardial ischemia-reperfusion injury. Aliskiren increased cardiac bradykinin levels and attenuated valsartan-induced increases in plasma angiotensin II levels. In vehicle-treated rats, myocardial infarct size (% area at risk, mean±SEM, n=7-13) was 43±3%. This was reduced to a similar extent by aliskiren, valsartan, and their combination to 24±3%, 25±3%, and 22±2%, respectively. Icatibant reversed the cardioprotective effects of aliskiren and the combination of aliskiren plus valsartan, but not valsartan alone, indicating that valsartan-induced cardioprotection was not mediated by the B2 receptor. PD123319 reversed the cardioprotective effects of aliskiren, valsartan, and the combination of aliskiren plus valsartan. Aliskiren protects the heart from myocardial ischemia-reperfusion injury via a B2 receptor- and AT2 receptor-mediated mechanism, whereas cardioprotection by valsartan is mediated via the AT2 receptor. In addition, aliskiren attenuates valsartan-induced increases in angiotensin II levels, thus preventing AT2 receptor-mediated cardioprotection by valsartan.

Neuronal and non-neuronal bradykinin receptors are involved in the contraction and/or relaxation to the pig bladder neck smooth muscle.

AIMS: The current study investigates the role played by bradykinin (BK) receptors in the contractility to the pig bladder neck smooth muscle. METHODS: Bladder neck strips were mounted in myographs for isometric force recordings and BK receptors expression was also determined by immunohistochemistry. RESULTS: B2 receptor expression was observed in the muscular layer and urothelium whereas B1 expression was consistent detected in urothelium. A strong B2 immunoreactivity was also observed within nerve fibers among smooth muscle bundles. On urothelium-denuded preparations basal tone, BK induced concentration-dependent contractions which were reduced in urothelium-intact samples, by extracellular Ca(2+) removal and by blockade of B2 receptors and voltage-gated Ca(2+) (VOC) and non-VOC channels, and increased by cyclooxygenase (COX) inhibition. On phenylephrine-precontracted denuded strips, under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. In urothelium-intact samples, the B1 receptor agonist kallidin promoted concentration-dependent relaxations which were reduced by blockade of B1 receptors, COX, COX-1 and large-conductance Ca(2+) -activated K(+) (BKCa ) channels and abolished in urothelium-denuded samples and in K(+) -enriched physiological saline solution-precontracted strips. CONCLUSIONS: These results suggest that BK produces contraction of pig bladder neck via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry via VOC and non-VOC channels with a minor role for intracellular Ca(2+) mobilization. Facilitatory neuronal B2 receptors modulating NANC inhibitory neurotransmission and urothelial B1 receptors producing relaxation via the COX-1 pathway and BKCa channel opening are also demonstrated. Neurourol. Urodynam. 33:558-565, 2014. © 2013 Wiley Periodicals, Inc.

Pulmonary oedema producing toxin from Mesobuthus tamulus venom augments cardio-respiratory reflexes through B2 kinin receptors.

The current study was undertaken to compare the effects of pulmonary oedema producing toxin (PO-Tx) isolated from Mesobuthus tamulus venom on cardio-respiratory reflexes with exogenously administered bradykinin (BK) and to delineate the type of BK receptors mediating these responses. Jugular venous injection of phenyldiguanide (PDG) in anaesthetized rats produced reflex bradycardia, hypotension and apnoea. The PDG-induced reflex was augmented (two folds) by PO-Tx. The pulmonary water content in PO-Tx treated group was also increased. The PO-Tx-induced reflex changes as well as pulmonary oedema were blocked by-Hoe-140 implicating the involvement of B2 kinin receptors. Exogenous BK also produced augmentation (two folds) of the PDG-induced reflexes and increased the pulmonary water content. The BK-induced augmentation was blocked by pre-treatment with des-Arg10 Hoe 140 (a B1 receptor antagonist) and Hoe 140 (B2 receptor antagonist). However, these antagonists did not prevent the development of BK-induced pulmonary oedema. Present results indicate that PO-Tx augmented the PDG-induced reflex responses similar to BK and the PO-Tx induced augmentation of reflexes is mediated through B2 receptors.

Kinin-B2 receptor exerted neuroprotection after diisopropylfluorophosphate-induced neuronal damage.

The kinin-B2 receptor (B2BKR) activated by its endogenous ligand bradykinin participates in various metabolic processes including the control of arterial pressure and inflammation. Recently, functions for this receptor in brain development and protection against glutamate-provoked excitotoxicity have been proposed. Here, we report neuroprotective properties for bradykinin against organophosphate poisoning using acute hippocampal slices as an in vitro model. Following slice perfusion for 10min with diisopropylfluorophosphate (DFP) to initiate the noxious stimulus, responses of pyramidal neurons upon an electric impulse were reduced to less than 30% of control amplitudes. Effects on synaptic-elicited population spikes were reverted when preparations had been exposed to bradykinin 30min after challenging with DFP. Accordingly, bradykinin-induced population spike recovery was abolished by HOE-140, a B2BKR antagonist. However, the kinin-B1 receptor (B1BKR) agonist Lys-des-Arg(9)-bradykinin, inducing the phosphorylation of mitogen-activated protein kinase (MEK/MAPK) and cell death, abolished bradykinin-mediated neuroprotection, an effect, which was reverted by the ERK inhibitor PD98059. In agreement with pivotal B1BKR functions in this process, antagonism of endogenous B1BKR activity alone was enough for restoring population spike activity. On the other hand pralidoxime, an oxime, reactivating acetylcholinesterase (AChE) after organophosphate poisoning, induced population spike recovery after DFP exposure in the presence of bradykinin and Lys-des-Arg(9)-bradykinin. Lys-des-Arg(9)-bradykinin did not revert protection exerted by pralidoxime, however when instead bradykinin and Ly-des-Arg(9)-bradykinin were superfused together, recovery of population spikes diminished. These findings again confirm the neuroprotective feature of bradykinin, which is, diminished by its endogenous metabolites, stimulating the B1BKR, providing a novel understanding of the physiological roles of these receptors.

Bradykinin B2 receptor contributes to the exaggerated muscle mechanoreflex in rats with femoral artery occlusion.

Static muscle contraction activates the exercise pressor reflex, which in turn increases sympathetic nerve activity (SNA) and blood pressure (BP). Bradykinin (BK) is considered as a muscle metabolite responsible for modulation of the sympathetic and cardiovascular responses to muscle contraction. Prior studies have suggested that kinin B2 receptor mediates the effects of BK on the reflex SNA and BP responses during stimulation of skeletal muscle afferents. In patients with peripheral artery disease and a rat model with femoral artery ligation, amplified SNA and BP responses to static exercise were observed. This dysfunction of the exercise pressor reflex has previously been shown to be mediated, in part, by muscle mechanoreflex overactivity. Thus, in this report, we determined whether kinin B2 receptor contributes to the augmented mechanoreflex activity in rats with 24 h of femoral artery occlusion. First, Western blot analysis was used to examine protein expression of B2 receptors in dorsal root ganglion tissues of control limbs and ligated limbs. Our data show that B2 receptor displays significant overexpression in ligated limbs as compared with control limbs (optical density: 0.94 ± 0.02 in control and 1.87 ± 0.08 after ligation, P < 0.05 vs. control; n = 6 in each group). Second, mechanoreflex was evoked by muscle stretch and the reflex renal SNA (RSNA) and mean arterial pressure (MAP) responses to muscle stretch were examined after HOE-140, a B2 receptors blocker, was injected into the arterial blood supply of the hindlimb muscles. The results demonstrate that the stretch-evoked reflex responses were attenuated by administration of HOE-140 in control rats and ligated rats; however, the attenuating effects of HOE-140 were significantly greater in ligated rats, i.e., after 5 µg/kg of HOE-140 RSNA and MAP responses evoked by 0.5 kg of muscle tension were attenuated by 43% and 25% in control vs. 54% and 34% in ligation (P < 0.05 vs. control group; n = 11 in each group). In contrast, there was no significant difference in B1 receptor expression in both experimental groups, and arterial injection of R-715, a B1 receptors blocker, had no significant effects on RSNA and MAP responses evoked by muscle stretch. Accordingly, results obtained from this study support our hypothesis that heightened kinin B2 receptor expression in the sensory nerves contributes to the exaggerated muscle mechanoreflex in rats with femoral artery occlusion.

Novel insights into the critical role of bradykinin and the kinin B2 receptor for vascular recruitment of circulating endothelial repair-promoting mononuclear cell subsets: alterations in patients with coronary disease.

BACKGROUND: Endothelial injury is considered critical for progression of atherosclerosis and its complications in coronary artery disease (CAD). The endothelial-supportive effects of bradykinin have mainly been attributed to activation of the resident endothelium. Here we newly investigate the role of bradykinin and its B2 receptor for the recruitment and functional activation of circulating mononuclear cell subsets with endothelial-repair promoting capacity, such as CD34(+)CXCR4(+)cells, at sites of arterial injury. METHODS AND RESULTS: Bradykinin-B2-receptor (B2R) blockade by icatibant substantially impaired recruitment of circulating CD34(+)CXCR4(+) mononuclear cells (expressing high levels of B2R) to endothelial cells in vitro and to injured arterial wall in vivo, whereas recruitment of CD14(hi) monocytes (expressing low levels of B2R) was unchanged. Moreover, the capacity of genetically B2R-deficient bone marrow cells to promote endothelial repair in vivo was markedly impaired as compared with wild-type bone marrow cells. B2R expression was reduced on CD34(+)CXCR4(+)mononuclear cells and endothelial repair-promoting early outgrowth cells, but not on CD14(hi)monocytes, from CAD patients as compared with healthy subjects. B2R stimulation induced CD18 activation in early outgrowth cells of healthy subjects, but not in early outgrowth cells of CAD patients. Adenoviral B2R overexpression enhanced in vivo vascular recruitment and rescued impaired endothelial repair capacity of early outgrowth cells from CAD patients. CONCLUSIONS: We newly report that bradykinin/B2R signaling may promote endothelial repair after arterial injury by selective recruitment and functional activation of B2R-expressing circulating mononuclear cell subsets. In CAD patients, B2R downregulation on endothelial repair-promoting circulating mononuclear cells substantially impairs the bradykinin-dependent endothelial repair, representing a novel mechanism promoting endothelial injury in CAD patients.