Bradykinin receptor participates in doxorubicin-induced cardiotoxicity by modulating iNOS signal pathway.

Doxorubicin (DOX), an effective and broad-spectrum anthracycline antibiotic, is widely used in the treatment of numerous malignancies. However, dose-dependent cardiotoxicity limits the clinical application of DOX, and the molecular mechanisms are still unknown. In this study, we used the BK receptor B1/B2 double-knockout (B1B2 -/- ) mice to observe the role of BK receptor in cardiotoxicity induced by DOX and the underlying mechanisms. DOX induced myocardial injury with increased serum levels of AST, CK, and LDH, upregulated tissue expression of bradykinin B1/B2 receptor, FABP4 and iNOS, and downregulated expression of eNOS. However, these altered releases of myocardial enzyme and the expression level of iNOS were significantly prevented in the B1B2-/- mice. We concluded that the activation of both B1 and B2 receptors of BK were involved in the DOX-induced acute myocardial injury, possibly mediated through iNOS signaling pathways.

In vivo near-infrared fluorescence and SPECT-CT imaging of colorectal Cancer using the bradykinin B2R-specific ligand icatibant.

Cancer molecular imaging using specific probes designed to identify target proteins in cancer is a powerful tool to guide therapeutic selection, patient management, and follow-up. We demonstrated that icatibant may be used as a targeting probe for the significantly upregulated bradykinin B2R in colorectal cancer (CRC). Icatibant-based probes with high affinity towards bradykinin B2R were identified. The near-infrared (NIR) fluorescent dye conjugate MPA-PEG3-k-Icatibant and radioconjugate [99mTc]Tc-HYNIC-PEG4-Icatibant exhibited favourable selective and specific uptake in tumours when the subcutaneous and orthotopic colorectal tumour-bearing mouse models were imaged using NIR fluorescence imaging and Single-Photon Emission Computed Tomography-Computed Tomography (SPECT-CT), respectively. The tracer of [99mTc]Tc-HYNIC-PEG4-Icatibant accumulated in tumours according to biodistribution studies and peaked at 4 h with an uptake value of 3.41 ± 0.27%ID/g in HT29 tumour-bearing nude mice following intravenous injection (i.v.). The tumour-to-colorectal signal ratios were 5.03 ± 0.37, 15.45 ± 0.32, 13.58 ± 1.19 and 11.33 ± 1.73 1, 2, 4 and 6 h after tail-veil injection, respectively. Overall, in the wake of rapid and precise tumour delineation and penetration characteristics, icatibant-based probes represent promising high-contrast molecular imaging probes for the detection of bradykinin B2R.

Function and structure of bradykinin receptor 2 for drug discovery.

Type 2 bradykinin receptor (B2R) is an essential G protein-coupled receptor (GPCR) that regulates the cardiovascular system as a vasodepressor. Dysfunction of B2R is also closely related to cancers and hereditary angioedema (HAE). Although several B2R agonists and antagonists have been developed, icatibant is the only B2R antagonist clinically used for treating HAE. The recently determined structures of B2R have provided molecular insights into the functions and regulation of B2R, which shed light on structure-based drug design for the treatment of B2R-related diseases. In this review, we summarize the structure and function of B2R in relation to drug discovery and discuss future research directions to elucidate the remaining unknown functions of B2R dimerization.

The Role of Bradykinin Receptors in Hereditary Angioedema Due to C1-Inhibitor Deficiency.

BACKGROUND: Hereditary angioedema (HAE) is a rare, genetic disease caused by the decreased level or function of the C1 inhibitor. The primary mediator of symptoms in HAE is bradykinin acting through its two receptors, namely receptors 1 (BR1) and 2 (BR2). Although BR2 is well characterized, the role of BR1 remains unclear. OBJECTIVE: To study the role of bradykinin receptors 1 (BR1) in the etiopathogenesis of HAE. METHODS: A total of 70 individuals, 40 patients with HAE, and 30 healthy subjects were recruited to the study. HAE was diagnosed in accordance with the international guideline. The level of bradykinin receptors was determined in populations of CD3+, CD4+, CD8+, and CD14++CD16-, CD14++CD16+ monocytes. In addition, the level of disease activity-specific markers was measured. RESULTS: There were statistically significant differences in the subpopulation of lymphocytes and monocytes between patients with HAE compared to healthy subjects. The level of BR1 and BR2 on PBMCs was comparable in healthy subjects and HAE patients during remission with significant overexpression of both receptors, triggered by HAE attack. Moreover, a significant increase in TNF-alpha and IL-1 plasma levels was observed among HAE patients. CONCLUSIONS: BR1 expression may play an important role in the pathomechanism of HAE.

Sex-specific Regulation of Prolactin Secretion by Pituitary Bradykinin Receptors.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Are noscapine and raloxifene ligands of the bradykinin B2 receptor? An assessment based on the human umbilical vein contractility assay.

The centrally acting antitussive opiate derivative, noscapine, has been claimed to be a non-competitive bradykinin B2 receptor antagonist. Raloxifene, a selective estrogen receptor modulator, was predicted to bind the bradykinin B2 receptor and to exert a partial agonist activity. These intriguing claims suggest that new molecular scaffolds ("chemotypes") may be identified for small molecule ligands of kinin receptors and that some off-target effects of noscapine or raloxifene may be mediated by bradykinin B2 receptors. An established contractile bioassay for ligands of the bradykinin B2 receptor, the isolated human umbilical vein, was exploited to characterize the inhibitory effect of noscapine and raloxifene on the B2 receptor-mediated contractile response to bradykinin. Observed effects were compared with those of the peptide antagonist icatibant, a potent, selective and competitive B2 receptor antagonist. Our results indicate that neither noscapine (2.5 µM) nor raloxifene (20 µM) behave as B2 receptor antagonists in concentrations that vastly exceeded an effective concentration of the control antagonist, icatibant; further, none of these drugs had direct contractile effects. It is suggested that the previously reported B2 receptor inhibitory effect of noscapine, a putative sigma-receptor agonist, might result from an indirect physiological antagonism, while raloxifene did not appear to have any significant affinity for the B2 receptors.

Raf kinase inhibitory protein reduces bradykinin receptor desensitization.

Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin-stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G-protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down-regulation increases BK receptor desensitization in real-time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2-sensitive G-protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.

A comprehensive review on current understanding of bradykinin in COVID-19 and inflammatory diseases.

Bradykinin, a member of the kallikrein-kinin system (KKS), is a potent, short-lived vasoactive peptide that acts as a vasodilator and an inflammatory mediator in a number of signaling mechanisms. Bradykinin induced signaling is mediated through kinin B1 (BDKRB1) and B2 (BDKRB2) transmembrane receptors coupled with different subunits of G proteins (Gαi/Gα0, Gαq and Gß1γ2). The bradykinin-mediated signaling mechanism activates excessive pro-inflammatory cytokines, including IL-6, IL-1ß, IL-8 and IL-2. Upregulation of these cytokines has implications in a wide range of clinical conditions such as inflammation leading to fibrosis, cardiovascular diseases, and most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In SARS-CoV-2 infection, bradykinin is found to be at raised levels and is reported to trigger a diverse array of symptoms. All of this brings bradykinin to the core point as a molecule of immense therapeutic value. Our understanding of its involvement in various pathways has expanded with time. Therefore, there is a need to look at the overall picture that emerges from the developments made by deciphering the bradykinin mediated signaling mechanisms involved in the pathological conditions. It will help devise strategies for developing better treatment modalities in the implicated diseases. This review summarizes the current state of knowledge on bradykinin mediated signaling in the diverse conditions described above, with a marked emphasis on the therapeutic potential of targeting the bradykinin receptor.

Kinin B1 receptor deficiency protects mice fed by cafeteria diet from abnormal glucose homeostasis.

The kallikrein-kinin system has been implicated in body weight and glucose homeostasis. Their major effectors act by binding to the kinin B2 and B1 receptors. It was assessed the role of the kinin B1 receptor in weight and glucose homeostasis in B1 receptor knockout mice (B1RKO) subjected to a cafeteria diet (CAF). Wild-type (WT) and B1RKO male mice (C57BL/6 background; 8 weeks old) were fed a standard diet (SD) or CAF for 14 weeks, ad libitum, and four groups were formed: WT-SD; B1RKO-SD; WT-CAF; B1RKO-CAF. Body weight and food intake were assessed weekly. It was performed glucose tolerance (GTT) and insulin tolerance tests (ITT), and HOMA-IR, HOMA-ß and HOMA-ß* 1/HOMA-IR were calculated. Islets from WT and B1RKO were isolated in order to measure the insulin secretion. Western blot was used to assess the hepatic AKT phosphorylation and qPCR to assess gene expression. CAF induced a higher body mass gain in B1RKO compared to WT mice. CAF diet increased epididymal fat depot mass, hepatic fat infiltration and hepatic AKT phosphorylation in both genotypes. However, B1RKO mice presented lower glycemic response during GTT when fed with CAF, and a lower glucose decrease in the ITT. This higher resistance was overcomed with higher insulin secretion when stimulated by high glucose, resulting in higher glucose uptake in the GTT when submitted to CAF, despite lower insulin sensitivity. Islets from B1RKO delivered 4 times more insulin in 3-month-old mice than islets from WT. The higher insulin disposition index and high insulin delivery of B1RKO can explain the decreased glucose excursion during GTT. In conclusion, CAF increased the ß-cell function in B1RKO mice, compensated by the diet-induced insulin resistance and resulting in a healthier glycemic response despite the higher weight gain.

Exogenous pancreatic kininogenase protects against tacrolimus-induced renal injury by inhibiting PI3K/AKT signaling: The role of bradykinin receptors.

BACKGROUND: Tissue kallikrein offers a wide spectrum of biological activity in the protection against various types of injury. However, information on its role in tacrolimus (TAC)-induced renal injury is limited. OBJECTIVES: This study aimed to assess the beneficial effects of pancreatic kininogenase (PK) in a rat model of chronic TAC nephrotoxicity and in vitro. METHODS: Sprague Dawley rats were treated daily with either TAC or PK or a combination of the two for four weeks. The influence of PK on renal injury was examined in terms of renal function, histopathology, cytokine expression, oxidative stress, intracellular organelles, programmed cell death, and PI3K/AKT signaling. Human kidney proximal tubular (HK-2) cells and mouse mesangial (SV40 MES13) cells treated with TAC and PK were also studied. RESULTS: PK treatment improved renal function and histopathology. This effect was paralleled by downregulation of proinflammatory and profibrotic cytokine expression. TAC-induced oxidative stress was closely associated with endoplasmic reticulum stress and mitochondrial dysfunction, resulting in excessive programmed cell death (apoptosis and autophagy) that was significantly abrogated by concurrent PK interference with PI3K/AKT signaling. PK also stimulated bradykinin receptor 1 (B1R) and B2R mRNA synthesis and increased bioactive nitric oxide (NO) and cAMP concentrations in TAC-treated kidneys. Blockade of either B1R or B2R eliminated the renoprotective effects of PK. In HK-2 and SV40 MES13 cells, PK decreased TAC-induced overproduction of intracellular reactive oxygen species and inhibited apoptotic cells, whereas cell viability was improved. Moreover, activated PI3K/AKT signaling in HK-2 cells was inhibited by PK and the PI3K inhibitor, LY294002. CONCLUSIONS: These findings indicate that PK treatment protects against chronic TAC nephrotoxicity via inhibition of PI3K/AKT signaling.

Remimazolam alleviates neuropathic pain via regulating bradykinin receptor B1 and autophagy.

OBJECTIVES: Neuropathic pain (NP) represents a broad scope of various pathological ramifications of the nervous system. Remimazolam is a proved sedative in treating neuropathic pain. Considering the Bradykinin receptor's vital role and the potentials of Bradykinin receptor B1 (BDKRB1) in the neuropathic pain-signalling pathway, we nominated them as a primary target for remimazolam. METHODS: In this study, rats were injected with complete freund's adjuvant (CFA) to construct NP models in vivo. BV2 microglia cells were treated with LPS to establish NP model in vitro. qRT-PCR, ELISA, western blot and immunofluorescence were applied to determine gene expression. KEY FINDINGS: Our findings revealed that BDKRB1 was overexpressed in NP models in vivo, while R715 (an antagonist of BDKRB1) suppressed the levels of BDKRB1 and inhibited the hyperpathia induced by spinal nerve litigation surgery. Moreover, remimazolam inactivated BDKRB1 signalling via suppressing NF-κB translocation and decreased the release of pro-inflammatory cytokines. Additionally, remimazolam suppressed the translocation of NF-κB, and inhibited autophagic lysosome formation in vivo and in vitro. However, R838 (an agonist of BDKRB1) reversed the effects of remimazolam. CONCLUSIONS: Remimazolam downregulated BDKRB1, inhibited BDKRB1/RAS/MEK signalling pathway and regulated the autophagic lysosome induction, exhibiting a better outcome in the NP.

Intermittent hypoxia changes the interaction of the kinin-VEGF system and impairs myocardial angiogenesis in the hypertrophic heart.

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension-related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein-kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1-5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.

Inhibitors of angiotensin I converting enzyme potentiate fibromyalgia-like pain symptoms via kinin receptors in mice.

Fibromyalgia is a potentially disabling chronic disease, characterized by widespread pain and a range of comorbidities such as hypertension. Among the mechanisms involved in fibromyalgia-like pain symptoms are kinins and their B1 and B2 receptors. Moreover, angiotensin I converting enzyme (ACE) inhibitors, commonly used as antihypertensive drugs, can enhance pain by blocking the degradation of peptides such as substance P and bradykinin, besides enhancing kinin receptors signalling. We investigated the effect of ACE inhibitors on reserpine-induced fibromyalgia-like pain symptoms and the involvement of kinins in this effect in mice. Nociceptive parameters (mechanical and cold allodynia and overt nociception) were evaluated after ACE inhibitors administration in mice previously treated with reserpine. The role of kinin B1 and B2 receptors was investigated using pharmacological antagonism. Additionally, bradykinin levels, as well as the activity of ACE and kininase I, were measured in the sciatic nerve, spinal cord and cerebral cortex of the mice. The ACE inhibitors enalapril and captopril enhanced reserpine-induced mechanical allodynia, and this increase was prevented by kinin B1 and B2 receptor antagonists. Substance P and bradykinin caused overt nociception and increased mechanical allodynia in animals treated with reserpine. Reserpine plus ACE inhibitors increased bradykinin-related peptide levels and inhibited ACE activity in pain modulation structures. Since hypertension is a frequent comorbidity affecting fibromyalgia patients, hypertension treatment with ACE inhibitors in these patients should be reviewed once this could enhance fibromyalgia-like pain symptoms. Thus, the treatment of hypertensive patients with fibromyalgia could include other classes of antihypertensive drugs, different from ACE inhibitors.

Blockade of bradykinin receptors or angiotensin II type 2 receptor prevents paclitaxel-associated acute pain syndrome in mice.

BACKGROUND: Paclitaxel (PCX) is the first-line choice for the treatment of several types of cancer, including breast, ovarian, and lung cancers. However, patients who receive even a single dose with PCX commonly develop mechanical and cold allodynia, a symptom known as PCX-associated acute pain syndrome (P-APS). Here, we assessed possible involvement of kinin-kallikrein and renin-angiotensin systems in P-APS in mice. METHODS: Male mice C57Bl/6 wild type (WT) and knockouts for bradykinin receptors, B1 (B1-/- ) and B2 (B2-/- ), were used. Mechanical and cold allodynia were evaluated by using von Frey filaments and acetone test, respectively. P-APS was induced by administration of PCX 4 mg/kg, i.v.. ACE inhibitors (captopril and enalapril), antagonists for angiotensin II type 1 (losartan) and type 2 ([AT2R]; PD123319 and EMA 401) receptors were administrated prior the treatment with PCX. RT-PCR was used to analyse the expression of mRNA for B1, B2 and AT2R receptors. RESULTS: Administration of PCX in B1-/- and B2-/- mice induced lower mechanical and cold allodynia compared to the WT. However, the pre-treatment with ACE inhibitors reduced the development of mechanical and cold allodynia in P-APS. Surprisingly, we found that mice pre-treatment with the PD123319 or EMA401, but not losartan, prevented the development of mechanical and cold allodynia induced by PCX. CONCLUSION: Our results demonstrated the involvement of bradykinin receptors B1 and B2 as well as AT2R in the induction of P-APS in mice, and suggest the use of AT2R antagonists as a potential therapy for the prevention of P-APS in humans. SIGNIFICANCE: Kinin-kallikrein and renin-angiotensin systems, through B1, B2 and AT2 receptors, potentiates paclitaxel-associated acute pain syndrome (P-APS) in mice. Antagonists for AT2R are potential alternatives to prevent P-APS.

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva.

The regulation of the kallikrein-kinin system is an important mechanism controlling vasodilation and promoting inflammation. We aimed to investigate the role of Toll-like receptor 2 (TLR2) in regulating kinin B1 and B2 receptor expression in human gingival fibroblasts and in mouse gingiva. Both P. gingivalis LPS and the synthetic TLR2 agonist Pam2CSK4 increased kinin receptor transcripts. Silencing of TLR2, but not of TLR4, inhibited the induction of kinin receptor transcripts by both P. gingivalis LPS and Pam2CSK4. Human gingival fibroblasts (HGF) exposed to Pam2CSK4 increased binding sites for bradykinin (BK, B2 receptor agonist) and des-Arg10-Lys-bradykinin (DALBK, B1 receptor agonist). Pre-treatment of HGF for 24 h with Pam2CSK4 resulted in increased PGE2 release in response to BK and DALBK. The increase of B1 and B2 receptor transcripts by P. gingivalis LPS was not blocked by IL-1ß neutralizing antibody; TNF-α blocking antibody did not affect B1 receptor up-regulation, but partially blocked increase of B2 receptor mRNA. Injection of P. gingivalis LPS in mouse gingiva induced an increase of B1 and B2 receptor mRNA. These data show that activation of TLR2 in human gingival fibroblasts as well as in mouse gingival tissue leads to increase of B1 and B2 receptor mRNA and protein.

Implication of the Kallikrein-Kinin system in neurological disorders: Quest for potential biomarkers and mechanisms.

Neurological disorders represent major health concerns in terms of comorbidity and mortality worldwide. Despite a tremendous increase in our understanding of the pathophysiological processes involved in disease progression and prevention, the accumulated knowledge so far resulted in relatively moderate translational benefits in terms of therapeutic interventions and enhanced clinical outcomes. Aiming at specific neural molecular pathways, different strategies have been geared to target the development and progression of such disorders. The kallikrein-kinin system (KKS) is among the most delineated candidate systems due to its ubiquitous roles mediating several of the pathophysiological features of these neurological disorders as well as being implicated in regulating various brain functions. Several experimental KKS models revealed that the inhibition or stimulation of the two receptors of the KKS system (B1R and B2R) can exhibit neuroprotective and/or adverse pathological outcomes. This updated review provides background details of the KKS components and their functions in different neurological disorders including temporal lobe epilepsy, traumatic brain injury, stroke, spinal cord injury, Alzheimer's disease, multiple sclerosis and glioma. Finally, this work will highlight the putative roles of the KKS components as potential neurotherapeutic targets and provide future perspectives on the possibility of translating these findings into potential clinical biomarkers in neurological disease.

[Significance of kallikrein-kinin system in central nervous system diseases].

Kallikreins cleave kininogens to release kinins. Kinins exert their biological effect by activating constitutive bradykinin receptor-2 (BR2) and inducible by inflammatory cytokines bradykinin receptor-1 (BR1). Studies in animal models and some clinical observations indicate tat the activation of kallikrein - kinin system may have relevance to central nervous system (CNS) diseases, including multiple sclerosis, Alzheimer's disease, epilepsy as well as cerebral ischemia and neoplasmatic tumors. The actions of kinins include vasodilatation and increased vascular permeability may contribute to blood-brain barrier disruption. Kinins evoke pain, and stimulate of endothelial cells, white blood cells, astrocytes and microgia cells to release of prostanoids, cytokines, free radicals, nitric oxide. Kinins stimulate angiogenesis and proliferation of tumor cells. These events lead to neural tissue damage and long lasting disturbances in blood-brain barrier function. In animal models the overexpression of genes and proteins of tissue kallikrens, kininogen as well as RB1 and RB2 has been observed. Kinin receptors antagonists, especially B1R blockade decreased morphological and biochemical features of CNS inflammation. On the other hand in brain tumor models RB1 and RB2 activation has been shown to mediate reversible blood-brain barrier permeability to enhance anti-cancer drug delivery, which may have therapeutic potential.

A cyclic pathway of P2 × 7, bradykinin, and dopamine receptor activation induces a sustained articular hyperalgesia in the knee joint of rats.

OBJECTIVE: We investigated whether: (1) P2 × 7 receptor activation by its agonist (BzATP) induces articular hyperalgesia in the rat's knee joint via inflammatory mechanisms and (2) activation of P2 × 7 receptors by endogenous ATP contributes to the articular hyperalgesia induced by bradykinin, TNF-α, IL-1ß, CINC-1, PGE2, and dopamine. METHODS: The articular hyperalgesia was quantified using the rat knee joint incapacitation test. The knee joint inflammation, characterized by the concentration of pro-inflammatory cytokines and by neutrophil migration, was quantified in the synovial lavage fluid by ELISA and myeloperoxidase enzyme activity assay, respectively. RESULTS: BzATP induced a dose-dependent articular hyperalgesia in the rat's knee joint that was significantly reduced by the selective antagonists for P2 × 7, bradykinin B1 or B2 receptors, ß1 or ß2 adrenoceptors, and by pre-treatment with Indomethacin. BzATP induced a local increase of TNF-α, IL-1ß, IL-6, and CINC-1 concentration and neutrophil migration into the knee joint. The co-administration of the selective P2 × 7 receptor antagonist A-740003 significantly reduced the articular hyperalgesia induced by bradykinin and dopamine, but not by TNF-α, IL-1ß, CINC-1, and PGE2. CONCLUSIONS: P2 × 7 receptor activation induces articular hyperalgesia mediated by the previous inflammatory mediator release. P2 × 7 receptor-induced articular hyperalgesia is sustained by the involvement of this purinergic receptor in bradykinin and dopamine-induced hyperalgesia in the knee joint.

Bradykinin system is involved in endometriosis-related pain through endothelin-1 production.

BACKGROUND: Endometriosis is a gynaecological disease exhibiting severe pelvic pain, but the mechanism of pain production remains unknown. Bradykinin (BK) is known as an inflammatory mediator, and shows elevated levels in inflammatory diseases such as rheumatoid arthritis. In the present study, we evaluated whether BK is involved in endometriosis-related pain. METHODS: Endometriotic lesions were used for immunohistochemistry. Primary cultures of endometriotic stromal cells (ESC) were stimulated with IL-1ß and/or BK. Quantitative RT-PCR was used to evaluate the mRNA expressions of BK receptors (BKR) and endothelin-1 in ESC. The concentration of endothelin-1 in cystic fluid of endometrioma or non-endometrioma was measured with ELISA. The conditioned medium of ESC stimulated with IL-1ß and/or BK was injected intraplantarly in mice, and evaluated whether pain-related licking behaviour was elicited. RESULTS: The expressions of BK and BKR in endometriotic lesions were observed by immunohistochemistry. In vitro experiments showed that IL-1ß induced BKR-B1 and B2 on ESC. Activation of these receptors by BK significantly induced endothelin-1 expression in ESC, which was negated completely by HOE-140, a BKR-B2 antagonist. The cystic fluid of endometrioma contained higher amount of endothelin-1 compared to non-endometrioma. Intraplantar injection of the conditioned medium of ESC treated with IL-1ß and BK significantly induced licking behaviour, which was suppressed with BQ-123, an endothelin type-A receptor antagonist. CONCLUSIONS: The present study demonstrated the presence and the function of the BK axis in endometriosis, and established a potential new therapy target for endometriosis-related pain. SIGNIFICANCE: The present study demonstrated (1) the presence and the function of the BK system in endometriosis, (2) activation of BKR induced endothelin-1 in endometriotic lesion and (3) blocking endothelin-1 was effective to decrease pain.

Effect of C1-INH on ischemia/reperfusion injury in a porcine limb ex vivo perfusion model.

Revascularization of an amputated limb within 4-6h is essential to avoid extensive ischemia/reperfusion (I/R) injury leading to vascular leakage, edema and tissue necrosis. I/R injury is a pathological inflammatory condition that occurs during reperfusion of an organ or tissue after prolonged ischemia. It is characterized by a complex crosstalk between endothelial cell activation and the activation of plasma cascades. Vasculoprotective pharmacological intervention to prevent I/R injury might be an option to prolong the time window between limb amputation and successful replantation. We used C1-easterase inhibitor (C1-INH) in this study because of its known inhibitory effects on the activation of the complement, coagulation and kinin cascades. Forelimbs of 8 large white pigs were amputated, subjected to ischemia, and then reperfused with autologous whole blood. All limbs were exposed to 9h of cold ischemia at 4°C. After 2h of cold ischemia the limbs were either perfused with of C1-INH (1U/ml in hydroxyethyl starch, n=8) or hydroxyethyl starch alone (n=7). After completion of the 9-h ischemia period, all limbs were ex vivo perfused with heparinized autologous whole blood for 12h using a pediatric heart lung machine to simulate in vivo revascularization. Our results show that I/R injury in the control group led to a significant elevation of tissue deposition of IgG and IgM, complement C3b/c, C5b-9 and MBL. Also, activation of the kinin system was significantly increased, namely bradykinin in plasma, and expression of bradykinin receptors 1 and 2 in tissue. In addition, markers for endothelial integrity like expression of CD31, VE-cadherin and heparan sulfate proteoglycans were decreased in reperfused tissue. Limb I/R injury also led to activation of the coagulation cascade with a significant elevation of fibrin and thrombin deposition and increased fibrinogen-like protein-2 expression. C1-INH treated limbs showed much less activation of plasma cascades and better protection of endothelial integrity compared to the reperfused control limbs. In conclusion, the use of the cytoprotective drug C1-INH significantly reduced I/R injury by protecting the vascular endothelium as well as the muscle tissue from deposition of immunoglobulins, complement and fibrin.