Bradykinin B1 Receptor Expression Research Articles

Tissue Kallikrein-1 Suppresses Type I Interferon Responses and Reduces Depressive-Like Behavior in the MRL/lpr Lupus-Prone Mouse Model.

Excessive production and response to Type I interferons (IFNs) is a hallmark of systemic lupus erythematosus (SLE). Neuropsychiatric lupus (NPSLE) is a common manifestation of human SLE, with major depression as the most common presentation. Clinical studies have demonstrated that IFNα can cause depressive symptoms. We have shown that the kallikrein-kinin system (KKS) [comprised of kallikreins (Klks) and bradykinins] and angiotensin-converting enzyme inhibitors suppressed Type I IFN responses in dendritic cells from lupus-prone mice and human peripheral blood mononuclear cells. Tissue Klk genes are decreased in patients with lupus, and giving exogenous Klk1 ameliorated kidney pathology in mice. We retro-orbitally administered mouse klk1 gene-carrying adenovirus in the Murphy Roths Large lymphoproliferative (MRL/lpr) lupus-prone mice at early disease onset and analyzed immune responses and depressive-like behavior. Klk1 improved depressive-like behavior, suppressed interferon-responsive genes and neuroinflammation, and reduced plasma IFNα levels and proinflammatory cytokines. Klk1 also reduced IFNAR1 and JAK1 protein expression, important upstream molecules in Type I IFN signaling. Klk1 reduced bradykinin B1 receptor expression, which is known to induce proinflammatory response. Together, these findings suggest that Klk1 may be a potential therapeutic candidate to control IFNα production/responses and other inflammatory responses in SLE and NPSLE.

Electroacupuncture alleviates diabetes-induced mechanical allodynia and downregulates bradykinin B1 receptor expression in spinal cord dorsal horn.

Diabetic neuropathic pain (DNP) is one of the most prevalent symptoms of diabetes. The alteration of proteins in the spinal cord dorsal horn (SCDH) plays a significant role in the genesis and the development of DNP. Our previous study has shown electroacupuncture could effectively relieve DNP. However, the potential mechanism inducing DNP's genesis and development remains unclear and needs further research. This study established DNP model rats by intraperitoneally injecting a single high-dose streptozotocin; 2 Hz electroacupuncture was used to stimulate Zusanli (ST36) and Kunlun (BL60) of DNP rats daily from day 15 to day 21 after streptozotocin injection. Behavioral assay, quantitative PCR, immunofluorescence staining, and western blotting were used to study the analgesic mechanism of electroacupuncture. The bradykinin B1 receptor (B1R) mRNA, nuclear factor-κB p65 (p65), substance P, and calcitonin gene-related peptide (CGRP) protein expression were significantly enhanced in SCDH of DNP rats. The paw withdrawal threshold was increased while body weight and fasting blood glucose did not change in DNP rats after the electroacupuncture treatment. The expression of B1R, p65, substance P, and CGRP in SCDH of DNP rats was also inhibited after the electroacupuncture treatment. This work suggests that the potential mechanisms inducing the allodynia of DNP rats were possibly related to the increased expression of B1R, p65, substance P, and CGRP in SCDH. Downregulating B1R, p65, substance P, and CGRP expression levels in SCDH may achieve the analgesic effect of 2 Hz electroacupuncture treatment.

Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice.

Bradykinin B1 receptor (B1R) has garnered attention as a cancer therapeutic and diagnostic target. Several reports on radiolabelled derivatives of B1R antagonists have shown favourable properties as imaging agents in cells highly expressing hB1R following transfection. In the present study, we assessed whether radiolabelled probes can detect B1R endogenously expressed in cancer cells. To this end, we evaluated 111In-labelled derivatives of a B1R antagonist ([111In]In-DOTA-Ahx-R954) using glioblastoma cell lines (U87MG and U251MG) with different B1R expression levels. Cellular uptake studies showed that the specific accumulation of [111In]In-DOTA-Ahx-R954 in U87MG was higher than that in U251MG, which correlated with B1R expression levels. Tissue distribution in U87MG-bearing mice revealed approximately 2-fold higher radioactivity in tumours than in the muscle in the contralateral leg. The specific accumulation of [111In]In-DOTA-Ahx-R954 in the tumour was demonstrated by the reduction in the tumour-to-plasma ratios in nonlabelled R954-treated mice. Moreover, ex vivo autoradiographic images revealed that the intratumoural distribution of [111In]In-DOTA-Ahx-R954 correlated with the localisation of B1R-expressing glioblastoma cells. In conclusion, we demonstrated that [111In]In-DOTA-Ahx-R954 radioactivity correlated with B1R expression in glioblastoma cells, indicating that radiolabelled derivatives of the B1R antagonist could serve as promising tools for elucidating the involvement of B1R in cancer.

B2R-Targeting Radiotracer for PET/MR Imaging of Hepatocellular Carcinoma and Guiding Anti-B2R Therapy.

The bradykinin B2 receptor (B2R) is overexpressed in a wide variety of tumors and is a well-defined target for tumor imaging and therapy. The hybrid positron emission tomography/magnetic resonance imaging (PET/MRI) scanner is considered a noninvasive and advanced instrument for precise tumor imaging. In this work, we developed a novel B2R-targeting radiotracer, 68Ga-DOTA-icatibant, for quantifying B2R expression. 68Ga-DOTA-icatibant showed high stability, fast clearance and specific binding to B2R. PET/MR imaging revealed excellent tumor accumulation, and the uptake in tumors could be blocked by DOTA-icatibant. Icatibant-mediated anti-B2R therapy downregulated B2R expression in tumor cells and inhibited the growth of HepG2 tumors, and the decrease in tumor uptake was monitored by timely PET/MR imaging. Hematoxylin and eosin (H&E) and immunohistochemical staining results further demonstrated that the efficacy of anti-B2R could be accurately monitored with the developed PET/MR imaging radiotracer. 68Ga-DOTA-icatibant can be utilized to noninvasively determine B2R expression and dynamically and sensitively monitor the efficacy of anti-B2R therapy.

Deletion of AT1a Receptors Selectively in Renomedullary Interstitial Cells Attenuates Basal, Angiotensin II-, or Vasopressin-induced Vasoactive Factors' mRNA Expression in The Mouse Kidney Medulla

We recently shown that deletion of the vasoactive peptide angiotensin II (Ang II) AT1a receptors selectively in renomedullary interstitial cells (RMICs) of the kidney medulla significantly lowered basal blood pressure and impaired urine concentration in mice. However, the underlying signaling mechanisms involved remains poorly understood. In the present study, AT1a receptors were selectively deleted in the RMICs of the mouse kidney medulla using tamoxifen-inducible Tenascin-C-CreER2/Agtr1af/f approach. Three groups of adult male wild-type (WT) and RMIC- Agtr1a−/− mice (n=6-10) were pretreated with or without tamoxifen (150 mg/kg, i.p., for 5 days) before Ang II was infused to induce Ang II-dependent hypertension (~0.75 mg/kg/day, i.p., for 2 weeks). Systolic, diastolic, and mean arterial blood pressure and urine osmolality were determined before and after Ang II treatment. At the end of the experiment, the kidneys were harvested, and the inner and outer medulla were dissected out for the measurement of the mRNA expression of key vasoactive factors that are associated with the regulation of blood pressure and urine concentration using digital droplet PCR. Deletion of AT1a receptors in RMICs significantly decreased AT1a receptor mRNA expression by 41% (WT: 554 ± 49 vs. RMIC- Agtr1a−/−: 327 ± 10 copies/ng RNA; P<0.01) and bradykinin B2 receptor mRNA expression by 52% (WT: 10.10 ± 0.75 vs. RMIC- Agtr1a−/−: 4.90 ± 0.56 copies/ng RNA; P<0.01), without altering the expression of endothelin ETB (WT: 466 ± 21 vs. RMIC- Agtr1a−/−: 393 ± 24 copies/ng RNA; n.s.) and vasopressin V2 receptor mRNAs in the kidney medulla (WT: 580 ± 60 vs. RMIC- Agtr1a−/−: 586 ± 61 copies/ng RNA; n.s.). Deletion of AT1a receptors in RMICs also significantly decreased the expression of eNOS (WT: 87.0 ± 3.0 vs. RMIC- Agtr1a−/−: 61.2 ± 2.8 copies/ng RNA; P<0.01) and aquaporin 2 (AQP2) mRNAs (WT: 12463 ± 2015 vs. RMIC- Agtr1a−/−: 6613 ± 692 copies/ng RNA; P<0.01). Interestingly, deletion of AT1a receptors in RMICs did not alter the mRNA expression of cyclooxygenase-2 (COX-2, n.s.), prostaglandin E2 (PGE2, n.s.), epithelial sodium channel (ENaC, n.s.) or prorenin receptors (PRR, n.s.) in the kidney medulla. In response to Ang II infusion, deletion of AT1a receptors in RMICs significantly attenuated Ang II-induced expression of bradykinin B2 receptors, COX-2, eNOS, and AQP2 mRNAs ( P<0.01), respectively. Furthermore, deletion of AT1a receptors in RMICs also significantly attenuated vasopressin-induced bradykinin B2 receptor, eNOS, and AQP2 mRNA expression in the kidney medulla ( P<0.01). We concluded that AT1a receptors in RMICs in the kidney medulla play an important role in maintaining physiological blood pressure homeostasis and urine concentration process likely via AT1a receptor/bradykinin B2 receptor/eNOS/AQP2-mediated signaling pathways. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway

Apelin receptor (APJ) and bradykinin B2 receptor (B2R) play an important role in many physiological processes and share multiple similar characteristics in distribution and functions in the cardiovascular system. We first identified the endogenous expression of APJ and B2R in human umbilical vein endothelial cells (HUVECs) and their co-localization on human embryonic kidney (HEK) 293 cells membrane. A suite of bioluminescence and fluorescence resonance energy transfer (BRET and FRET), proximity ligation assay (PLA), and co-immunoprecipitation (Co-IP) was exploited to demonstrate formation of functional APJ and B2R heterodimer in HUVECs and transfected cells. Stimulation with apelin-13 and bradykinin (BK) increased the phosphorylation of the endothelial nitric oxide synthase (eNOS) in HUVECs, which could be inhibited by the silencing of APJ or B2R, indicating the APJ-B2R dimer is critical for eNOS phosphorylation in HUVECs. Furthermore, the increase of NOS and extracellular signal regulated kinases1/2 (ERK1/2) phosphorylation mediated by APJ/B2R dimer can be inhibited by U0126 and U73122, respectively, suggesting that the heterodimer might activate the PLC/ERK1/2/eNOS signaling pathway, and finally leading to a significant increase in cell proliferation. Thus, we uncovered for the first time the existence of APJ-B2R heterodimer and provided a promising new target in cardiovascular therapeutics.

Down-regulation of B2R contributes to preeclampsia by inhibiting human trophoblast cell invasion and angiogenesis.

Bradykinin B2 receptor (B2R) was decreased in early chorionic villi of pregnancies who progressed to severe preeclampsia (PE), suggesting downregulation of B2R may be involved in the pathogenesis of PE. The aim of this study was to investigate the possible roles of B2R in the pathophysiology of PE and its function in trophoblastic cells. The expression of B2R in placentas from patients with early-onset severe PE (sPE) and LPS induced PE-like rats were detected. The roles of B2R in HTR-8/SVneo cells migration and invasion were analyzed through transfecting B2R overexpressing plasmid vector or B2R-specific siRNA. The effect of HTR-8/SVneo cells culture supernatant with high and low expressing B2R on human umbilical vein endothelial cells (HUVEC) capillary formation ability was also investigated. We found that B2R expression was significantly decreased in placentas of patients with sPE and PE-like rats. In addition, siRNA-mediated down-regulation of B2R markedly inhibited the migration and invasion of HTR-8/SVneo cells. Conversely, over-expression of B2R significantly promoted the migration and invasion of HTR-8/SVneo cells. Furthermore, the culture supernatant from B2R-overexpressed-HTR-8/SVneo cells promoted the capillary formation of HUVEC through increasing placental growth factor (PlGF) levels, while the culture supernatant from si-B2R-HTR-8/SVneo cells had the opposite effects. The decrease of B2R in placentas leads to the dysfunction of invasion, migration and angiogenesis of trophoblasts, which may be involved in the pathogenesis of PE.

Evaluation of long-term rt-PA effects on bEnd.3 endothelial cells under ischemic conditions; changes in ZO-1 expression and glycosylation of the bradykinin B2 receptor

Recombinant tissue plasminogen activator (rt-PA) has proven effective in the treatment of acute ischemic stroke, despite the increased risk of hemorrhagic transformation (HT), its major associated complication. Although it is known that HT is related to blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. We assessed time-dependent effects of rt-PA on the bEnd.3 murine brain endothelial cell line subjected either to normoxia or to 2.5 h of oxygen and glucose deprivation (OGD), evaluating a longer period than has previously been done, beyond 6 h post-reoxygenation. Parameters of cell viability, metabolic activity, ionic and transcellular permeability, as well as levels of claudin-5, zonula occludens-1 (ZO-1) and bradykinin B2 receptor (B2R) protein expression were analyzed at 24, 48 and 72 h post-reoxygenation with or without the administration of rt-PA. rt-PA treatment increased both the ionic and transcellular permeability until 72 h and did not modify cell viability or metabolic activity or the expression of claudin-5, ZO-1 and B2R under normoxia at any analyzed time. Under OGD conditions, rt-PA exacerbated OGD effects on metabolic activity from 48 to 72 h, increased transcellular permeability from 24 to 72 h, significantly decreased ZO-1 protein levels at the plasma membrane and increased B2R glycosylation at 72 h post-reoxygenation. Our findings suggest that a long-term analysis is necessary to elucidate time-dependent molecular mechanisms associated to BBB breakdown due to rt-PA administration under ischemia. Thus, protective BBB therapies after ischemic stroke and rt-PA treatment should be explored at least until 72 h after OGD and rt-PA administration.

Expression of B2 Receptor on Circulating CD34-Positive Cells and Outcomes of Myocardial Infarction.

Background Bradykinin B2 receptor (B2R) is a widely expressed cell surface receptor. The relationship between B2R expression on circulating CD34+ cells and prognosis of myocardial infarction remains unknown. Methods We analyzed the expression of B2R on circulating CD34-positive cells and plasma VEGF concentration in 174 myocardial infarction patients. All involved patients were divided into two groups: high B2R group and low B2R group according to the median B2R expression percentage. 48 months of follow-up was performed. The endpoints were heart failure and revascularization. Results The plasma level of VEGF in the low B2R group is 67 ± 12 pg/mL, whereas the high B2R group has significantly elevated VEGF levels of 145 ± 27 pg/mL (P < 0.001). The concentration of VEGF has correlated with expression of B2R (r = 0.574, P < 0.001). During the 48 months of follow-up, low expression of B2 receptor on circulating CD34-positive cells indicates the high incidence of heart failure (hazard ratio: 2.247; 95% confidence interval: 1.110-4.547; P = 0.024) and revascularization (hazard ratio: 2.335; 95% confidence interval: 1.075-5.074; P = 0.032). Kaplan-Meier survival analysis showed that the cumulative hazard of heart failure (P = 0.014) and revascularization (P = 0.032) has significant differences between low B2R and high B2R. Conclusion Low expression of B2R on circulating progenitor cells indicated the poor outcomes of myocardial infarction.

MASP-1 Increases Endothelial Permeability.

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs—for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development.

Effects and potential mechanisms of bradykinin B2 receptor on growth and biological function of human extravillous trophoblast cells

Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells). Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized. HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group), pcDNA3.1-B2R (B2R expression plasmid group), siRNA negative control and B2R-specific siRNA, respectively. Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R, matrix metalloproteinase-2, matrix metalloproteinase-9, cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells. Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle, respectively. Cell migration assay and cell invasion assay were used to detect cell migration and invasion, respectively. Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells. All data were analyzed with t test. Results (1) Compared with the blank plasmid group, expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28, t=7.226, P=0.002) and protein levels (1.34±0.07 vs 1.00±0.05, t=3.727, P=0.006). And the expression of B2R in HTR-8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17, t=3.667, P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05, t=4.097, P=0.006) comparing with the siRNA negative control group. (2) Compared with the blank plasmid group, HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50±0.03 vs 1.34±0.04) promoting G0/G1 to S phase transition; compared with the siRNA negative control group, B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06±0.04 vs 1.20±0.02) and arrested the cell cycle at G0/G1 phase (all P<0.05). (3) Compared with the blank plasmid group, B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm], the number of cells penetrating matrigel gel (360.70±12.33 vs 268.70±14.45) and the number of cells having tube-like structures (28.20±2.47 vs 14.00±1.67), while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance: (56.00±3.51) vs (87.00±1.53) μm, number of cells penetrating matrigel gel: 143.30±12.91 vs 252.30±17.07; number of tube-like structures: 6.25±1.49 vs 15.75±2.02; all P<0.05]. (4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level, and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group, and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P<0.05). Conclusions B2R might enhance the activity, migration, invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2, matrix metalloproteinase-9, cyclin D1 and vascular endothelial growth factor-A. Key words: Receptor, bradykinin B2; Trophoblasts; Cell line; Cell proliferation

Role of selective blocking of bradykinin B1 receptor in attenuating immune liver injury in trichloroethylene-sensitized mice

Trichloroethylene (TCE) is able to induce trichloroethylene hypersensitivity syndrome (THS) with multi-system immune injuries. In our previous study, we found kallikrein-kinin system (KKS) activation, including the bradykinin B1 receptor (B1R), which contributed to immune organ injury in TCE sensitized mice. However, the mechanism of B1R mediating immune dysfunction is not clarified. The present study initiates to investigate the potential mechanism of B1R on liver injury. We establish a TCE sensitized BALB/c mouse model to explore the mechanism with or without a B1R inhibitor R715. We found B1R expression was increased in TCE sensitization-positive mice. As expect, hepatocyte intracellular organelles and mitochondria disappeared, glycogen particles reduced significantly as well in TCE sensitization-positive mice via the transmission electron microscopic examination, meanwhile, R715 alleviated the deteriorate above. The blockade of B1R resulted in a significant decreased p-ERK1/2 and increased p-AKT expression. The expression of CD68 kupffer cell and its relative cytokine, including IL-6 and TNF-α, increased in TCE sensitization-positive mice and decreased in R715 pretreatment TCE sensitization-positive mice. Together, the results demonstrate B1R plays a key role in ERK/MAPK and PI3K/AKT signal pathway activation and inflammation cytokine expression in immune liver injury induced by TCE. B1R exerts a pivotal role in the development of TCE induced liver injury.

Pretreatment with simvastatin upregulates expression of BK-2R and CD11b in the ischemic penumbra of rats.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductases, collectively known as statins, have been shown to minimize cerebral ischemic events in patients. We assessed the mechanisms of simvastatin pretreatment in preventing cerebral ischemia/reperfusion injury in rats using a model of middle cerebral artery occlusion (MCAO). Rats were pretreated with simvastatin 14 days prior to MCAO induction. At 3, 24, and 48 hours after reperfusion, bradykinin levels in the ischemic penumbra were assayed by ELISA, mRNA levels of bradykinin B2 receptors (BK-2Rs) and CD11b were measured by fluorescent quantitative real-time PCR (RT-PCR), and co-expression of microglia and BK-2Rs was determined by immunofluorescence. Simvastatin had no effect on bradykinin expression in the ischemic penumbra at any time point. However, the levels of BK-2R and CD11b mRNA in the ischemic penumbra, which were significantly decreased 3 hours after ischemia-reperfusion, were increased in simvastatin-pretreated rats. Moreover, the co-expression of BK-2Rs and microglia was confirmed by immunofluorescence analysis. These results suggest that the beneficial effects of simvastatin pretreatment before cerebral ischemia/reperfusion injury in rats may be partially due to increased expression of BK-2R and CD11b in the ischemic penumbra.

Bradykinin does not acutely sensitize the reflex pressor response during hindlimb skeletal muscle stretch in decerebrate rats.

Hindlimb skeletal muscle stretch (i.e., selective activation of the muscle mechanoreflex) in decerebrate rats evokes reflex increases in blood pressure and sympathetic nerve activity. Bradykinin has been found to sensitize mechanogated channels through a bradykinin B2 receptor-dependent mechanism. Moreover, bradykinin B2 receptor expression on sensory neurons is increased following chronic femoral artery ligation in the rat (a model of simulated peripheral artery disease). We tested the hypothesis that injection of bradykinin into the arterial supply of a hindlimb in decerebrate, unanesthetized rats would acutely augment (i.e., sensitize) the increase in blood pressure and renal sympathetic nerve activity during hindlimb muscle stretch to a greater extent in rats with a ligated femoral artery than in rats with a freely perfused femoral artery. The pressor response during static hindlimb muscle stretch was compared before and after hindlimb arterial injection of 0.5 µg of bradykinin. Injection of bradykinin increased blood pressure to a greater extent in "ligated" (n = 10) than "freely perfused" (n = 10) rats. The increase in blood pressure during hindlimb muscle stretch, however, was not different before vs. after bradykinin injection in freely perfused (14 ± 2 and 15 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.62) or ligated (15 ± 3 and 14 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.80) rats. Likewise, the increase in renal sympathetic nerve activity during stretch was not different before vs. after bradykinin injection in either group of rats. We conclude that bradykinin did not acutely sensitize the pressor response during hindlimb skeletal muscle stretch in freely perfused or ligated decerebrate rats.

Novel signaling of dynorphin at κ-opioid receptor/bradykinin B2 receptor heterodimers

The κ-opioid receptor (KOR) and bradykinin B2 receptor (B2R) are involved in a variety of important physiological processes and share many similar characteristics in terms of their distribution and functions in the nervous system. We first demonstrated the endogenous expression of KOR and B2R in human SH-SY5Y cells and their co-localization on the membrane of human embryonic kidney 293 (HEK293) cells. Bioluminescence and fluorescence resonance energy transfer and the proximity ligation assay were exploited to demonstrate the formation of functional KOR and B2R heteromers in transfected cells. KOR/B2R heteromers triggered dynorphin A (1−13)-induced Gαs/protein kinase A signaling pathway activity, including upregulation of intracellular cAMP levels and cAMP-response element luciferase reporter activity, resulting in increased cAMP-response element-binding protein (CREB) phosphorylation, which could be dampened by the protein kinase A (PKA) inhibitor H89. This indicated that the co-existence of KOR and B2R is critical for CREB phosphorylation. In addition, dynorphin A (1–13) induced a significantly higher rate of proliferation in HEK293-KOR/B2R and human SH-SY5Y cells than in the control group. These results indicate that KOR can form a heterodimer with B2R and this leads to increased protein kinase A activity by the CREB signaling pathway, leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of dynorphin in the regulation of neuroprotective effects.

Synthesis and evaluation of a 68Ga-labeled bradykinin B1 receptor agonist for imaging with positron emission tomography

A novel 68Ga-labeled bradykinin B1 receptor (B1R) agonist, 68Ga-Z01115, was synthesized and evaluated for imaging with positron emission tomography (PET). Z01115 exhibited good binding affinity (Ki=25.4±5.1nM) to hB1R. 68Ga-Z01115 was prepared in 74±5 decay-corrected radiochemical yield with >99% radiochemical purity and 155±89GBq/µmol (4.2±2.4Ci/μmol) specific activity. 68Ga-Z01115 was stable in vitro in mouse plasma (93% remaining intact after 60min incubation), and relatively stable in vivo (51±5% remaining intact at 5min post-injection). PET imaging and biodistribution studies in mice showed that 68Ga-Z01115 cleared rapidly from nontarget tissues/organs, and generated high target-to-nontarget contrast images. The uptake of 68Ga-Z01115 in B1R-positive (B1R+) tumor was 5.65±0.59%ID/g at 1h post-injection. Average contrast ratios of B1R+ tumor-to-B1R− tumor, -to-blood and -to-muscle were 24.3, 24.4 and 82.9, respectively. Uptake of 68Ga-Z01115 in B1R+ tumors was reduced by ∼90% with co-injection of cold standard, confirming it was mediated by B1R. Our data suggest that 68Ga-Z01115 is a promising tracer for imaging the expression of B1R that is overexpressed in a variety of cancers.

Tissue kallikrein protects SH-SY5Y neuronal cells against oxygen and glucose deprivation-induced injury through bradykinin B2 receptor-dependent regulation of autophagy induction.

Previous studies have demonstrated that tissue kallikrein (TK) protects against cerebral ischemia injury mainly through inhibition of apoptosis via bradykinin B2 receptor (B2R). In this study, we proposed that autophagy induction contributed to the neuroprotective mechanism of TK. To validate this hypothesis, we investigated TK-induced autophagy and its signaling mechanisms in human SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD). We found that TK treatment enhanced autophagy induction, reflected by augmented LC3 conversion and Beclin1 expression, decreased p62 levels and increased monomeric red fluorescent protein-LC3 puncta formation. Green fluorescent protein-monomeric red fluorescent protein-LC3 adenovirus assay indicated that TK maintained autophagic flux. Moreover, bafilomycin A1 (Baf.A1) caused obvious LC3-II accumulation either in the presence or absence of TK. Autophagy inhibition by Beclin1 knockdown or Baf.A1 treatment abrogated the neuroprotective effects of TK. Mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase (ERK)1/2 and AMP-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) signaling were induced by OGD stress and enhanced by TK. MEK/ERK inhibitor U0126 alone elevated autophagy in OGD conditions, but impaired TK-induced autophagy. Blockade of AMPK/TSC2/mTOR signaling by AMPK inhibitor compound C and shRNA mediated the knockdown of AMPK α1 and TSC2 but abolished autophagy in SH-SY5Y cells exposed to OGD treated either with or without TK. Moreover, B2R expression was up-regulated by OGD exposure. B2R knockdown attenuated autophagy and suppressed MEK1/2/ERK1/2 and AMPK/TSC2/mTOR signaling in OGD conditions in either the presence or absence of TK. In sum, we revealed the significance of B2R-mediated MEK/ERK and AMPK signaling in autophagy induction under OGD stress, and proposed novel mechanisms involved in the neuropotective function of TK through B2R-dependent regulation of autophagy. We propose the depicted model for the neuroprotective mechanism of tissue kallikrein (TK) during OGD stress: TK enhances bradykinin B2 receptor (B2R)-mediated MEK1/2/ERK1/2 and AMPK/TSC2/mTOR signaling, thus inducing protective autophagy. The findings reported in this study should provide new evidence for the pro-survival role of B2R-mediated autophagy in cerebral ischemia.

Radiolabeled B9958 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography: Effect of the Radiolabel-Chelator Complex on Biodistribution and Tumor Uptake.

Bradykinin B1 receptor (B1R), which is upregulated in a variety of malignancies, is an attractive cancer imaging biomarker. In this study we optimized the selection of radiolabel-chelator complex to improve tumor uptake and tumor-to-background contrast of radiolabeled analogues of B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-d-Tic-Cpg), a potent B1R antagonist. Peptide sequences were assembled on solid phase. Cold standards were prepared by incubating DOTA-/NODA-conjugated peptides with GaCl3, and by incubating AlOH-NODA-conjugated peptide with NaF. Binding affinities were measured via in vitro competition binding assays. (68)Ga and (18)F labeling experiments were performed in acidic buffer and purified by HPLC. Imaging/biodistribution studies were performed in mice bearing both B1R-positive (B1R+) HEK293T::hB1R and B1R-negative (B1R-) HEK293T tumors. Z02176 (Ga-DOTA-Pip-B9958; Pip: 4-amino-(1-carboxymethyl)piperidine), Z02137 (Ga-NODA-Mpaa-Pip-B9958; Mpaa: 4-methylphenylacetic acid), and Z04139 (AlF-NODA-Mpaa-Pip-B9958) bound hB1R with high affinity (Ki = 1.4-2.5 nM). (68)Ga-/(18)F-labeled peptides were obtained on average in ≥32% decay-corrected radiochemical yield with >99% radiochemical purity and 100-261 GBq/μmol specific activity. Biodistribution/imaging studies at 1 h postinjection showed that all tracers cleared rapidly from background tissues (except kidneys) and were excreted predominantly via the renal pathway. Only kidneys, bladders, and B1R+ tumors were clearly visualized in PET images. Uptake in B1R+ tumor was higher by using (68)Ga-Z02176 (28.9 ± 6.21 %ID/g) and (18)F-Z04139 (22.6 ± 3.41 %ID/g) than (68)Ga-Z02137 (14.0 ± 4.86 %ID/g). The B1R+ tumor-to-blood and B1R+ tumor-to-muscle contrast ratios were also higher for (68)Ga-Z02176 (56.1 ± 17.3 and 167 ± 57.6) and (18)F-Z04139 (58.0 ± 20.9 and 173 ± 42.9) than (68)Ga-Z02137 (34.3 ± 15.2 and 103 ± 30.2). With improved target-to-background contrast (68)Ga-Z02176 and (18)F-Z04139 are promising for imaging B1R expression in cancers with PET.

Bradykinin B2 receptor expression in the bronchial mucosa of allergic asthmatics: the role of NF-kB.

Bradykinin (BK) mediates acute allergic asthma and airway remodelling. Nuclear factor-kappa B (NF-kB) is potentially involved in BK B2 receptor (B2R) regulation. In this observational cross-sectional study, B2R and NF-kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls, examining the role of NF-kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects (HNBFb and HABFb). B2R and NF-kB (total and nuclear) expression was analysed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 h after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5(+) and αSMA(+) mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/HNBFb incubated with interleukin (IL)-4/IL-13 with/without BK, and after NF-kB inhibitor, by Western blotting. Bronchial mucosa B2R and nuclear NF-kB expression was higher in asthmatics after diluent (B2R only) and allergen challenge than in controls (P < 0.05), while B2R and NF-kB (total and nuclear) increased after allergen compared with after diluent (P < 0.05). Allergen exposure increased B2R expression in 5B5(+) and αSMA(+) cells. Constitutive B2R protein expression was higher in HABFb than in HNBFb (P < 0.05) and increased in both cell types after IL-13 or IL-4/IL-13 and BK treatment. This increase was suppressed by a NF-kB inhibitor (P < 0.05). Bronchial B2R expression is constitutively elevated in allergic asthma and is further increased after allergen exposure together with NF-kB expression. NF-kB inhibitor blocked IL-4/IL-13-induced increase in B2R expression in cultured fibroblasts, suggesting a role as potential anti-asthma drug.

Utero-placental cellular and nuclear expression of bradykinin B2 receptors in normal and preeclamptic pregnancies.

The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation.