Bradykinin B1 Receptor Agonist Research Articles

The Bradykinin B2 Receptor Agonist (NG291) Causes Rapid Onset of Transient Blood-Brain Barrier Disruption Without Evidence of Early Brain Injury.

Background: The blood–brain barrier (BBB) describes the brain’s highly specialized capillaries, which form a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, by preventing the entry of potentially harmful circulating molecules into the brain. However, this specialized function is challenging for the development of CNS therapeutics. Several strategies to facilitate drug delivery into the brain parenchyma via disruption of the BBB have been proposed. Bradykinin has proven effective in disrupting mechanisms across the blood–tumor barrier. Unfortunately, bradykinin has limited therapeutic value because of its short half-life and the undesirable biological activity elicited by its active metabolites.Objective: To evaluate NG291, a stable bradykinin analog, with selective agonist activity on the bradykinin-B2 receptor and its ability to disrupt the BBB transiently.Methods: Sprague Dawley rats and CD-1 mice were subjected to NG291 treatment (either 50 or 100 μg/kg, intravenously). Time and dose-dependent BBB disruption were evaluated by histological analysis of Evans blue (EB) extravasation. Transcellular and paracellular BBB leakage were assessed by infiltration of 99mTc-albumin (66.5 KDa) and 14C-sucrose (340 Da) radiolabeled probes into the brains of CD-1 mice treated with NG291. NG291 influence on P-glycoprotein (P-gp) efflux pump activity was evaluated by quantifying the brain accumulation of 3H-verapamil, a known P-gp substrate, in CD-1 mice.Results: NG291-mediated BBB disruption was localized, dose-dependent, and reversible as measured by EB extravasation. 99mTc-albumin leakage was significantly increased by 50 μg/kg of NG291, whereas 100 μg/kg of NG291 significantly augmented both 14C-sucrose and 99mTc-albumin leakage. NG291 enhanced P-gp efflux transporter activity and was unable to increase brain uptake of the P-gp substrate pralidoxime. NG291 did not evoke significant short-term neurotoxicity, as it did not increase brain water content, the number of Fluoro-Jade C positive cells, or astrocyte activation.Conclusion: Our findings strongly suggest that NG291 increases BBB permeability by two different mechanisms in a dose-dependent manner and increases P-gp efflux transport. This increased permeability may facilitate the penetration into the brain of therapeutic candidates that are not P-gp substrates.

ACE2 expression is affected by Angiotensin‐II type I receptor (AT 1 R) and kinin B1 receptor (B 1 R) in primary human aortic endothelial cells

Angiotensin Converting Enzyme type 2 (ACE2) acts as a critical player in the renin-angiotensin system (RAS) by converting Angiotensin (Ang)-II to Ang-(1-7) and in the kallikrein-kinin system (KKS) by converting Des-Arg9-Bradykinin (DABK) into inactive peptide. We previously reported in Neuro-2A cells that Ang-II mediates ACE2 internalization through its type 1 receptor (AT1R) via ubiquitination and ultimately degradation in lysosomes. In addition, we recently identified UBR1 and BRCC3, as potential ubiquitinase and deubiquitinase, respectively that may modulate ACE2 ubiquitination and internalization in hypertensive conditions. Here, we hypothesize that both AT1R and the bradykinin B1 receptor (B1R) promote ACE2 ubiquitination and degradation in human aortic endothelial cells. Protein expression for ACE2, UBR1 and BRCC3 was assessed using capillary western in endothelial cells pre-treated with media (control group), an AT1R antagonist (Telmisartan,10 μM) or a B1R antagonist (R-715, 10 μM), one hour prior to stimulation with a B1R agonist DABK (1 μM) or Ang-II (100 nM) for 4 hours. Also, immunocytochemistry against ACE2 along with a plasma marker was performed to determine the localization of ACE2. ACE2 protein expression was significantly decreased with Ang-II (~50 %) or DABK (~40 %) treatments in comparison to untreated cells (n=6; Dunnett's test, p<0.05), while Telmisartan and R-715 fully blocked the effects of Ang-II and DABK. In addition, UBR1 protein level was significantly increased with Ang-II (~25 %) or DABK (~50 %) treatments (n=3; Dunnett's test, p<0.05). Moreover, treatment with DABK and Ang-II produced a synergistic effect resulting in a 4-fold increase in UBR1 expression. On the other hand, both agonists produced a significant reduction of BRCC3 (n=3; Dunnett's test, p<0.05) while simultaneous administration resulted in a paradoxical 3-fold upregulation. Immunocytochemistry showed that Ang-II and DABK treatments resulted in a similar reduction of ACE2 colocalization on the plasma membrane. In summary, stimulation with DABK or Ang-II increases the protein expression of the ubiquitinase UBR1 while decreasing the deubiquitinase BRCC3. These effects were associated with a reduction of ACE2 expression on the plasma membrane, mediated by both AT1R and B1R. These data suggest that hypertension and inflammation may contribute to the loss of ACE2 compensatory activity on the plasma membrane in endothelial cells. Therefore, designing treatments targeting AT1R and B1R as well as intracellular components such as UBR1 and BRCC3, involved in the regulation of ACE2 expression may be effective for the treatment of cardiovascular and pulmonary diseases related to ACE2.

Bradykinin stimulates prostaglandin E2 release in human skeletal muscular fibroblasts

Local mediator prostaglandins and bradykinin are involved in inflammation and pain. We explored bradykinin effects on prostaglandin E2 (PGE2) release from fibroblasts derived from human skeletal muscular biopsies. Bradykinin induced PGE2 release through bradykinin B2 receptors, since PGE2 release was blocked by the bradykinin B2 receptor selective antagonist FR173657 and B2 receptor agonist (Hyp3)-bradykinin showed effects comparable to bradykinin. Consistently, bradykinin induced both mRNA cyclooxygenase 2 (COX-2) and protein. Bradykinin also induced ERK1/2 and p38 phosphorylation and provoked the translocation from the cytosol to the nucleus of p65/NF-kB. The release of PGE2 by bradykinin could be blocked inhibiting COX-2 and p65/NF-kB, ERK1/2 or p38 activation. Both ERK1/2 and p38 were upstream to NF-kB inasmuch siRNAs significantly blocked the p65/NF-kB activation induced by bradykinin. Thus, bradykinin, acting via B2 receptors, induced PGE2 release through ERK1/2 and p38-dependent pathways and consequent p65/NF-kB translocation to nucleus. p65/NF-kB induced COX-2 transcription. The release of PGE2 provide a possible explanation for the role of bradykinin in inflammatory diseases.

Impact of Chronic Angiotensin Treatment on the Responses of Isolated Cardiac Fibroblasts to Acute Pro‐Fibrotic Stimuli

Pathological cardiac remodeling involving fibrosis can lead to heart failure in people with hypertension. We have recently demonstrated that angiotensin II (AngII)‐induced increases in left ventricular collagen content and macrophage infiltration are attenuated by concomitant bradykinin‐B1 receptor (B1R) antagonism in Sprague‐Dawley (SD) rats. Given that AngII has been shown to upregulate B1R, the goal of the present study was to determine the impact of B1R stimulation on fibroblast physiology following chronic (in vivo) or acute (in vitro) treatment with AngII. Adult male SD rats were implanted with osmotic mini‐pumps that delivered AngII (200 ng/kg/min) or saline for 4 weeks. Cardiac fibroblasts were isolated from the left ventricle of saline and AngII‐treated rats and passaged to P1. Cells were treated in vitro with AngII (1uM), a selective B1R agonist (Lys‐ [Des‐Arg9], 100 nM), AngII+B1R agonist or vehicle for 48 hr. Transforming growth factor beta 1 (TGF‐β1) and monocyte chemoattractant protein 1 (MCP‐1) were assessed in culture media. Expression of the matricellular protein periostin and the pro‐oxidant NADPH oxidase 2 (Nox2) were determined in cell lysate by western blot. Fibroblasts isolated from rats chronically treated with AngII secreted elevated levels of MCP‐1 in vitro (p&lt;0.05), when compared to control fibroblasts. Although MCP‐1 secretion was not further modified by acute AngII treatment, stimulation with the B1R agonist tended to reduce MCP‐1 levels. TGF‐β1 release was not different from cells isolated from saline or AngII infused rats. However, in vitro incubation with AngII significantly increased secretion of TGF‐β1 to a similar degree in fibroblasts isolated from saline or AngII treated rats. B1R stimulation had no impact on TGF‐β1 levels. Periostin expression tended to be increased in fibroblasts isolated from AngII infused rats, while Nox2 expression was not different. Acutely, AngII tended to increase Nox2 expression in vitro to a greater degree in fibroblasts that were isolated from saline‐infused rats. Selective B1R stimulation in vitro resulted in a significant 1.77‐fold upregulation of Nox2 only in fibroblasts isolated from saline treated rats (p&lt;0.05). Combined AngII+B1R stimulation did not further increase Nox2 expression. Taken together, these data demonstrate that upregulation of MCP‐1 production and periostin induced by chronic AngII persist at least to P1 in isolated cardiac fibroblasts. Moreover, chronic AngII resulted in an attenuated increase in Nox2 in response to acute AngII stimulation. Notably, we demonstrate for the first time that selective B1R activation results in a marked increase in Nox2 expression in cardiac fibroblasts. Although B1R antagonism has been shown to offset fibrotic and inflammatory effects of AngII in vivo, acute effects of AngII stimulation are not enhanced in the presence of B1R stimulation. Future studies will determine the extent to which in vivo or in vitro B1R antagonism prevents AngII‐induced fibroblast activation. Investigation of oxidative stress and the nature of Nox2 regulation by B1R activation may provide greater insight into the impact of bradykinin on fibroblast physiology.Support or Funding InformationSpringboard, UA COM‐PThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

In Vivo Effects of Bradykinin B2 Receptor Agonists with Varying Susceptibility to Peptidases.

We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B2 receptor (B2R) agonists. Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK), BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer’s inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation (increased Doppler shift signal). Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be extracted from prodrug peptides that behave as peptidase-activated B2R agonists.

Differential effect of intranasally administrated kinin B1 and B2 receptor antagonists in Alzheimer's disease mice.

An Increasing body of evidence supports a critical role of brain inflammation in the pathogenesis of Alzheimer's disease. A principal aspect of the brain immune response to inflammation is the activation of microglia. It has been shown that the kinin system is activated during brain inflammation and previously we demonstrated that bradykinin B1 receptor agonist reduced microglial activation in vitro. The aim of the present study was to investigate the effects of bradykinin B1 or B2 receptor antagonists on microglial release of pro-inflammatory factors in BV2 microglia. In vivo, we focused on the effects of intranasally given kinin antagonists on amyloid burden and microglia/macrophage marker expression in brains of 5X familial Alzheimer's disease mice. The present data show that pharmacological antagonism of B1 receptor (R-715) but not B2 receptor (HOE-140) markedly increased nitric oxide and tumor necrosis factor alpha release from BV2 microglial cells. We also showed that intranasal treatment with R-715 but not HOE-140 of Alzheimer's mice enhanced amyloid beta burden and microglia/macrophages activation. Taken together, our data reveal a possible role for the bradykinin B1 receptor in neuroinflammation and in the control of Abeta accumulation in transgenic mice, possibly through regulation of glial cell responses.

Abstract P113: Angiotensin Converting Enzyme 2 Modulates Bradykinin B1 Receptor Function During DOCA-Salt Hypertension

DOCA-salt hypertension is associated with reduced angiotensin converting enzyme 2 (ACE2) and increased bradykinin B1 receptor (B1R) expression in the brain. ACE2 hydrolyzes des-Arg(9)-bradykinin, the endogenous B1R agonist, into inactive metabolites. Therefore, we hypothesized that ACE2 overexpression or deletion modulates B1R function in the brain during neurogenic hypertension. To test this hypothesis, we used mice overexpressing ACE2 in neurons (SA), ACE2 knockout and B1R knockout mice. Blood pressure (BP) was monitored using telemetry probes in conscious animals. While baseline BP was not different between strains, DOCA-salt treatment (1 mg/g body weight DOCA, 1% NaCl for 3 weeks) resulted in a significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to wildtype (WT) mice (138 ±3 mmHg, n=8). DOCA-salt hypertension resulted in 27% decrease (74 ±6 vs. 54 ±2 Fluorescence Units (FU)/min/μg of protein, p&lt;0.05) in ACE2 activity in the hypothalamus, but not in B1R knockout mice with DOCA (69 ±6 vs. 65 ±3 FU/min/μg of protein). In DOCA-treated WT mice, B1R mRNA (Real time PCR) and protein expression (Western blot) in the hypothalamus were increased by 3 and 2 fold (n=6, p&lt;0.01), respectively. This increased B1R expression was blunted in SA mice with DOCA (3.2 ±0.4 vs. 0.9 ±0.1 fold, p&lt;0.001) but not in ACE2 knockout mice with DOCA (3.2 ±0.4 vs. 3.1 ±0.9 fold). Moreover, DOCA-salt treatment resulted in an increased expression of pro-inflammatory ADAM17 in the brain by 2.2 fold (n=6, p&lt;0.01 vs. WT). ACE2 overexpression or B1R knockdown blunted this ADAM17 expression (1.4 ±0.1 and 1.1 ±0.6 fold, respectively, n=3, p&lt;0.01 vs. WT+DOCA), while it was remain increased in ACE2 knockout mice (2.6 ±0.7 fold, n=3, p&lt;0.01) compared to control mice. Together, our data provide novel evidence to support a role for ACE2 in the modulation of central B1R function in the development of DOCA-salt hypertension.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167–384 nM; Emax = 32–86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g. IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60–84 nM; Emax = 29–44% relative to max. BK-induced effects). FR-190997 (0.3–300 μg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g. 34.5 ± 7.5% decrease; 6 h post-dose of 30 μg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

Differential regulation of astrocyte prostaglandin response by kinins: Possible role for mitogen activated protein kinases

The role of kinins, well known as peripheral inflammatory mediators, in the modulation of brain inflammation is not completely understood. The present data show that bradykinin, a B2 receptor agonist, enhanced both basal and lipopolysaccharide (LPS)-induced cyclooxygenase-2 mRNA and protein levels and prostaglandin E2 synthesis in primary rat astrocytes. By contrast, Lys-des-Arg9-bradykinin, which is a bradykinin breakdown product and a selective kinin B1 receptor agonist, attenuated both basal and LPS-induced astrocyte cyclooxygenase-2 mRNA levels and prostaglandin E2 production. Pre-treating the cells with p42/p44 MAPK but not with JNK or p38 inhibitors completely abrogated PGE2 synthesis in cells stimulated with LPS in the presence of bradykinin or bradykinin B1 receptor agonist. Bradykinin, but not the bradykinin B1 receptor agonist, augmented p42/p44 MAPK phosphorylation. The phosphorylation of JNK and p38 was not altered upon exposure to Bradykinin or the bradykinin B1 receptor agonist. These results suggest that the dual delayed effect of kinins on PGE2 synthesis may be due to differential regulation of COX-2 and signaling molecules such as p42/p44 MAPKs. Thus, kinins may exert opposing actions on brain inflammation and neurodegenerative diseases.

The effect of bradykinin on the electrical activity of rat myenteric neurons

Bradykinin is a mediator involved in inflammatory processes in the gut. Here we investigated the effect of bradykinin on the electrical activity of rat myenteric neurons, the key players for regulation of gastrointestinal motility. Bradykinin (2×10−8mol/l) induced a biphasic increase in frequency of action potentials measured with microelectrode arrays. This increase was mirrored by a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i), which was observed in about 40% of the myenteric neurons. The bradykinin B1 receptor agonist des-arg9-bradykinin as well as the bradykinin B2 receptor agonist hyp3-bradykinin induced a similar effect on [Ca2+]i. Immunocytochemical stainings confirmed the expression of both receptor types by myenteric ganglionic cells. Real time PCR showed that the inducible B1 receptor was upregulated during cell culture. The inhibition of cyclooxygenases with piroxicam reduced the effect of bradykinin on the electrical activity of myenteric neurons. The suppression of the glial growth on microelectrode arrays did not affect the bradykinin-induced change in frequency of action potentials. This suggests that prostaglandins, which probably mediate the effect of bradykinin, are not exclusively released from glial cells.The bradykinin-induced increase in [Ca2+]i was dependent on the presence of extracellular Ca2+ and was inhibited by Co2+, Cd2+, and Ni2+, blockers of voltage-dependent Ca2+ channels, indicating a stimulation of the influx of extracellular Ca2+ by the kinin. Consequently, bradykinin induces a Ca2+ influx in myenteric neurons via Ca2+ channels in the plasma membrane.

Bifunctional epitope-agonist ligands of the bradykinin B2 receptor

Two bradykinin (BK) B(2) receptor agonists N-terminally extended with the myc epitope were synthesized and evaluated: myc-KPG-BK and myc-KGP-B-9972. The latter was modeled on the inactivation-resistant agonist B-9972 (D-Arg(0), Hyp(3), Igl(5), Oic(7), Igl(8)-BK) and is also resistant to endosomal inactivation. Despite a large loss of affinity relative to the parent peptide, the tagged analogs are conventional agonists in the umbilical vein contractility assay and compete for [(3)H]BK binding at the rabbit B(2) receptor. Endocytosed myc-KGP-B-9972 most effectively carried AlexaFluor-488-conjugated anti-myc monoclonal antibodies into intact cells expressing the B(2) receptor. Results support the prospects of functionally-active cargoes entering cells in a pharmacologically controlled manner.

Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist

We recently identified a novel human B2 receptor (B2R) agonist [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp(3),Thi(5),(4-Me)Tyr(8)(ΨCH(2)NH)Arg(9)]-BK) (RMP-7) were also tested. Contraction assays using isolated mouse stomachs (containing kinin B1R, B2R, and kininase I- and II-like activities) showed that NG291 is a more potent contractant than BK and is inhibited by HOE-140 (B2R antagonist) but unaffected by R954 (B1R antagonist), whereas both decreased the potency of BK. In stomach tissues from B2R knockout mice, BK maintained its activity via B1R, whereas NG291 had no contractile effect, indicating that it was selective for B2R. Unlike BK, NG291 was not degraded by rabbit lung ACE. Comparing intravenously administered BK and NG291 revealed that NG291 exhibited more potent and prolonged hypotensive action and greater antithrombotic and profibrinolytic activities. These effects were of comparable magnitude to RMP-7 and were absent in B2R knockout mice. We concluded that NG291 is a novel biostable B2R-selective agonist that may prove suitable for investigating the (pre)clinical cardioprotective efficacy of B2R activation.

Bradykinin‐induced cell migration and COX‐2 production mediated by the bradykinin B1 receptor in glioma cells

Bradykinin is produced and acts at the site of injury and inflammation. Recent reports have also shown that bradykinin selectively modulates blood-tumor barrier permeability. However, the molecular mechanisms and pathologic roles underlying bradykinin-induced glioma migration remain unclear. Glioma is the most common primary adult brain tumor, with a poor prognosis because of the ease with which tumor cells spread to other regions of the brain. In this study, we found that bradykinin increases the cell migration and expression of cyclo-oxygenase-2 (COX-2) in glioma cells. Bradykinin-mediated migration was attenuated by the selective COX-2 inhibitor NS-398. Moreover, increased motility of glioma cells and expression of COX-2 were mimicked by a bradykinin B1 receptor (B1R) agonist and markedly inhibited by a B1R antagonist. Bradykinin-mediated migration was attenuated by phosphoinositide 3-kinase (PI-3 kinase)/AKT inhibitors LY 294002 and wortmannin. Bradykinin stimulation also increased the phosphorylation of the p85 subunit of PI-3 kinase and serine 473 of AKT. Treatment of bradykinin with AP-1 inhibitors Tanshinone IIA and curcumin also reduced COX-2 expression and glioma cell migration. Moreover, treatment of bradykinin also induced phosphorylation of c-Jun in glioma cells. AP-1 promoter analysis in the luciferase reporter construct showed that bradykinin increased AP-1 transcription activity and was inhibited by LY 294002 and wortmannin. One mechanism underlying bradykinin-directed migration is transcriptional up-regulation of COX-2 and activation of the B1R receptor, PI-3 kinase, AKT, c-Jun, and AP-1 pathways.

Metabolically stable bradykinin B2 receptor agonists enhance transvascular drug delivery into malignant brain tumors by increasing drug half-life.

BackgroundThe intravenous co-infusion of labradimil, a metabolically stable bradykinin B2 receptor agonist, has been shown to temporarily enhance the transvascular delivery of small chemotherapy drugs, such as carboplatin, across the blood-brain tumor barrier. It has been thought that the primary mechanism by which labradimil does so is by acting selectively on tumor microvasculature to increase the local transvascular flow rate across the blood-brain tumor barrier. This mechanism of action does not explain why, in the clinical setting, carboplatin dosing based on patient renal function over-estimates the carboplatin dose required for target carboplatin exposure. In this study we investigated the systemic actions of labradimil, as well as other bradykinin B2 receptor agonists with a range of metabolic stabilities, in context of the local actions of the respective B2 receptor agonists on the blood-brain tumor barrier of rodent malignant gliomas.MethodsUsing dynamic contrast-enhanced MRI, the pharmacokinetics of gadolinium-diethyltriaminepentaacetic acid (Gd-DTPA), a small MRI contrast agent, were imaged in rodents bearing orthotopic RG-2 malignant gliomas. Baseline blood and brain tumor tissue pharmacokinetics were imaged with the 1st bolus of Gd-DTPA over the first hour, and then re-imaged with a 2nd bolus of Gd-DTPA over the second hour, during which normal saline or a bradykinin B2 receptor agonist was infused intravenously for 15 minutes. Changes in mean arterial blood pressure were recorded. Imaging data was analyzed using both qualitative and quantitative methods.ResultsThe decrease in systemic blood pressure correlated with the known metabolic stability of the bradykinin B2 receptor agonist infused. Metabolically stable bradykinin B2 agonists, methionine-lysine-bradykinin and labradimil, had differential effects on the transvascular flow rate of Gd-DTPA across the blood-brain tumor barrier. Both methionine-lysine-bradykinin and labradimil increased the blood half-life of Gd-DTPA sufficiently enough to increase significantly the tumor tissue Gd-DTPA area under the time-concentration curve.ConclusionMetabolically stable bradykinin B2 receptor agonists, methionine-lysine-bradykinin and labradimil, enhance the transvascular delivery of small chemotherapy drugs across the BBTB of malignant gliomas by increasing the blood half-life of the co-infused drug. The selectivity of the increase in drug delivery into the malignant glioma tissue, but not into normal brain tissue or skeletal muscle tissue, is due to the inherent porous nature of the BBTB of malignant glioma microvasculature.

The role of the renal kallikrein–kinin system in diabetic nephropathy

Diabetic nephropathy is one of the most common complications in diabetes mellitus. Multiple pathogenic mechanisms are now believed to contribute to this disease, including inflammatory cytokines, autacoids and oxidative stress. Numerous studies have shown that the kallikrein-kinin system may be involved in these mechanisms. This review focuses on recent research advance on the potential role of the kallikrein-kinin system in the development of diabetic nephropathy, and its clinical relevance. A collection of recent studies has shown that angiotensin-converting enzyme inhibitors, which inhibit angiotensin II formation and degradation of bradykinin, and vasopeptidase inhibitors attenuated the development of diabetic nephropathy in experimental animals and clinical settings. The role of the kallikrein-kinin system in diabetes is further supported by findings that diabetic nephropathy is worsened in diabetic mice lacking bradykinin B2 receptors. Although long-acting bradykinin B2 receptor agonists have been shown to have renal protective effects, their therapeutic benefits have not been well studied. Current experimental investigations demonstrated that pharmacological intervention of the kallikrein-kinin system improved renal conditions in diabetes mellitus. These findings suggest that the kallikrein-kinin system may be a therapeutic target in preventing and treating diabetic nephropathy.

Bradykinin modulates pacemaker currents through bradykinin B2 receptors in cultured interstitial cells of Cajal from the murine small intestine

We studied the modulation of pacemaker activities by bradykinin in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied bradykinin produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. Pretreatment with bradykinin B1 antagonist did not block the bradykinin-induced effects on pacemaker currents. However, pretreatment with bradykinin B2 antagonist selectively blocked the bradykinin-induced effects. Also, only externally applied selective bradykinin B2 receptor agonist produced tonic inward pacemaker currents and ICC revealed a colocalization of the bradykinin B2 receptor and c-kit immunoreactivities, but bradykinin B1 receptors did not localize in ICC. External Na(+)-free solution abolished the generation of pacemaker currents and inhibited the bradykinin-induced tonic inward current. However, a Cl(-) channel blocker (DIDS) did not block the bradykinin-induced tonic inward current. The pretreatment with Ca(2+)-free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the bradykinin-induced action. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the bradykinin-induced effects on pacemaker currents. These results suggest that bradykinin modulates the pacemaker activities through bradykinin B2 receptor activation in ICC by external Ca(2+) influx and internal Ca(2+) release via protein kinase C- or cyclooxygenase-independent mechanism. Therefore, the ICC are targets for bradykinin and their interaction can affect intestinal motility.

Discovery of Non‐Peptide Bradykinin B2 Receptor Antagonists and Agonists

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Hydrolysis of Angiotensin Peptides by Human Angiotensin I–Converting Enzyme and the Resensitization of B 2 Kinin Receptors

We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I-converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B2 receptors coupled to green fluorescent protein (B2GFP) or to express only coupled B2GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B2 receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18x slower than Ang I and &30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although micromol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B2GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-alpha, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B2 activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B2 receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Treatment of severe pulmonary hypertension: A bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy

Bradykinin is an important modulator of endothelial cell function and has also a powerful cardioprotective effect. Here we report that treatment of severely pulmonary hypertensive rats (that recapitulate several of the physiological and pathological characteristics of the human pulmonary vascular disease, including dramatic right ventricular hypertrophy, pericardial effusion and death) with a newly synthesized long-acting bradykinin B2 receptor agonist B9972 caused reduction of the pulmonary artery pressure (PAP = 51 ± 2.0 versus 68 ± 2.8 of untreated animals) and of right ventricular hypertrophy ( R v/ L v + S = 0.55 ± 0.02 versus 0.73 ± 0.03 of untreated rats) and activation of Akt. Long-term stimulation with B9972 in our animal model of SPH resulted in decreased expression of the B2 receptor in lung vasculature. Treatment with B9972 decreased the number of plexiform lesions in the lungs by inducing cell apoptosis in the obliterated vessels and by restoring caveolin-1 expression. B9972 also promoted eNOS activation. In vitro B9972 caused activation of caspase-3 as well as Erk and induction of prostacyclin production in rat pulmonary microvascular EC. Taken together our data suggest that a stable bradykinin B2 agonist B9972 demonstrates the capacity to reduce severe pulmonary hypertension, right ventricular hypertrophy and induce apoptosis of hyperproliferative cells in pre-capillary pulmonary arterioles.