Bradykinin Type Research Articles

A Role for Activation of the Kallikrein-Kinin System in the Homing Property of Endothelial Progenitor Cells.

The plasma kallikrein–kinin system (KKS) plays an important role in inflammation, thrombosis and vascular pathology. Activation of the KKS results in bradykinin release, which increases vascular permeability and modulates inflammatory responses. Circulating endothelial progenitor cells (EPCs) home to ischemic and inflamed tissues and participate in the vasculogenesis. However, the homing mechanism remains poorly understood. We now report that activation of the KKS regulates the homing property of EPCs using in vivo and in vitro approaches. We have previously shown that the Lewis rat, which displays a mutant kininogen (Ser511Ala), is susceptible to peptidoglycan-polysaccharide (PG-PS)- induced systemic inflammation including arthritis and displays activation of the KKS and increased bradykinin levels. CD34-positive EPCs were isolated from bone marrow of disease-free Lewis rats by Ficoll gradient centrifugation. After culture in EGM2 on collagen surfaces for 7 days, a single colony with cobblestone-like morphology appeared and was subjected to large-scale expansion. Compared with differentiated rat lung microvessel endothelial cells (rLMECs), rat EPCs expressed similar levels of endothelial cell lineage marker mRNA such as VE-cadherin, CD31, vWF and uPAR. EPCs exclusively expressed hematopoietic progenitor cell markers, Sca-1 and CD34. Both of EPCs and rLMECs expressed bradykinin type 2 receptor (B2R) but not B1R. To observe the in vivo mobilization of EPCs, EPCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and injected into Lewis rats in which inflammation/arthritis was induced by PG-PS. Two weeks after PG-PS injection, pannus formed in the synovial tissues, representing the entry into the chronic phase of arthritis. The fluorescently-labeled EPCs were selectively detected in the inflamed synovium of PG-PS-treated rats, but not that of disease-free rats. As detected by confocal microscopy, implanted EPCs formed new blood vessels in the inflamed synovial tissues. To further investigate the mechanism by which the KKS regulates EPC mobilization, we observed that bradykinin (a B2R agonist), but not des-Arg9-bradykinin (a B1R agonist), stimulated EPC transmigration through confluent rLMEC layer at 100 and 1000 nM (P<0.01 and P<0.001, respectively). Bradykinin did not affect EPC adhesion to rLMECs. Priming by tumor necrosis factor a (TNFa), a major inflammatory cytokine, induced transendothelial migration of EPCs by 100 nM des-Arg9- bradykinin (168.56 ± 9.56 vs 96.02 ± 10.23, P<0.001). The priming effect resulted from TNFa induction of B1R mRNA. As determined by RT-PCR, rat EPCs expressed mRNA of multiple homing receptors including chemokine (C-X-C motif) receptor 4, CD49d and CD62-E, but not CD11a. Thus, activation of the KKS regulates the homing property of EPCs, which constitutes an additional mechanism for involvement of the KKS in the vasculogenesis. B2R predominantly mediates transendothelial migration of normal EPCs, whereas B1R seems responsible for that of inflamed EPCs. Kinins and cytokines such as TNFa may synergize to regulate the mobilization of EPCs. These observations provide a new insight into understanding the role of the KKS in vascular pathology and inflammation.

Abstract 5955: Role of Bradykinin and Chymase in Cardiorenal Function Under Long-Term ACE Inhibition in Heart Failure

Renal insufficiency is relatively common in patients with heart failure (HF) and patients with chronic kidney disease are known to have a significant increase in cardiovascular morbidity and mortality. Angiotensin (Ang) II plays an important role in water and sodium (Na) balance and renal injury. Although ACE inhibitors (ACEIs) suppress the formation of Ang II and are useful in the treatment of HF, the agents also prevent the breakdown of bradykinin (BK), which exerts its vasodilative action through BK type 2 (B2) receptors. On the other hand, over 50% of renal Ang II formation in HF is mediated by ACE independent chymase pathway. The roles of BK and chymase in the cardiorenal function under chronic treatment of ACEI has not been fully elucidated as yet. We therefore evaluated the cardiorenal effects of a specific B2 receptor antagonist, FR173657 (FR, 0.3mg/kg/day) or a specific chymase inhibitor, (ChyI, 100mg/kg/day) with an ACEI, enalapril (1mg/kg/day) compared to those of enalapril alone in dogs with tachycardia induced HF (22days). Cardiac contractile function markedly deteriorated and body fluid retention and impaired renal function accompanied with upregulation of renal ACE and chymase mRNA levels after the induction of severe HF. However, enalapril significantly improved the ejection fraction (vehicle 19% vs. enalapril 36%, p&lt;0.01) and increased urine excretion (vehicle 0.36 vs. enalapril 0.55 ml/min, p&lt;0.05) associated with increases in the glomerular filtration rate (GFR) and renal plasma flow (RPF) (vehicle 24 vs. enalapril 60 ml/min, and 74 vs. 169 ml/min, respectively p&lt;0.05) in HF. There were no differences observed in blood pressure and LV ejection fraction among three treatment groups. FR did not statistically affect urinary Na excretion, GFR and RPF compared to the ACE inhibition alone, however, the chymase inhibition has more significantly increased water and Na excretion rate (0.70 vs. 0.55 ml/min and 56 vs. 26 μEq/min, respectively p&lt;0.05) and GFR (70 vs. 59 ml/min, p&lt;0.05) compared to the ACE inhibition alone. In conclusion, endogenous BK does not likely contributes to the renoprotective effects of ACEI, however, combining ACE and chymase inhibition has greater therapeutic potential in the amelioration of renal function in HF.

C-Src-mediated phosphorylation of AP-2 reveals a general mechanism for receptors internalizing through the clathrin pathway

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of β2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of β2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with βarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on β2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both βarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the β2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on β2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.

Segmental Expression of the Bradykinin Type 2 Receptor in Rat Efferent Ducts and Epididymis and Its Role in the Regulation of Aquaporin 91

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

The effect of C1 inhibitor on intestinal ischemia and reperfusion injury

Complement activation and neutrophil stimulation are two major components in events leading to ischemia and reperfusion (IR) injury. C1 inhibitor (C1INH) inhibits activation of each of the three pathways of complement activation and of the contact system. It is also endowed with anti-inflammatory properties that are independent of protease inhibition. The goal of these studies was to investigate the role and mechanism of C1INH in alleviating IR-induced intestinal injury. C57BL/6, C1INH-deficient (C1INH(-/-)), bradykinin type 2 receptor-deficient (Bk2R(-/-)), and C3-deficient mice (C3(-/-)) were randomized into three groups: sham operated control, IR, and IR + C1INH-treated groups. Ischemia was generated by occlusion of the superior mesenteric artery followed by reperfusion. C1INH or reactive center-cleaved inactive C1INH (iC1INH) was injected intravenously before reperfusion. IR resulted in intestinal injury in C57BL/6, C1INH(-/-), Bk2R(-/-), and C3(-/-) mice with significantly increased neutrophil infiltration into intestinal tissue. In each mouse strain, C1INH treatment reduced intestinal tissue injury and attenuated leukocyte infiltration compared with the untreated IR group. C1INH inhibited leukocyte rolling in the mesenteric veins of both C57BL/6 and C3-deficient mice subjected to IR. C1INH treatment also improved the survival rate of C57BL/6 and C1INH(-/-) mice following IR. Similar findings were observed in the IR animals treated with iC1INH. These studies emphasize the therapeutic benefit of C1INH in preventing intestinal injury caused by IR. In addition to the protective activities mediated via inhibition of the complement system, these studies indicate that C1INH also plays a direct role in suppression of leukocyte transmigration into reperfused tissue.

Expression and Functional Role of the Bradykinin Type 2 Teceptor in Epididymal Principal Cells.

Expression and Functional Role of the Bradykinin Type 2 Teceptor in Epididymal Principal Cells.

Lipopolysaccharide activates the kallikrein–kinin system in mouse choroid plexus cell line ECPC4

Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.

Bradykinin Type 2 Receptor BE1 Genotype Influences Bradykinin-Dependent Vasodilation During Angiotensin-Converting Enzyme Inhibition

To test the hypothesis that the bradykinin receptor 2 (BDKRB2) BE1+9/-9 polymorphism affects vascular responses to bradykinin, we measured the effect of intra-arterial bradykinin on forearm blood flow and tissue-type plasminogen activator (t-PA) release in 89 normotensive, nonsmoking, white American subjects in whom degradation of bradykinin was blocked by enalaprilat. BE1 genotype frequencies were +9/+9:+9/-9:-9/-9=19:42:28. BE1 genotype was associated with systolic blood pressure (121.4+/-2.8, 113.8+/-1.8, and 110.6+/-1.8 mm Hg in +9/+9, +9/-9, and -9/-9 groups, respectively; P=0.007). In the absence of enalaprilat, bradykinin-stimulated forearm blood flow, forearm vascular resistance, and net t-PA release were similar among genotype groups. Enalaprilat increased basal forearm blood flow (P=0.002) and decreased basal forearm vascular resistance (P=0.01) without affecting blood pressure. Enalaprilat enhanced the effect of bradykinin on forearm blood flow, forearm vascular resistance, and t-PA release (all P<0.001). During enalaprilat, forearm blood flow was significantly lower and forearm vascular resistance was higher in response to bradykinin in the +9/+9 compared with +9/-9 and -9/-9 genotype groups (P=0.04 for both). t-PA release tended to be decreased in response to bradykinin in the +9/+9 group (P=0.08). When analyzed separately by gender, BE1 genotype was associated with bradykinin-stimulated t-PA release in angiotensin-converting enzyme inhibitor-treated men but not women (P=0.02 and P=0.77, respectively), after controlling for body mass index. There was no effect of BE1 genotype on responses to the bradykinin type 2 receptor-independent vasodilator methacholine during enalaprilat. In conclusion, the BDKRB2 BE1 polymorphism influences bradykinin type 2 receptor-mediated vasodilation during angiotensin-converting enzyme inhibition.

Inferring the Lifetime of Endosomal Protein Complexes by Fluorescence Recovery after Photobleaching

Cellular signal transduction is dynamic, with signaling proteins continually associating and dissociating into and from protein complexes. Here we present a fluorescence recovery after photobleaching technique to determine the lifetime of protein complexes on intracellular vesicles. We use Bayesian inference based on a model that includes the diffusion of cytosolic proteins and their interaction with membrane-bound receptors. Our analysis is general: we incorporate prior information on protein diffusion, measurement error in determining fluorescence intensities, corrections for photobleaching, and variation in the concentration of receptors between vesicles. We apply our method to the complexes formed on endosomes by G-protein-coupled receptors and the protein β-arrestin. The lifetime of these complexes determines the recycling rate of the receptors. We find in mammalian cells that the bradykinin type 2 receptor and β-arrestin2 complex has a lifetime of ∼2 min, while the angiotensin II type 1A receptor and β-arrestin2 complex has a lifetime of ∼6 min. As well as allowing quantitative comparisons between experiments, our method provides in vivo parameters for systems biology simulations of signaling networks.

New therapies for hereditary angioedema: Disease outlook changes dramatically

Hereditary angioedema (HAE) is an autosomal dominant disease associated with episodic attacks of nonpitting edema that may affect any external or mucosal body surface. Attacks most often affect the extremities, causing local swelling, the GI tract, leading to severe abdominal pain, and the mouth and throat, at times causing asphyxiation. Most patients with HAE have low levels of the plasma serine protease inhibitor C1 inhibitor. The edema in these patients is caused by unregulated generation of bradykinin. Effective chronic therapy of patients with impeded androgens or plasmin inhibitors has been available for decades, but in the United States, we do not have therapy for acute attacks. Five companies have completed or are in the process of conducting phase 3 clinical trials, double-blind, placebo-controlled studies of products designed to terminate acute attacks or to be used in prophylaxis. Two companies, Lev Pharmaceuticals and CSL Behring, have preparations of C1 inhibitor purified from plasma that have been used in Europe for decades (trade names Cinryze and Berinert P, respectively). One company, Pharming, has developed a recombinant C1 inhibitor preparation. One company, Dyax, is testing a kallikrein inhibitor (ecallantide), and one company, Jerini, is completing testing of a bradykinin type 2 receptor antagonist (Icatibant). Although little has been published thus far, all of these products may prove effective. It is likely that HAE treatment will change dramatically within the next few years.

Lovastatin induces the expression of bradykinin type 2 receptors in cultured human coronary artery endothelial cells

Cardioprotective bradykinin type-2 receptors (BK-2Rs) are downregulated in the myocardial endothelium of both human and rat failing hearts. Statins are cardioprotective drugs that reduce the level of plasma cholesterol but also exert cholesterol-independent pleiotropic effects. Here we examined the effect of lovastatin on BK-2R expression in cultured human coronary artery endothelial cells. The effect of lovastatin on the expression of BK receptors in human coronary artery endothelial cells (HCAECs) was examined by real-time PCR, Western blot analysis and immunocytochemistry. Lovastatin induced a time- and concentration-dependent increase in both BK-2R and BK-1R mRNA expression in the cultured HCAECs. Also, the number of functional BK-2Rs capable of inducing BK-mediated NO production and cGMP signaling was increased in the lovastatin-treated HCAECs. Mevalonate, the direct metabolite of HMG-CoA reductase, reversed the effect of lovastatin. Furthermore, lovastatin inhibited Rho activation and a selective inhibitor of Rho-associated kinases, Y-27632, induced a similar increase in BK-2R expression as lovastatin. In contrast, a specific inhibitor of COX-2, NS398, significantly inhibited the lovastatin-induced expression of BK-2Rs. Here we show for the first time that lovastatin induces the expression of BK-2Rs in cultured human coronary artery endothelial cells through a novel cholesterol-independent pleiotropic mechanism that involves RhoA kinase inhibition and COX-2 activation. Thus, reported beneficial effects of statins in cardiovascular diseases may be partly mediated by an increased expression of cardioprotective BK-2Rs in the endothelial cells of the coronary tree. Moreover, the use of COX-2 inhibitors may affect the level of endothelial BK-2Rs in a negative fashion.

Increased expression of profibrotic neutral endopeptidase and bradykinin type 1 receptors in stenotic aortic valves

In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS. Stenotic aortic valves (n = 86) were obtained at valve replacement surgery and control valves (n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves (P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-alpha induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-beta1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist. NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.

Calcium-activated potassium channels mediated blood-brain tumor barrier opening in a rat metastatic brain tumor model

BackgroundThe blood-brain tumor barrier (BTB) impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases.ResultsIn this study, we examined the function and regulation of calcium-activated potassium (KCa) channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R) expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain), human brain microvessel endothelial cells (HBMEC) and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening.ConclusionThese findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.

Efficacy of Lozartan and Enalapril in mild-to-moderate hypertensive patients with different polymorphisms of type 2 bradykinin receptor and angiotensinogene genes

The aim of the study was to investigate the influence of bradykinin type 2 receptor and angiotensinogen genes polymorphism on clinical features of hypertension and efficacy of the angiotensin II receptor blocker (Losartan) and ACE inhibitor (enalapril) in patients with mild-to-moderate hypertension. We examined 140 hypertensive patients; mean duration of hypertension constituted 9,3±2,7 years. Ambulatory blood pressure monitoring, ECHO-cardiography and genes polymorphism definition were done in all patients, with the repeated blood pressure monitoring used as a criteria of treatment efficacy. As a result it was determined, that blood pressure was higher in patients with T/T polymorphism of angiotensinogen gene and +9/+9 polymorphism of type 2 bradykinin receptor gene. Left ventricular mass index was higher in patients with M/M and +9/+9 polymorphisms, and the antihypertensive effect of the drugs was higher in patients with +9/+9 polymorphism.

The Dynamic regulation of ßarrestin trafficking in endosomes

ßarrestins bind agonist-activated G protein-coupled receptors (GPCRs) to mediate their desensitization and internalization, and to regulate the rate at which receptors resensitize. GPCRs exhibit different patterns of agonist-induced ßarrestin interaction. Some receptors dissociate from ßarrestin, at or near the plasma membrane and recycle faster than those which are trafficking in endosomes with ßarrestin. It is unknown however what controls the dissociation of ßarrestin from receptors. We have previously reported that the bradykinin type 2 receptor (B2R) forms a stable complex with ßarrestin in endosomes and recycles rapidly to the plasma membrane despite its high avidity to ßarrestin. Here we show using FRET and FRAP approaches that although ßarrestin2 interacts with B2R in endosomes, it dissociated rapidly from the receptors. Although both B2R and the Angiotensin II type 1 receptor (AT1R) internalize with ßarrestin in endosomes, the rate of ßarrestin2 dissociation was faster for B2R than for AT1R. We also tested the effect of a ßarrestin2 mutant, which has an increased avidity for B2R, and showed that it dissociated slower from the receptor than wild-type. Finally we demonstrate that ERK2 signaling in endosomes is important in regulating the ßarrestin2/B2R complex. These experiments give new insight about the mechanisms regulating receptor trafficking, which impact receptor recycling and resensitization.

+9/+9 Homozygosity of the bradykinin receptor gene polymorphism is associated with reduced fat-free mass in chronic obstructive pulmonary disease

The etiology of muscle wasting in chronic obstructive pulmonary disease (COPD) is incompletely understood. We previously showed that the D rather than the I polymorphic variant of the angiotensin-converting enzyme (ACE) gene is associated with preserved quadriceps strength in COPD. If the ACE D allele influences skeletal muscle through increased ACE-related kinin degradation [and reduced activity at the bradykinin type 2 receptor (BK(2)R)], we might expect a similar association with the +9 BK(2)R genotype in this population as well. The objective was to test the hypothesis that the BK(2)R gene polymorphism is a determinant of fat-free mass and quadriceps strength in patients with COPD. In a cross-sectional design we determined BK(2)R genotype, fat-free mass, and quadriceps strength in 110 COPD patients with a mean (+/-SD) predicted forced expiratory volume in 1 s of 34.3 +/- 16.4% and in 104 healthy age-matched control subjects. The mean (+/-SD) fat-free mass index (in kg/m(2)) was significantly lower in 37 patients homozygous for the +9 allele than in carriers of the -9 allele (15.7 +/- 1.8 compared with 16.7 +/- 2.3; P = 0.038); the same pattern was true for quadriceps maximal voluntary force (30.8 +/- 10.4 and 36.4 +/- 12.8 kg; P = 0.02), respectively. No significant effect of BK(2)R genotype on inspiratory muscle strength or on any variable in control subjects was observed. There was no interaction between the effect of the BK(2)R and ACE genotypes on quadriceps strength. The genotype associated with reduced BK(2)R expression is associated with reduced fat-free mass and quadriceps strength in COPD. However, alterations in the activity at the BK(2)R do not seem to account for the previously identified association of quadriceps strength with ACE genotype.

Bradykinin Signaling Counteracts cAMP-Elicited Aquaporin 2 Translocation in Renal Cells

Bradykinin (BK) is one of the most important peptides regulating vascular tone, water, and ionic balance in the body, playing a key role in controlling BP. It is interesting that patients with essential hypertension excrete less BK than normotensive individuals. For elucidating the mechanism by which BK regulates renal water transport that contributes to its antihypertensive effect, aquaporin 2 (AQP2)-transfected collecting duct CD8 cells, expressing the BK type II receptor (BK2R), were used as an experimental model. In CD8 cells, BK pretreatment impaired forskolin-induced AQP2 translocation to the apical plasma membrane. For clarifying the signal transduction cascade associated with this effect, whether BK induced an increase in cytosolic calcium, via the G protein Gq, known to be coupled to BK2R, first was investigated. Spectrofluorometry using fura-2-AM revealed that 100 nM BK elicited a significant increase in Ca(i), which was abolished by the receptor antagonist HOE-140. BK acts through BK2R coupled to both Gq and Galpha13, a known upstream effector of Rho protein. In CD8 cells, BK causes an increase in Rho activity, likely as a result of Galpha13 activation. This results in stabilization of the cortical F-actin network, thus impairing AQP2 trafficking. These effects counteract physiologic vasopressin stimulation, which instead has an opposite effect on actin network organization through Rho inactivation.

Angiotensin-(1–7) Acts as a Vasodepressor Agent Via Angiotensin II Type 2 Receptors in Conscious Rats

Given that angiotensin-(1-7) (Ang-[1-7]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(1-7) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of angiotensin II type 1 (AT1) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT1 receptor (AT1R) antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, Ang-(1-7) (5 pmol/min) alone, candesartan plus Ang-(1-7), and candesartan plus Ang-(1-7) and angiotensin II type 2 (AT2) receptor (AT2R) antagonist PD123319 (50 microg/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(1-7), or candesartan alone caused no significant alteration in MAP, whereas Ang-(1-7) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(1-7) during AT1R blockade was observed in SHR. Moreover, the AT(2)R antagonist PD123319 markedly attenuated the enhanced depressor response evoked by the Ang-(1-7)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(1-7) antagonist A-779 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE 140 (100 microg/kg IV) or the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (1 mg/kg IV) abolished the depressor effect of Ang-(1-7) in the presence of candesartan. Collectively, these results suggest that Ang-(1-7) evoked a depressor response during AT1R blockade via activation of AT2R, which involves the bradykinin-NO cascade.

Dissociation of β-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of β-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with β-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, β-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for β-arrestin, B2R remains associated with β-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a β-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/β-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of β-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.

Endogenous Bradykinin Suppresses Myocardial Fibrosis through the Cardiac-generated Endothelin System Under Chronic Angiotensin-converting Enzyme Inhibition in Heart Failure

In congestive heart failure, angiotensin-converting enzyme inhibitors (ACEIs) may prevent cardiac fibrosis via interaction with both angiotensin II and endothelin-1, which enhance myocardial collagen synthesis. However, whether endogenous bradykinin with an ACEI modifies the cardiac collagen architecture, affecting the endothelin system, has not yet been fully elucidated. We evaluated the changes in circulating hormonal factors, myocardial fibrosis and cardiac gene expression closely linked with heart failure, using an orally active specific bradykinin type 2 receptor antagonist, FR173657 (0.3 mg/kg/day, n = 6), with an ACEI, enalapril (1 mg/kg/day), in dogs with tachycardia-induced congestive heart failure (270 p.p.m., 22 days) and compared the effects with enalapril alone (n = 6). Although there were no differences observed in blood pressure, plasma renin activity, aldosterone and endothelin-1 levels, combined FR173657 significantly increased the cardiac expression of preproendothelin- 1 mRNA (P < 0.05) and collagen type I and type III mRNA (P < 0.05), and cardiac collagen deposits (P < 0.05), and decreased eNOS gene expression (P < 0.05) in the left ventricle compared with the ACEI-treated group. Furthermore, there was a significant negative correlation between the expression of preproendothelin- 1 and eNOS mRNA levels (r = -0.708, P < 0.001). In conclusion, bradykinin may prevent cardiac fibrosis in part via suppression of the local endothelin system in the failing heart through the enhancement of nitric oxide production under chronic angiotensin-converting enzyme inhibition.