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

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

Inhibition of AT2R and Bradykinin Type II Receptor (BK2R) Compromises High K+ Intake-Induced Renal K+ Excretion.

The inhibition of Type II angiotensin II receptor (AT2R) or BK2R (bradykinin type II receptor) stimulates basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT) and activates thiazide-sensitive NCC (Na-Cl cotransporter). The aim of the present study is to examine the role of AT2R and BK2R in mediating the effect of HK (high dietary K+) intake on the basolateral K+ channels, NCC, and renal K+ excretion. Feeding mice (male and female) with HK diet for overnight significantly decreased the basolateral K+ conductance, depolarized the DCT membrane, diminished the expression of pNCC (phosphorylated NCC) and tNCC (total NCC), and decreased thiazide-sensitive natriuresis. Overnight HK intake also increased the expression of cleaved ENaC-α and -γ subunits but had no effect on NKCC2 expression. Pretreatment of the mice (male and female) with PD123319 and HOE140 stimulated the expression of tNCC and pNCC, augmented hydrochlorothiazide-induced natriuresis, and increased the negativity of the DCT membrane. The deletion of Kir4.1 not only decreased the NCC activity but also abolished the stimulatory effect of PD123319 and HOE140 perfusion on NCC activity. Moreover, the effect of overnight HK loading on Kir4.1/Kir5.1 in the DCT and NCC expression/activity was compromised in the mice treated with AT2R/BK2R antagonists. Renal clearance study showed that inhibition of AT2R and BK2R impairs renal K+ excretion in response to overnight HK loading, and the mice pretreated with PD123319 and HOE140 were hyperkalemic during HK intake. We conclude that synergistic activation of AT2R and BK2R is required for the effect of overnight HK diet on Kir4.1/Kir5.1 in the DCT and NCC activity.

Pulsatile antagonism on bradykinin 2-receptor (BK2R) by icatibant triggers the most effective kinin-dependent post-conditioning on rat hearts.

OBJECTIVE: Pharmacological post-conditioning (PC) by intermittent but not continuous administration of exogenous bradykinin (BK) reduces ischemia/reperfusion (I/R) injury via the Reperfusion Injury Salvage Kinase (RISK) pathway activation. We evaluated whether intermittent administration with icatibant (HOE140), a BK2R antagonist, may represent an effective PC strategy, with the advantage of limiting the potential risks of supra-physiologic BK activity. MATERIALS AND METHODS: Hearts from male Sprague-Dawley (SD) rats on a Langendorff system were exposed to I/R injury (30/120 min). BK (100 nM) and HOE140 (1 µM) were administered post-ischemically during the first 3 min of reperfusion, under continuous or intermittent infusion (10 s/each). Hearts were randomly assigned to 5 groups: 1) I/R alone (n=5); 2) continuous HOE140 (cHOE n=6); 3) intermittent HOE140 (iHOE n=6); 4) continuous BK (cBK n=6); 5) intermittent BK (iBK n=6). End-diastolic left ventricular pressure (LVEDP), developed left ventricular pressure (dLVP) and coronary flow (CF) were monitored throughout reperfusion. Left ventricular infarct mass (IM) was quantified together with the phosphorylated levels of Akt and GSK3ß (RISK pathway kinases) at the end of reperfusion. RESULTS: IM was not significantly changed in cBK or cHOE groups (vs. I/R). Conversely, both iBK and iHOE groups showed a significant limitation in IM (vs. I/R, p<0.05, p<0.01, respectively). Akt and GSK3ß phosphorylation levels were higher in iBK and iHOE groups (vs. I/R, p<0.05). When compared to I/R group, both LVEDP values (p<0.05, first 60-min reperfusion), as well as dLVP values (p<0.01) were improved only in iHOE group. CF values did not vary among all groups. CONCLUSIONS: In isolated rat hearts, intermittent modulation of the endogenous kallikrein-kinin system by a selective BK2R antagonist mediates PC cardioprotection via RISK signaling.

Bradykinin contributes to immune liver injury via B2R receptor-mediated pathways in trichloroethylene sensitized mice: A role in Kupffer cell activation.

We have previously shown trichloroethylene (TCE) induced occupational medicamentosa-like dermatitis due to TCE (OMLDT) with immune liver injury, and kallikrein-kinin system (KKS) activation as a probably mechanism underlying the immune damage. Bradykinin (BK) is an important active component of KKS system function, but the specific role of BK in the immune liver injury has never been examined. The present study aimed to explore the important role of BK and mechanisms of action in immune liver injury induced by TCE. TCE sensitization significantly increased the expression of BK receptor (B2R) in the liver. Compared to blank and vehicle control group, TCE sensitization positive mice developed exacerbated liver injury evidenced by elevated AST, ALT levels and hepatocyte damage. TCE sensitization also stimulated MAPK and STAT3 activation in liver tissue. B2R antagonist HOE140 ameliorated these changes. Kupffer cells (KCs) of the liver were also activated following TCE sensitization; both CD68+ KCs and CD16/CD32+ M1 type KCs were increased in TCE positive group. Further experiments isolated the KCs from the liver in each group and showed that TCE sensitization resulted activation of MAPK signal pathway which in turn caused release of the pro-inflammatory cytokines, IL-1ß, IL-6, TNF-α, in KCs; the antagonist HOE140 again decreased these changes in KCs. These results uncover a novel role of BK and B2R cross-talk in KCs activation in TCE sensitized mice, mediated by pro-inflammatory cytokine release via MAPK and STAT3 activation, contributing to the immune liver injury.

Expression and distribution of kinin B2 receptor in medulla and spinal cord tissues of Rats treated with capsaicin / Expresión y distribución del receptor B2 de cininas en la médula y tejido medular espinal de ratones tratados con capsaicina

Kinins are vasoactive peptides that promote pain and inflammation, yet centrally believed to participate to cardiovascular defensive reflexes produced by noxious stimuli. These peptides signal through the activation of two transmembrane G-protein-coupled receptors named B1 and B2 receptors (B1R and B2R). The B2R is constitutive in healthy tissues and animals. The aim of the study was to measure the gene and protein expression of B2R kinin receptors in central and peripheral tissues isolated from control rats and rats were pre-treated with capsaicin on the second day of life (50 mg/kg, s.c.) or two weeks prior to sacrifice (125 mg/kg over three days, s.c.). The same treatment with saline was made in control animals. Levels of mRNA for B2R were measured by quantitative RT-PCR and Qualitative while receptor binding sites were measured on tissue sections with the radioligands 125I-HPP-Hoe 140 (B2R). B2R was expressed in all studied tissues (hypothalamus, paratrigeminal nucleus, nucleus of solitary tract, spinal cord, aorta and liver) and treatment capsaicin neonates when compared to controls, did not affect its level of expression. Capsaicin had no significant effect on the expression of B2R in some tissues on binding sites. The synthesis of B2R kinin receptor is not associated with sensory C-fibre and tissues showed no significant difference indicating that B2R was regulated by distinct mechanisms.

Las cininas son péptidos vaso activos que participan de mecanismos de dolor e inflamación, con la promoción de reflejos cardiovasculares defensivos producidos por estímulos nocivos. Estos péptidos señalizan por medio de la activación de dos receptores transmembrana acoplados a proteína G llamados receptores B1 y B2 (B1R y B2R). El B2R es constitutivo en tejidos saludables y en animales. El objetivo de este estudio consistió en tomar las medidas de la expresión del gen y la proteína del receptor B2R de cinina en tejidos centrales y periféricos aislados de ratones control y ratones pre tratados con capsaicina en el segundo día de vida (50 mg/kg, s.c.) o con dos semanas antes del sacrificio (125 mg/kg por tres días, s.c.). El mismo tratamiento fue hecho con solución salina en los animales control. Fueron medidos niveles de mRNA para B2R por RT-PCR en forma cuantitativa y cualitativa, en cuanto los sitios de unión del receptor fueron medidos en secciones de tejido con radioligante 125I-HPP-Hoe 140 (B2R). B2R fue expresado en todos los tejidos estudiados (hipotálamo, núcleo paratrigeminal, núcleo del tracto solitario, médula espinal, aorta e hígado). El tratamiento con capsaicina en ratones neonatos comparados con controles no presentaron afectación del nivel de expresión. Por otro lado, la capsaicina no tuvo efecto significante en la expresión de los sitios de unión de B2R en los tejidos. La síntesis del receptor B2R de cininas no está asociado con fibras C sensoriales así como los tejidos no mostraron diferencias significativas indicando que B2R es regulado por mecanismos distintos.

Bradykinin -induced vasodilatation: Role of age, ACE1-inhibitory peptide, mas- and bradykinin receptors.

Bradykinin exerts its vascular actions via two types of receptors, the non-constitutively expressed bradykinin receptor type 1 (BR1) and the constitutive type 2 receptor (BR2). Bradykinin-induced vasorelaxation is age-dependent, a phenomenon related to the varying amounts of BR1 and BR2 in the vasculature. Isoleucine-proline-proline (Ile-Pro-Pro), a bioactive tripeptide, lowers elevated blood pressure and improves impaired endothelium-dependent vasorelaxation in hypertensive rats. It inhibits angiotensin converting enzyme 1 (ACE1). Other mechanisms of action have also been postulated. The aims of the study were to clarify the underlying mechanisms of the age-dependency of bradykinin-induced vasodilatation such as the roles of the two bradykinin receptors, the mas-receptor and synergism with Ile-Pro-Pro. The vascular response studies were conducted using mesenteric artery and aorta rings from normotensive 6 wk. (young) and 22 wk. (old) Wistar rats. Cumulative dosing of acetylcholine, bradykinin and angiotensin(1-7) (Ang(1-7))were tested in phenylephrine-induced vasoconstriction with or without 10min pre-incubation with antagonists against BR1-, BR2- or mas-receptors, Ang(1-7) or ACE1-inhibitors captopril and Ile-Pro-Pro. The bradykinin-induced vasorelaxation in vitro was age-dependent and it was improved by pre-incubation with Ile-Pro-Pro, especially in old rats with endothelial dysfunction. The mas-receptor antagonist, D-Pro7-Ang(1-7) abolished bradykinin-induced relaxation totally. Interestingly, BR1 and BR2 antagonists only slightly reduced bradykinin-induced vasorelaxation, as an evidence for the involvement of other mechanisms in addition to receptor activation. In conclusion, bradykinin-induced vasorelaxation was age-dependent and Ile-Pro-Pro improved it. Mas receptor antagonist abolished relaxation while bradykinin receptor antagonist only slightly reduced it, suggesting that bradykinin-induced vasorelaxation is regulated also by other mechanisms than the classical BR1/BR2 pathway.

Heteromerization Between the Bradykinin B2 Receptor and the Angiotensin-(1-7) Mas Receptor: Functional Consequences.

Bradykinin B2 receptor (B2R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B2R and MasR formed a constitutive heteromer, which was not modified by their agonists. B2R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B2R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B2R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2R antagonist. Concerning serine/threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2R-MasR but not in B2R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties.

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.

The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents.

AIMS: The isolated human umbilical vein is a robust contractile bioassay for ligands of the bradykinin (BK) B2 receptor (B2R), also extendable to B1 receptor (B1R) pharmacology. We hypothesized that, as a freshly isolated vessel, it also contains traces of plasma proteins that may confer responses to exogenous proteases via the formation of kinins. MAIN METHODS: Rings of human umbilical veins were mounted in organ baths containing Krebs buffer maintained at 37°C and purified proteases were introduced in the bathing fluid along with additional drugs/proteins that permit mechanistic analysis of effects. KEY FINDINGS: The previously described contractile response to human recombinant tissue kallikrein (KLK-1, 1-10nM) is not influenced by metabolic inhibitors, suggesting its dependence on a preexisting reservoir of low molecular weight-kininogen (LK). Active plasma kallikrein (apK, ≤5nM) was inactive in fresh tissues, unless high molecular weight-kininogen (HK, 39-197nM) replenishment was applied. The effects of KLK-1 and HK+apK are abolished by pretreating tissues with icatibant, but not with tranexamic acid. C1-esterase inhibitor inhibited only HK+apK. Purified plasmin and neutrophil proteinase-3 produced small contractions in the presence of HK only, and tissue plasminogen activator, none. B1R stimulation was pharmacologically evidenced in response to KLK-1 if LK was supplied. SIGNIFICANCE: The pharmacology of KLK-1 and HK+apK in the human isolated umbilical vein is essentially based on the activity of locally generated kinins and this assay models the inhibitory action of some therapeutic agents active in angioedema states. Proteases that indirectly generate kinins have little activity in the system.

Icatibant as acute treatment for hereditary angioedema in adults.

INTRODUCTION: Hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) is a rare disease, characterized by recurrent, unpredictable episodes of cutaneous and/or mucosal edema. Bradykinin, released by the activation of the contact system, binds to bradykinin B2 receptors on the endothelial cell surface to enhance vascular permeaility, which leads to angioedema. AREAS COVERED: C1-INH-HAE therapy is aimed at the inhibition of bradykinin release, as well as at the blockage of its effects mediated by its receptor. Three controlled trials, three open-label extensions, and two open-label studies, and a prospective, observational study have confirmed the safety and efficacy of the bradykinin B2 receptor antagonist, icatibant administered as acute treatment for HAE attacks in adult patients with C1-INH-HAE. Expert commentary: The ready-to-use, pre-filled syringes of icatibant can be self-administered easily, effectively, safely and, importantly, conviently. - This has resulted in patients being able to quickly treat an attack and realize a dramatic change for the better in their lives.

Improvement of skin wound healing in diabetic mice by kinin B2 receptor blockade.

Impaired skin wound healing is a major medical problem in diabetic subjects. Kinins exert a number of vascular and other actions limiting organ damage in ischaemia or diabetes, but their role in skin injury is unknown. We investigated, through pharmacological manipulation of bradykinin B1 and B2 receptors (B1R and B2R respectively), the role of kinins in wound healing in non-diabetic and diabetic mice. Using two mouse models of diabetes (streptozotocin-induced and db/db mice) and non-diabetic mice, we assessed the effect of kinin receptor activation or inhibition by subtype-selective pharmacological agonists (B1R and B2R) and antagonist (B2R) on healing of experimental skin wounds. We also studied effects of agonists and antagonist on keratinocytes and fibroblasts in vitro. Levels of Bdkrb1 (encoding B1R) and Bdkrb2 (encoding B2R) mRNAs increased 1-2-fold in healthy and wounded diabetic skin compared with in non-diabetic skin. Diabetes delayed wound healing. The B1R agonist had no effect on wound healing. In contrast, the B2R agonist impaired wound repair in both non-diabetic and diabetic mice, inducing skin disorganization and epidermis thickening. In vitro, B2R activation unbalanced fibroblast/keratinocyte proliferation and increased keratinocyte migration. These effects were abolished by co-administration of B2R antagonist. Interestingly, in the two mouse models of diabetes, the B2R antagonist administered alone normalized wound healing. This effect was associated with the induction of Ccl2 (encoding monocyte chemoattractant protein 1)/Tnf (encoding tumour necrosis factor α) mRNAs. Thus stimulation of kinin B2 receptor impairs skin wound healing in mice. B2R activation occurs in the diabetic skin and delays wound healing. B2R blockade improves skin wound healing in diabetic mice and is a potential therapeutic approach to diabetic ulcers.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice.

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin-angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Kinin B2 receptor can play a neuroprotective role in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by cognitive decline, presence of amyloid-beta peptide (Aß) aggregates and neurofibrillary tangles. Kinins act through B1 and B2 G-protein coupled receptors (B1R and B2R). Chronic infusion of Aß peptide leads to memory impairment and increases in densities of both kinin receptors in memory processing areas. Similar memory impairment was observed in C57BL/6 mice (WTAß) but occurred earlier in mice lacking B2R (KOB2Aß) and was absent in mice lacking B1R (KOB1Aß). Thus, the aim of this study was to evaluate the participation of B1R and B2R in Aß peptide induced cognitive deficits through the evaluation of densitiesof kinin receptors, synapses, cell bodies and number of Aß deposits in brain ofWTAß, KOB1Aß and KOB2Aß mice. An increase in B2R density was observed in both WTAß and KOB1Aß in memory processing related areas. KOB1Aß showed a decrease in neuronal density and an increase in synaptic density and, in addition, an increase in Aß deposits in KOB2Aß was observed. In conclusion, memory preservation in KOB1Aß, could be due to the increase in densities of B2R, suggesting a neuroprotective role for B2R, reinforced by the increased number of Aß plaques in KOB2Aß. Our data point to B2R as a potential therapeutic target in AD.

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus.

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1ß and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus.

Intracellular and nuclear bradykinin B2 receptors.

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

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

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

Interruption of the ionic lock in the bradykinin B2 receptor results in constitutive internalization and turns several antagonists into strong agonists.

The DRY motif with the highly conserved R3.50 is a hallmark of family A G protein-coupled receptors (GPCRs). The crystal structure of rhodopsin revealed a salt bridge between R135(3.50) and another conserved residue, E247(6.30), in helix 6. This ionic lock was shown to maintain rhodopsin in its inactive state. Thus far, little information is available on how interruption of this ionic bond affects signaling properties of nonrhodopsin GPCRs, because the focus has been on mutations of R3.50, although this residue is indispensable for G protein activation. To investigate the importance of an ionic lock for overall receptor activity in a nonrhodopsin GPCR, we mutated R128(3.50) and E238(6.30) in the bradykinin (BK) B(2) receptor (B(2)R) and stably expressed the constructs in HEK293 cells. As expected, mutation of R3.50 resulted in lack of G protein activation. In addition, this mutation led to considerable constitutive receptor internalization. Mutation of E6.30 (mutants E6.30A and E6.30R) also caused strong constitutive internalization. Most intriguingly, however, although the two E6.30 mutants displayed no increased basal phosphatidylinositol hydrolysis, they gave a response to three different B(2)R antagonists that was almost comparable to that obtained with BK. In contrast, swapping of R3.50 and E6.30, thus allowing the formation of an inverse ionic bond, resulted in rescue of the wild type phenotype. These findings demonstrate for the first time, to our knowledge, that interruption of the ionic lock in a family A GPCR can have distinctly different effects on receptor internalization and G protein stimulation, shedding new light on its role in the activation process.

Angiotensin-(1-7) reduces the perfusion pressure response to angiotensin II and methoxamine via an endothelial nitric oxide-mediated pathway in cirrhotic rat liver.

Recent studies have shown that, in cirrhosis, portal angiotensin-(1-7) [Ang-(1-7)] levels are increased and hepatic expression of angiotensin converting enzyme 2 (ACE2) and the Mas receptor are upregulated, but the effects of Ang-(1-7) on hepatic hemodynamics in cirrhosis have not been studied. This study investigated the effects of Ang-(1-7) on vasoconstrictor-induced perfusion pressure increases in cirrhotic rat livers. Ang II or the alpha 1 agonist methoxamine (MTX) were injected in the presence or absence of Ang-(1-7), and the perfusion pressure response was recorded. Denudation of vascular endothelial cells with sodium deoxycholate was used to investigate the contribution of endothelium to the effects of Ang-(1-7). Ang-(1-7) alone had no effect on perfusion pressure. However, it reduced the maximal vasoconstriction response and area under the pressure response curve to Ang II and MTX by >50% (P < 0.05). This effect of Ang-(1-7) was not blocked by Mas receptor inhibition with A779 or by Ang II type 1 and type 2 receptor and bradykinin B(2) receptor blockade and was not reproduced by the Mas receptor agonist AVE0991. D-Pro(7)-Ang-(1-7), a novel Ang-(1-7) receptor antagonist, completely abolished the vasodilatory effects of Ang-(1-7), as did inhibition of endothelial nitric oxide synthase (eNOS) with N(G)-nitro-L-arginine methyl-ester, guanylate cyclase blockade with ODQ and endothelium denudation. The functional inhibition by D-Pro(7)-Ang-(1-7) was accompanied by significant (P < 0.05) inhibition of eNOS phosphorylation. This study shows that Ang-(1-7) significantly inhibits intrahepatic vasoconstriction in response to key mediators of increased vascular and sinusoidal tone in cirrhosis via a receptor population present on the vascular endothelium that is sensitive to D-Pro(7)-Ang-(1-7) and causes activation of eNOS and guanylate cyclase-dependent NO signaling pathways.

Bradykinin-induced leukocyte- and platelet-endothelium interactions in the cerebral microcirculation.

Bradykinin is known for its pathophysiological role as mediator of inflammation. Following cerebral ischemia, bradykinin promotes the secondary brain damage through an increase of vascular permeability and brain edema formation, again hallmarks of inflammation. It is not clear, whether bradykinin also activates inflammatory cells and regulates microcirculatory blood flow in the brain. The purpose of the study is to investigate the reaction of bradykinin upon cerebral leukocyte- and thrombocyte-endothelium interactions as well as microvascular perfusion. Intravital fluorescence microscopy of pial blood vessels was performed in gerbils. Intracarotid injection of bradykinin resulted in increased numbers of rolling and adherent leukocytes as well as rolling platelets at the venular endothelium. This was reversed by administration of a bradykinin B2 receptor antagonist. In contrast, after additional administration of a B1 receptor antagonist, microvascular blood-flow and capillary density was decreased. We conclude that bradykinin initiates leukocyte- and platelet-endothelium interactions in the cerebral microcirculation via activation of B2 receptors. Activation of B1 receptors ensures regular cerebral perfusion. Thus, to attenuate secondary brain damage, inhibition of B2 but not B1 receptors might be of therapeutical benefit.

XJP-1, a novel ACEI, with anti-inflammatory properties in HUVECs.

AIM: We investigated whether 8-dihydroxy-3-methyl-isochromanone (XJP-1), a novel angiotensin-converting enzyme inhibitor (ACEI), exhibited inhibitory activity to lipopolysaccharide (LPS)-accelerated vascular inflammation. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and cultured. The direct effect of XJP-1 on the activation of endothelial cells was measured using MTT assay. Nitric oxide (NO) in the culture medium was measured using Griess method. The expression of cell adhesion molecules (ICAM-1 and VCAM-1) was determined by flow cytometry and RT-PCR. The protein expression levels of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1, and endothelin-1 (ET-1) secretion were measured using ELISA. Quantitative analysis of eNOS, iNOS, inhibitory factor NF-κB (IκB) and MAPKs were determined using Western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined using immunofluorescence. RESULTS: XJP-1 significantly inhibited LPS-mediated endothelial cell dysfunction, as measured by NO production, iNOS expression, adhesion molecule (ICAM-1, VCAM-1) expression, and chemokine (TNF-α, MCP-1) production in vitro. It up-regulated eNOS expression in the same experimental setting. XJP-1 alone was found non-cytotoxic at the concentration up to 1000µM. The mechanistic investigations of XJP-1 suppression LPS-induced inflammation in HUVECs revealed that XJP-1 blocked NF-κB nuclear entry in an IκB-dependent manner, as well as inhibited MAPK activation induced by LPS. XJP-1 reduced endothelin-1 secretion and increased nitric oxide metabolite production by HUVECs. However, the effect of XJP-1 on nitric oxide and endothelin-1 metabolite production is mediated by the activation of bradykinin B(2) receptor being counteracted, at least in part, by a specific antagonist. CONCLUSION: XJP-1 inhibited LPS-induced cytotoxicity and inflammatory response. The mechanism underlying this protective effect might be related to the inhibition of MAPK and NF-κB signaling pathway activation, suggesting the potential inhibition of the atherosclerotic process by suppressing the expression of chemoattractant molecules and monocyte adhesion. XJP-1 also has an effect in improving endothelin-1 through activating bradykinin B(2) receptor. These findings indicated that XJP-1 is potentially a novel therapeutic candidate for the treatment of atherosclerosis.