Effect of the bradykinin 1 receptor antagonist SSR240612 after oral administration in Mycobacterium tuberculosis-infected mice.

The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B1R-/-, B2R-/- and double B1R/B2R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B1R and B2R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B2R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B1R/B2R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B1R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg9-BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B1R (des-Arg9-BK and SSR240612) and B2R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis.

Kinin B1 receptors as a therapeutic target for inflammation.

INTRODUCTION: Kinins are peptide mediators exerting their pro-inflammatory actions by the selective stimulation of two distinct G-protein coupled receptors, termed BKB1R and BKB2R. While BKB2R is constitutively expressed in a multitude of tissues, BKB1R is hardly expressed at baseline but highly inducible by inflammatory mediators. In particular, BKB1R was shown to be involved in the pathogenesis of numerous inflammatory diseases. Areas covered: This review intends to evaluate the therapeutic potential of substances interacting with the BKB1R. To this purpose we summarize the published literature on animal studies with antagonists and knockout mice for this receptor. Expert Opinion: In most cases the pharmacological inhibition of BKB1R or its genetic deletion was beneficial for the outcome of the disease in animal models. Therefore, several companies have developed BKB1R antagonists and tested them in phase I and II clinical trials. However, none of the developed BKB1R antagonists was further developed for clinical use. We discuss possible reasons for this failure of translation of preclinical findings on BKB1R antagonists into the clinic.

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.

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.

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 IL1β 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. .

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.

Bradykinin promotes migration and invasion of human immortalized trophoblasts.

Having demonstrated that the bradykinin B2 receptor (B2R) is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK) on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L) for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L) incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L) for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8), modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies.

Angiotensin I-converting enzyme inhibitors are allosteric enhancers of kinin B1 and B2 receptor function.

The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE to decrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R and B2R) signaling, which may underlie some of their therapeutic usefulness. They can indirectly potentiate the actions of bradykinin (BK) and ACE-resistant BK analogs on B2Rs to elevate arachidonic acid and NO release in laboratory experiments. Studies indicate that ACE inhibitors and some Ang metabolites increase B2R functions as allosteric enhancers by inducing a conformational change in ACE. This is transmitted to B2Rs via heterodimerization with ACE on the plasma membrane of cells. ACE inhibitors are also agonists of the B1R, at a Zn-binding sequence on the second extracellular loop that differs from the orthosteric binding site of the des-Arg-kinin peptide ligands. Thus, ACE inhibitors act as direct allosteric B1R agonists. When ACE inhibitors enhance B2R and B1R signaling, they augment NO production. Enhancement of B2R signaling activates endothelial NO synthase, yielding a short burst of NO; activation of B1Rs results in a prolonged high output of NO by inducible NO synthase. These actions, outside inhibiting peptide hydrolysis, may contribute to the pleiotropic therapeutic effects of ACE inhibitors in various cardiovascular disorders.

In vivo up-regulation of kinin B1 receptors after treatment with Porphyromonas gingivalis lipopolysaccharide in rat paw.

It has been demonstrated that kinin B(1) receptors are highly up-regulated under several stressful stimuli, such as infection. However, there is no evidence indicating whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) might lead to B(1) receptor up-regulation. In this study, we demonstrate that Pg-LPS injection into the rat paw resulted in a marked functional up-regulation of B(1) receptors (as measured by an increase of B(1) receptor-induced edema), which was preceded by a rapid rise in B(1) receptor mRNA expression. The local administration of Pg-LPS also resulted in a prominent production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), followed by an increase of neutrophil influx; both events were observed at periods before B(1) receptor induction. The functional and molecular Pg-LPS-elicited B(1) receptor up-regulation was significantly reduced by the glucocorticoid dexamethasone (0.5 mg/kg s.c.), and to a lesser extent by the chimeric anti-TNF-alpha antibody infliximab (1 mg/kg s.c.). Of high relevance, we show for the first time that a single administration of the proresolution lipid mediator (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (resolvin E1; 300 ng/rat i.p.) was able to markedly down-regulate Pg-LPS-driven B(1) receptor expression, probably by inhibiting TNF-alpha production and neutrophil migration. Collectively, the present findings clearly suggest that Pg-LPS is able to induce the up-regulation of B(1) receptors through mechanisms involving TNF-alpha release and neutrophil influx, which are largely sensitive to resolvin E1. It is tempting to suggest that kinin B(1) receptors might well represent a pivotal pathway for the inflammatory responses evoked by P. gingivalis and its virulence factors.

Fluorescent ligands of the bradykinin B1 receptors: pharmacologic characterization and application to the study of agonist-induced receptor translocation and cell surface receptor expression.

Unlike the widely distributed and preformed B(2) receptors, the bradykinin B(1) receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B(1) receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-epsilon-aminocaproyl-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-bradykinin] and the agonist B-10378 [5(6)CF-epsilon-aminocaproyl-Lys-des-Arg(9)-bradykinin]. B-10376 has a K(i) of 10 to 20 nM to displace [(3)H]Lys-des-Arg(9)-bradykinin from rabbit or human recombinant B(1) receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA(2), 7.49). B-10378 was a full agonist at the naturally expressed B(1) receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K(i) of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B(1) receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B(1) receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B(1) receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B(1) receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).

Effects of angiotensin-1 converting enzyme inhibition on oxidative stress and bradykinin receptor expression during doxorubicin-induced cardiomyopathy in rats.

To evaluate the mechanisms and the impact of the angiotensin-converting enzyme inhibitor perindopril (P) in a model of doxorubicin (D)-induced cardiotoxicity, male Wistar rats received D (1 mg/kg/d, IP for 10 days), P (2 mg/kg/d by gavage from day 1 to day 18), D (for 10 days) + P (for 18 days) or saline. D decreased systolic blood pressure and body and heart weights. Left ventricular diastolic diameter was increased by D (P < 0.01), but it was not attenuated by P. D decreased plasma vitamin C (P < 0.05) and increased the ascorbyl radical/vitamin C ratio (P < 0.01). This ratio was attenuated by P. No difference was found among groups in cardiac troponin I, brain natriuretic peptide concentrations, and tissue oxidative stress (OS). Myocardial MCP-1 expression was higher in the D group. Cardiac kinin receptor (B1R and B2R) expression was not affected by D, yet binding sites for B2R and B1R were increased in D+P and P groups, respectively (P < 0.05). In conclusion, D induced cardiac functional alterations, inflammation and plasma OS whereas tissue OS, and cardiac kinin receptors expression were not modified. P did not improve cardiac performance, but it modulated kinin receptor expression and enhanced antioxidant defense.

A fluorescent version of the bradykinin B2 receptor antagonist B-9430: pharmacological characterization and use in live cell imaging.

B-9430 (d-Arg-[Hyp3, Igl5, D-Igl7, Oic8]-bradykinin), where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine and Oic is (3as, 7as)-octahydroindol-2-yl-carbonyl is a high affinity bradykinin B2 receptor antagonist with effects extended to the B1 receptors at high concentrations. The N-terminus of B-9430 has been extended with d-biotinyl (B-10330) or 5(6)-carboxyfluorescein-epsilon-aminocaproyl (B-10380) to derive fluorescent receptor probes. The pharmacological profile of B-10380 was similar to that of B-9430 with a minor loss of potency (a competitive antagonist of bradykinin at the B2 receptors of the human isolated umbilical vein, pA2 6.83; an insurmountable antagonist at the B2 receptors in the rabbit jugular vein; a weak competitive antagonist of the B1 receptors in the rabbit aorta, pA2 5.95). B-10330 and B-10380 displaced the binding of [3H]bradykinin from rabbit B2 receptors with a potency slightly inferior to that of B-9430 (larger gap at the rat B2 receptor). Treatment with B-10330 and fluorescent streptavidin did not support imaging of recombinant B2 receptors. However, the plasma membrane of HEK 293a cells that transiently expressed recombinant rabbit B2 receptors, but not B1 receptors, was labeled with 5-50 nM B-10380 (epifluorescence microscopy). B-10380 staining was not observed in nontransfected cells and was abolished by co-treating receptor-expressing cells with a nonpeptide antagonist. The N-terminal extension of a potent peptide antagonist of the bradykinin B2 receptor with a fluorophore produced a fluorescent probe suitable for live cell imaging and other applications at the expense of a minor loss of affinity.

Kinin B1 and B2 receptor expression in osteoblasts and fibroblasts is enhanced by interleukin-1 and tumour necrosis factor-alpha. Effects dependent on activation of NF-kappaB and MAP kinases.

Pro-inflammatory mediators formed by the kallikrein-kinin system can stimulate bone resorption and synergistically potentiate bone resorption induced by IL-1 and TNF-alpha. We have shown that the effect is associated with synergistically enhanced RANKL expression and enhanced prostaglandin biosynthesis, due to increased cyclooxygenase-2 expression. In the present study, the effects of osteotropic cytokines and different kinins on the expression of receptor subtypes for bradykinin (BK), des-Arg10-Lys-BK (DALBK), IL-1beta and TNF-alpha have been investigated. IL-1beta and TNF-alpha enhanced kinin B1 and B2 receptor binding in the human osteoblastic cell line MG-63 and the mRNA expression of B1 and B2 receptors in MG-63 cells, human gingival fibroblasts and intact mouse calvarial bones. Kinins did not affect mRNA expression of IL-1 or TNF receptors. EMSA showed that IL-1beta and TNF-alpha activated NF-kappaB and AP-1 in MG-63 cells. IL-1beta stimulated NF-kappaB via a non-canonical pathway (p52/p65) and TNF-alpha via the canonical pathway (p50/p65). Activation of AP-1 involved c-Jun in both IL-1beta and TNF-alpha stimulated cells, but c-Fos only in TNF-alpha stimulated cells. Phospho-ELISA and Western blots showed that IL-1beta activated JNK and p38, but not ERK 1/2 MAP kinase. Pharmacological inhibitors showed that NF-kappaB, p38 and JNK were important for IL-1beta induced stimulation of B1 receptors, and NF-kappaB and p38 for B2 receptors. p38 and JNK were important for TNF-alpha induced stimulation of B1 receptors, whereas NF-kappaB, p38 and JNK were involved in TNF-alpha induced expression of B2 receptors. These data show that IL-1beta and TNF-alpha upregulate B1 and B2 receptor expression by mechanisms involving activation of both NF-kappaB and MAP kinase pathways, but that signal transduction pathways are different for IL-1beta and TNF-alpha. The enhanced kinin receptor expression induced by the pro-inflammatory cytokines IL-1beta and TNF-alpha might be one important mechanism involved in the synergistic enhancement of prostaglandin formation caused by co-treatment with kinins and one of the two cytokines. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including periodontitis and rheumatoid arthritis.

Interleukin-1beta enhances the action of bradykinin in rat myenteric neurons through up-regulation of glial B1 receptor expression.

Interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) are released under pathological conditions in the gastrointestinal tract such as inflammatory bowel diseases (IBD). We examined the effects of IL-1beta and TNFalpha on bradykinin (BK) -induced increases in the intracellular Ca(2+) concentration ([Ca(2+)]i) and prostaglandin (PG) E(2) release in neonatal rat myenteric plexus cells. BK evoked a [Ca(2+)]i increase in myenteric neurons and glial cells, both of which were potentiated by treatment with IL-1beta but not TNFalpha. In both cell types, the [Ca(2+)]i responses to BK were abolished by D-Arg(0)[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-BK (HOE140), a B2R antagonist, but not affected by des-Arg(9)-HOE140, a B1R antagonist. After culture with IL-1beta, however, the B1R antagonist suppressed the BK-induced [Ca(2+)]i increase. Only in glial cells did the B1R agonists des-Arg(9)-BK and BK fragment 1-8 evoke a [Ca(2+)]i rise in a dose-dependent manner. Real time RT-PCR and immunocytochemical analyses showed that IL-1beta treatment increased expression of B1R mRNA in myenteric plexus cells and B1R protein in glial cells, respectively. Either indomethacin or an EP1 receptor antagonist suppressed the increased [Ca(2+)]i response to BK invoked by treatment with IL-1beta. IL-1beta treatment increased BK-induced PGE(2) release from cultured myenteric plexus cells. These results suggest that IL-1beta promotes up-regulation of B1R expression in glial cells, resulting in the potentiation of neural responses to BK through the elevation of PGE(2) released from glial cells. The alteration of phenotypes of glial cells may be the cause of the changes in neural function in the enteric nervous system in IBD.

Double-stranded RNA increases kinin B1 receptor expression and function in human airway epithelial cells.

Increased levels of kinins have been detected within the airways during upper respiratory viral infections (URIs). Rhinovirus, the major URI associated with acute exacerbations of asthma, is an ssRNA virus that primarily infects the airway epithelium and produces dsRNA during replication. We asked whether dsRNA could increase the expression of kinin receptors in airway epithelial cells, thereby potentiating the inflammatory consequences of kinin generation. Human airway epithelial cell line BEAS-2B was stimulated with the dsRNA analog Poly I:C and kinin receptor expression detected by quantitative RT-PCR as well as radioligand binding. Poly I:C induced an increase in B1 and B2 receptor mRNA levels in BEAS-2B and primary human normal bronchial epithelial cells. At the cell surface, only B1 receptor expression was increased by Poly I:C. Furthermore, pretreatment of BEAS-2B cells with Poly I:C enhanced the induction of phospho-ERK following B1 receptor ligand stimulation. To investigate whether these finding had potential in vivo relevance, we assessed B1 receptor expression in nasal tissue obtained from 8 normal human subjects with URIs and 3 control subjects. Five of the URI subjects demonstrated increased B1 receptor mRNA compared to the 3 control subjects. We suggest that increased expression of B1 receptor in the human airway following a URI could increase the risk of an exacerbation of asthma by contributing to increased inflammation in the airway.

Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw.

BACKGROUND AND PURPOSE: alpha-Humulene and trans-caryophyllene are sesquiterpene compounds identified in the essential oil of Cordia verbenacea which display topical and systemic anti-inflammatory effects in different experimental models. However, the molecular mechanisms through which they exert their anti-inflammatory activity still remain unclear. Here, we evaluate the effects of alpha-humulene and trans-caryophyllene on the acute inflammatory responses elicited by LPS. EXPERIMENTAL APPROACH: The biological activities of alpha-humulene and trans-caryophyllene were investigated in a model of acute inflammation in rat paw, induced by LPS and characterized by paw oedema, neutrophil recruitment, cytokine production, activation of MAP kinases and NF-kappaB and up-regulated expression of kinin B(1) receptors. KEY RESULTS: Treatment with either alpha-humulene or trans-caryophyllene effectively reduced neutrophil migration and activation of NF-kappaB induced by LPS in the rat paw. However, only alpha-humulene significantly reduced the increase in TNF-alpha and IL-1beta levels, paw oedema and the up-regulation of B(1) receptors following treatment with LPS. Both compounds failed to interfere with the activation of the MAP kinases, ERK, p38 and JNK. CONCLUSIONS AND IMPLICATIONS: Both alpha-humulene and trans-caryophyllene inhibit the LPS-induced NF-kappaB activation and neutrophil migration, although only alpha-humulene had the ability to prevent the production of pro-inflammatory cytokines TNF-alpha and IL-1beta and the in vivo up-regulation of kinin B(1) receptors. These data provide additional molecular and functional insights into the beneficial effects of the sesquiterpenes alpha-humulene and trans-caryophyllene isolated from the essential oil of Cordia verbenacea as agents for the management of inflammatory diseases.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg9BK (DBK). Our aim was to determine the potential expression of kinin B1 and B2 receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD2) calculated from the curves was 7.0 ± 0.1 for BK and 7.3 ± 0.2 for DBK. The efficacy was 51 ± 2 percent for BK and 30 ± 1 percent for DBK when compared to 1 æM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 æM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B1 and B2 subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta.

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Does zaltoprofen antagonize the bradykinin receptors?

Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B(2) receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B(2) receptor antagonists (contractility, human isolated umbilical vein) or B(1) receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 microM) failed to antagonize BK or des-Arg(9)-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 microM) abated des-Arg(9)-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 microM) did not inhibit kinin-stimulated phospholipase A(2) activity in HEK 293 cells expressing recombinant forms of the rabbit B(1) or B(2) receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems.

Antagonist, partial agonist and antiproliferative actions of B-9870 (CU201) as a function of the expression and density of the bradykinin B1 and B2 receptors.

BACKGROUND AND PURPOSE: A bradykinin (BK) B2 receptor (B2R) antagonist, B-9870 (CU201), has been proposed to behave as a 'biased agonist' at B2Rs and to exert anti-neoplasic effects. It was unclear whether these effects were determined by the activation of B2Rs by the drug. B-9870 was evaluated for antagonism or stimulation of several responses mediated by the rabbit B2R or B1 receptor (B1R); its anti-proliferative activity was also characterized. EXPERIMENTAL APPROACH AND KEY RESULTS: B-9870 was an insurmountable B2R antagonist in the rabbit jugular vein contractility assay, but a partial agonist in HEK 293 cells expressing the rabbit B2R or a green fluorescent protein (GFP) conjugate of the latter (ERK1/2 phosphorylation, [Ca2+]i, [3H]-arachidonate release, endocytosis). The agonist-like effects of B-9870 were inhibited by the B2R antagonist LF 16.0687 and absent in untransfected cells. In addition, B-9870 was a surmontable antagonist of the rabbit B1R in the aorta contractility assay, and blocked Lys-des-Arg9-BK-induced ERK1/2 phosphorylation in HEK 293 cells expressing a fluorescent B1R conjugate. B-9870 inhibited the growth of MDA-MB-231 cells. The latter effect was not influenced by B1R or B2R antagonists and was not apoptotic. MDA-MB-231 cells expressed a small population of B2Rs but no B1Rs; they responded to BK (small calcium transients) and B-9870 behaved as an antagonist. CONCLUSION AND IMPLICATIONS: B-9870 is a dual B1R and B2R antagonist with confirmed stimulating effects at the B2R in high expression systems only. Its cell type-specific anti-proliferative effect occurs at a high concentration, independently from kinin receptors and apoptosis.