Identification of a Potent and Orally Active Non-peptide C5a Receptor Antagonist

The anaphylatoxin C5a is a potent chemotactic factor for neutrophils and other leukocytes, and functions as an important inflammatory mediator. Through a high capacity screening followed by chemical optimization, we identified a novel non-peptide C5a receptor antagonist,N-[(4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4-tetrahydronaphthalen-1- carboxamide hydrochloride (W-54011). W-54011 inhibited the binding of125I-labeled C5a to human neutrophils with aKivalue of 2.2 nm. W-54011 also inhibited C5a-induced intracellular Ca2+mobilization, chemotaxis, and generation of reactive super oxide species in human neutrophils with IC50values of 3.1, 2.7, and 1.6 nm, respectively. In C5a-induced intracellular Ca2+mobilization assay with human neutrophils, W-54011 did not show agonistic activity at up to 10 μmand shifted rightward the concentration-response curves to C5a without depressing the maximal responses. Examination on the species specificity of W-54011 revealed that it was able to inhibit C5a-induced intracellular Ca2+mobilization in neutrophils of cynomolgus monkeys and gerbils but not mice, rats, guinea pigs, rabbits, and dogs. In gerbils, oral administration of W-54011 (3–30 mg/kg) inhibited C5a-induced neutropenia in a dose-dependent manner. The present report is the first description of an orally active non-peptide C5a receptor antagonist that could contribute to the treatment of inflammatory diseases mediated by C5a.

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Adenosine A1receptor-mediated antiadrenergic effects are modulated by A2a receptor activation in rat heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.276.2.h341 ◽

1999 ◽

Vol 276 (2) ◽

pp. H341-H349 ◽

Cited By ~ 15

Author(s):

Gavin R. Norton ◽

Angela J. Woodiwiss ◽

Robert J. McGinn ◽

Mojca Lorbar ◽

Eugene S. Chung ◽

...

Keyword(s):

Receptor Antagonist ◽

Receptor Agonist ◽

Ventricular Myocytes ◽

Physiological Role ◽

Receptor Activation ◽

Dependent Manner ◽

Receptor Antagonists ◽

Rat Hearts ◽

Intracellular Ca ◽

Perfused Rat Hearts

Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10−8 M)-elicited contractile responses (+dP/d t max) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10−7 M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10−7 M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10−5 M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 × 10−7 M)-elicited increases in intracellular Ca2+concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]itransients in the absence of exogenous adenosine. These results indicate that adenosine A2areceptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.

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Bradykinin augments fibroblast-mediated contraction of released collagen gels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.1.l164 ◽

2001 ◽

Vol 281 (1) ◽

pp. L164-L171 ◽

Cited By ~ 15

Author(s):

Tadashi Mio ◽

Xiangde Liu ◽

Myron L. Toews ◽

Yuichi Adachi ◽

Debra J. Romberger ◽

...

Keyword(s):

Receptor Antagonist ◽

Type I Collagen ◽

Inflammatory Diseases ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Type I ◽

Dependent Manner ◽

Collagen Gels ◽

Protein Kinase C Inhibitors

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9 and 10−8 M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

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Neural and myogenic effects of leukotrienes C4, D4, and E4 on canine bronchial smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.4.l414 ◽

1994 ◽

Vol 266 (4) ◽

pp. L414-L425 ◽

Cited By ~ 2

Author(s):

A. Abela ◽

E. E. Daniel

Keyword(s):

Smooth Muscle ◽

Receptor Antagonist ◽

Contractile Activity ◽

Neuromuscular Control ◽

Dependent Manner ◽

Gamma Glutamyl Transpeptidase ◽

Response Curves ◽

Bronchial Smooth Muscle ◽

Concentration Dependent Manner

The leukotrienes (LTs), referred to as the slow-reacting substance of anaphylaxis (SRS-A), are reported to have little or no activity in the canine airway. The objective of this study was to determine whether LTC4, LTD4, and LTE4 (10(-10)-10(-7) M) play a role in neuromuscular control of third- to fifth-order canine bronchi. In the presence of 1 microM indomethacin (Indo), canine bronchial smooth muscle contracted and was depolarized in a concentration-dependent manner by LTC4 or LTD4 but not by LTE4. LTC4 and LTD4 concentration-response curves were not significantly affected when conducted in the presence of any of the following: 10(-7) M propranolol (beta-adrenoceptor antagonist), 10(-6) M chlorpheniramine (H1-receptor antagonist), 10(-6) M ketanserin (nonselective 5-hydroxytryptamine receptor antagonist), 10(-7) M atropine (muscarinic receptor antagonist), and 10(-6) M tetrodotoxin (sodium channel blocker). LTC4 and LTD4 also potentiated electrical field-stimulated (EFS) excitatory junction potentials (EJPs), suggesting a possible prejunctional enhancement of acetylcholine release. In the absence of Indo, no postjunctional responses to LTC4 and LTD4 occurred. Endogenous prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha (a stable metabolite of PGI2) levels from canine bronchi were significantly reduced by Indo. In the presence of Indo, addition of > or = 10(-8) M of PGE2 suppressed contractions to LTC4 and LTD4. These data suggest that the decrease in PGE2 and PGI2 production by Indo is sufficient to unmask the excitatory postjunctional actions of LTC4 and LTD4 on bronchial smooth muscle. Serine borate (45 mM; an inhibitor of gamma-glutamyl transpeptidase, which prevents the conversion of LTC4 to LTD4) increased selectively the contractile activity of LTC4. L-Cysteine (3 mM; an inhibitor of an aminopeptidase, which prevents the conversion of LTD4 to LTE4) enhanced the contractile responses to LTD4. Serine borate increased the amplitude and duration of EFS contractions and potentiated the amplitude of EFS EJPs; the last effects were prevented by nordihydroguaiaretic acid. These and other studies suggest that LTs are synthesized by canine bronchi and have receptors on canine bronchial smooth muscle but that contractions to LTC4 and LTD4 in the canine airway are usually not observed because of the presence of inhibitory prostanoids (PGE2 and PGI2). We suggest that decreases in PGE2 and PGI2 in models of airway disease in combination with increases in LTC4, LTD4, and thromboxane A2 may contribute to airway hyperresponsiveness in vitro.

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C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G-proteins

Blood ◽

10.1182/blood.v83.11.3324.bloodjournal83113324 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3324-3331 ◽

Cited By ~ 3

Author(s):

J Elsner ◽

M Oppermann ◽

W Czech ◽

A Kapp

Keyword(s):

Pertussis Toxin ◽

Respiratory Burst ◽

Inflammatory Diseases ◽

Free Calcium ◽

Dependent Manner ◽

Ros Production ◽

C5a Receptor ◽

Flow Cytometric Measurement ◽

Guanine Nucleotide Binding

In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a- induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.

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Treatment With Endothelin-A Receptor Antagonist BQ123 Attenuates Acute Inflammation in Mice Through T-Cell-Dependent Polymorphonuclear Myeloid-Derived Suppressor Cell Activation

Frontiers in Immunology ◽

10.3389/fimmu.2021.641874 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ziyang Chen ◽

Xiaogang Zhang ◽

Shuaijun Lv ◽

Zhe Xing ◽

Mengyu Shi ◽

...

Keyword(s):

T Cell ◽

Receptor Antagonist ◽

Cell Activation ◽

Acute Inflammation ◽

Inflammatory Diseases ◽

Suppressor Cells ◽

Suppressor Cell ◽

Dependent Manner ◽

Endothelin A Receptor ◽

Endothelin A

The endothelin-A receptor antagonist BQ123 is an effective treatment agent for hypertension and obese cardiomyopathy. However, the role of BQ123 in controlling acute inflammatory diseases and its underlying mechanisms are not well understood. Here, we showed that BQ123 activated polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in mice and that the IL13/STAT6/Arg1 signaling pathway is involved in this process. Importantly, both treatment with BQ123 and the transfer of BQ123-induced PMN-MDSCs (BQ123-MDSCs) were effective in relieving inflammation, including dextran sulfate sodium (DSS)-induced colitis, papain-induced pneumonia, and concanavalin A (ConA)-induced hepatitis, in mice. The treatment effects were mediated by the attenuation of the inflammation associated with the accumulation of PMN-MDSCs in the colon, lung, and liver. However, concurrent injection of Gr1 agonistic antibody with BQ123 induced PMN-MDSC aggravated the observed acute inflammation. Interestingly, no remission of inflammation was observed in Rag2 knockout mice administered BQ123-MDSCs, but co-injection with CD3+ T cells significantly relieved acute inflammation. In summary, BQ123-induced PMN-MDSCs attenuated acute inflammation in a T cell-dependent manner, providing a novel potential strategy to prevent the occurrence of acute inflammation.

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C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G-proteins

Blood ◽

10.1182/blood.v83.11.3324.3324 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3324-3331 ◽

Cited By ~ 99

Author(s):

J Elsner ◽

M Oppermann ◽

W Czech ◽

A Kapp

Keyword(s):

Pertussis Toxin ◽

Respiratory Burst ◽

Inflammatory Diseases ◽

Free Calcium ◽

Dependent Manner ◽

Ros Production ◽

C5a Receptor ◽

Flow Cytometric Measurement ◽

Guanine Nucleotide Binding

Abstract In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a- induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.

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Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors

Blood ◽

10.1182/blood.v78.10.2708.2708 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2708-2714 ◽

Cited By ~ 94

Author(s):

A Yuo ◽

S Kitagawa ◽

T Kasahara ◽

K Matsushima ◽

M Saito ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Priming Effect ◽

Tumor Necrosis ◽

Optimal Concentration ◽

Interleukin 8 ◽

Human Neutrophils ◽

Dependent Manner ◽

Response Curves ◽

Gm Csf ◽

Necrosis Factor

Abstract Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time- courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM- CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

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Stimulation and priming of human neutrophils by interleukin-8: cooperation with tumor necrosis factor and colony-stimulating factors

Blood ◽

10.1182/blood.v78.10.2708.bloodjournal78102708 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2708-2714 ◽

Cited By ~ 1

Author(s):

A Yuo ◽

S Kitagawa ◽

T Kasahara ◽

K Matsushima ◽

M Saito ◽

...

Keyword(s):

Tumor Necrosis Factor ◽

Priming Effect ◽

Tumor Necrosis ◽

Optimal Concentration ◽

Interleukin 8 ◽

Human Neutrophils ◽

Dependent Manner ◽

Response Curves ◽

Gm Csf ◽

Necrosis Factor

Interleukin-8 (IL-8) stimulated an increase in cytoplasmic-free Ca2+ ([Ca2+]i) and intracellular pH (pHi) in parallel at low concentrations (0.5 to 5 ng/mL), and stimulated O2- release and membrane depolarization in parallel at high concentrations (50 to 5,000 ng/mL). IL-8-induced O2- release was potentiated by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in a dose-dependent manner, whereas it was inhibited by cyclic AMP agonists. These characteristics and the time- courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O2- release stimulated by FMLP. The priming effect of IL-8 was very rapid and was maximal within 5 minutes of preincubation. The dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i and pHi. The potency of the maximal priming effects on FMLP-induced O2- release was TNF greater than GM-CSF greater than IL-8 greater than G-CSF. The combination of IL-8 and the suboptimal concentrations of TNF or GM-CSF resulted in the additive priming effect, whereas the combination of the optimal concentration of IL-8 and the optimal concentration of TNF, GM- CSF, or G-CSF resulted in the effect of more potent priming agent alone. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF, or FMLP at the inflammatory sites.

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