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

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3324-3331 ◽  
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.

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

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3324-3331 ◽  
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.

Abstract 16440: Cu Transporter ATP7A Protects Against Fibrosis Through Limiting Endothelial to Mesenchymal Transition

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Seock-Won Youn ◽  
Sudhahar Varadarajan ◽  
Archita Das ◽  
Ronald D McKinney ◽  
Tohru Fukai ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Inflammatory Diseases ◽  
Shape Change ◽  
Atherosclerotic Lesion ◽  
Transport Proteins ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition

Background: Endothelial to mesenchymal transition (EndMT) is induced by inflammation and contributes to fibrosis; however, underlying mechanism is poorly understood. Cu plays an important role in physiological processes and pathophysiologies associated with inflammatory diseases. Since excess Cu is toxic, bioavailability of Cu is tightly controlled by Cu exporter ATP7A, which obtains Cu via Cu chaperone, Atox1, and exclude Cu. We reported that Atox1 also functions as a Cu dependent transcription factor. However, role of Cu transport proteins in EndMT is entirely unknown.[[Unable to Display Character: 
]] Results: Here we show that TNFα stimulation for 24hr in HUVEC significantly decreased ATP7A protein (80%) and increased intracellular Cu and Atox1 in nucleus, which was associated with shape change forming EndMT. ATP7A depletion with shRNA in EC significantly reduced EC markers (VE-cadherin and VEGFR2) and increased mesenchymal markers (αSMA, Calponin, SM22α, Collagen I/II). ATP7A siRNA also increased intracellular Cu and nuclear Atox1. These ATP7A knockdown-induced phenotype changes were inhibited by Cu chelators BCS and TTM. Mechanistically, microarray and qPCR based screening revealed that ATP7A knockdown in EC significantly increased miR21 (2.5 fold) and miR125b (1.5 fold) which induce EndMT in a Cu-dependent manner. Of note, promoters of both miR21 and miR125b have Cu dependent transcription factor Atox1 binding sites. Consistent with this, overexpression of Atox1 increased miR21 and miR125b expression as well as promoted EndMT. In vivo, ATP7A mutant (ATP7Amut) mice with reduced Cu export function showed impaired blood flow recovery and reduced arteriogenesis while increased αSMA+ cells and fibrosis in capillary network after ischemic injury. Moreover, ATP7Amut mice crossed with ApoE-/- mice with high fat diet (HFD) induced robust fibrosis and enhanced atherosclerotic lesion vs ApoE-/-/HFD mice.[[Unable to Display Character: 
]] Conclusions: ATP7A protects against fibrosis by preventing EndMT via nuclear Atox1-mediated upregulation of miR21 and miR125b which induce EndMT, in Cu dependent manner. These findings provide the foundation for novel protective role of Cu transport proteins against EndMT- and fibrosis-mediated cardiovascular diseases.

Carbachol inhibits stimulant-induced increases in fundic D-cell cytosolic Ca2+ concentration

1989 ◽  
Vol 257 (2) ◽  
pp. G308-G312 ◽  
Author(s):  
T. Chiba ◽  
T. Fujita ◽  
T. Yamada
Keyword(s):  
Pertussis Toxin ◽  
Gastric Fundus ◽  
Free Calcium ◽  
Guanine Nucleotide ◽  
Cytosolic Free Calcium ◽  
Guanine Nucleotide Binding Protein ◽  
Kinase C ◽  
D Cell ◽  
Guanine Nucleotide Binding ◽  
D Cells

We undertook these studies to examine the mechanisms by which carbachol inhibits somatostatin release. For these studies, we utilized cultured D-cells isolated from the canine gastric fundus. Carbachol inhibited somatostatin release induced by both pentagastrin and 12-O-tetradecanoyl-phorbol-13-acetate but did not alter the redistribution of protein kinase C induced by these agents. In contrast, carbachol diminished the increase in D-cell cytosolic free calcium levels ([Ca2+]i) induced by pentagastrin, and this effect was no longer evident after pretreatment of D-cells with pertussis toxin. Although carbachol by itself had no effect on [Ca2+]i, after pretreatment of D-cells with pertussis toxin, carbachol both enhanced [Ca2+]i and stimulated somatostatin release. These data indicate that carbachol activates signals in D-cells that result in both increase and decrease in [Ca2+]i. The latter effect, which appears to be mediated via a pertussis toxin-sensitive guanine nucleotide binding protein, may be one mechanism responsible for cholinergic inhibition of somatostatin release.

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic β-cells

2003 ◽  
Vol 285 (5) ◽  
pp. E1001-E1009 ◽  
Author(s):  
Yoshikazu Miura ◽  
Hisao Matsui
Keyword(s):  
Insulin Secretion ◽  
Adenylate Cyclase ◽  
Islet Cells ◽  
Free Calcium ◽  
Dependent Manner ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2′-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.

scholarly journals Apocynin: Molecular Aptitudes

2008 ◽  
Vol 2008 ◽  
pp. 1-10 ◽  
Author(s):  
J. Stefanska ◽  
R. Pawliczak
Keyword(s):  
Nadph Oxidase ◽  
Inflammatory Diseases ◽  
Experimental Models ◽  
Ros Production ◽  
Oxygen Species ◽  
Naturally Occurring ◽  
Activated Neutrophils ◽  
Anti Inflammatory ◽  
Insight Into

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. It can decrease the production of superoxide (O2−) from activated neutrophils and macrophages while the ability of phagocytosis remains unaffected. The anti-inflammatory activity of apocynin has been demonstrated in a variety of cell and animal models of inflammation. Apocynin, after metabolic conversion, inhibits the assembly of NADPH-oxidase that is responsible for reactive oxygen species (ROS) production. It is, therefore, extensively used to reveal the role of this enzyme in cell and experimental models. Although some of the ROS serve as signaling molecules in the cells, excessive production is damaging and has been implicated to play an important role in the progression of many disease processes. This is why in many studies apocynin presents a promising potential treatment for some disorders; however, its utility with inflammatory diseases remains to be determined. Since its mode of action is not well defined, we tried to get a more precise insight into the mechanisms by which apocynin exerts its activity. Considering the anti-inflammatory activities of apocynin, we may conclude that this compound definitely deserves further study.

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

2002 ◽  
Vol 277 (51) ◽  
pp. 49403-49407 ◽  
Author(s):  
Hiroshi Sumichika ◽  
Kei Sakata ◽  
Noriko Sato ◽  
Sanae Takeshita ◽  
Seigo Ishibuchi ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Inflammatory Diseases ◽  
Present Report ◽  
High Capacity ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Response Curves ◽  
C5a Receptor ◽  
Intracellular Ca ◽  
Orally Active

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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