Lysophosphatidylcholine transduces Ca2+ signaling via the platelet-activating factor receptor in macrophages

1997 ◽  
Vol 272 (1) ◽  
pp. H17-H24 ◽  
Author(s):  
T. Ogita ◽  
Y. Tanaka ◽  
T. Nakaoka ◽  
R. Matsuoka ◽  
Y. Kira ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Peritoneal Macrophages ◽  
Free Calcium ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Paf Receptor ◽  
Web 2086

To clarify the molecular mechanism underlying the lysophosphatidylcholine (LPC) signaling, we studied the effect of LPC on the intracellular free calcium concentration ([Ca2+]i) in murine peritoneal macrophages. LPC when added alone induced biphasic elevation of [Ca2+]i, which consisted of a rapid increase followed by sustained elevation. LPC, when added with equimolar cholesterol, induced only the rapid increase in [Ca2+]i, which was blocked by WEB-2086, a selective platelet-activating factor (PAF) receptor antagonist. These results suggest LPC exerts a specific Ca2+ signaling. The sustained elevation reflected the cell lysis. Furthermore, we confirmed its pathway in a more specific manner using cloned PAF receptors expressed in Chinese hamster ovary cells. LPC induced an elevation of [Ca2+]i in a concentration-dependent manner only when the PAF receptor had been expressed, and the elevation of [Ca2+]i was blocked by WEB-2086. Taken together, LPC transduces Ca2+ signaling via the PAF receptor. Activation of the PAF receptor by LPC may indicate its novel important role in the pathogenesis of atherosclerosis.

Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

2007 ◽  
Vol 292 (5) ◽  
pp. F1583-F1591 ◽  
Author(s):  
Ryan M. Pelis ◽  
Yodying Dangprapai ◽  
Theresa M. Wunz ◽  
Stephen H. Wright
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Inorganic Mercury ◽  
Chinese Hamster ◽  
Organic Cation Transporter ◽  
Tubular Secretion ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Transport Activity ◽  
Concentration Dependent Manner ◽  
Quadruple Mutant

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO2-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272–35280, 2006). Thus it was hypothesized that the environmental contaminant Hg2+(as HgCl2) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [3H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl2, with an IC50of 3.9 ± 0.11 μM. Treatment with 10 μM HgCl2caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg2+, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl2than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl2effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg2+with these residues causes reduced TEA transport activity.

scholarly journals Platelet-activating factor exerts mitogenic activity and stimulates expression of interleukin 6 and interleukin 8 in human lung fibroblasts via binding to its functional receptor.

1996 ◽  
Vol 184 (1) ◽  
pp. 191-201 ◽  
Author(s):  
M Roth ◽  
M Nauck ◽  
S Yousefi ◽  
M Tamm ◽  
K Blaser ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Tyrosine Kinases ◽  
Human Lung ◽  
Human Fibroblasts ◽  
Platelet Activating Factor ◽  
Lung Fibroblasts ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Human Lung Fibroblasts ◽  
Paf Receptor

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator of the lung. In this study, we demonstrate that PAF receptor mRNA and protein is expressed by human lung fibroblasts. Interaction of PAF with its specific receptor resulted in increases of tyrosine phosphorylation of several intracellular proteins, indicating that the PAF-receptor might be functionally active. PAF-induced transcription of protooncogenes c-fos and c-jun as well as of interleukin (IL)-6 and IL-8 genes in human fibroblasts. Transcription of the interleukins was followed by secretion of the respective proteins. Moreover, PAF enhanced proliferation of fibroblasts in a concentration-dependent manner. Using signaling inhibitors, we demonstrate that PAF-induced transcription of the c-fos, IL-6, and IL-8 genes, as well as proliferation, require activation of pertussis toxin-sensitive G proteins, tyrosine kinases, and protein kinase C (PKC). In contrast, transcription of c-jun was blocked by pertussis toxin, but not by inhibitors for tyrosine kinases or PKC. These data suggest that PAF stimulates distinct signaling pathways in human lung fibroblasts. In addition, the activation of human fibroblasts by PAF leads to enhanced proliferation and to the expression of proinflammatory cytokines, which may contribute to the pathophysiological changes in pulmonary inflammation.

scholarly journals Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail

1997 ◽  
Vol 327 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Bo LIU ◽  
Shigeru NAKASHIMA ◽  
Takahito ADACHI ◽  
Yuzuru ITO ◽  
Tomoko TAKANO ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Mrna Levels ◽  
Primary Role ◽  
Ovary Cells ◽  
Protein Kinase Cα ◽  
Paf Receptor

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Cα (PKCα) to membrane was similar to that of DG formation. In WT-H cells, PKCα was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCα was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCα. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCα, which might play an important role in the expression of c-fos and c-jun.

scholarly journals Agonist-induced aggregation of Chinese hamster ovary cells coexpressing the human receptors for fibrinogen (integrin αIIbβ3) and the platelet-activating factor: dissociation between adhesion and aggregation

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2819-2827 ◽  
Author(s):  
Susana Larrucea ◽  
Consuelo González-Manchón ◽  
Nora Butta ◽  
Elena G. Arias-Salgado ◽  
Linnan Shen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activating Factor ◽  
Chinese Hamster ◽  
Cellular Adhesion ◽  
Dependent Manner ◽  
Cellular Aggregation ◽  
Induced Aggregation ◽  
Stimulation Of

Abstract This work reports the establishment of a Chinese hamster ovary (CHO) cell line stably coexpressing the human αIIbβ3 integrin and the platelet-activating factor receptor (PAFR). These cells aggregate in response to PAF in a Ca++, αIIbβ3, and soluble fibrinogen (Fg)–dependent manner that is prevented by PAF antagonists or αIIbβ3 blockade. The aggregating response is accompanied by enhanced binding of fibrinogen and the activation-dependent IgM PAC1. This model has permitted us to identify, for the first time, intracellular signals distinctly associated with either αIIbβ3-mediated adhesion or aggregation. Nonreceptor activation of protein kinase C (PKC) by phorbol ester produced cellular adhesion and spreading onto immobilized Fg, but it was not a sufficient signal to provoke cellular aggregation. Moreover, inhibition of PKC impeded the PAF stimulation of cellular adhesion, whereas the aggregation was not prevented. The PAF-induced cellular aggregation was distinctly associated with signaling events arising from the liganded Fg receptor and the agonist-induced stimulation of a calcium/calmodulin-dependent signaling pathway. Sustained tyrosine phosphorylation of both mitogen-activated protein kinase (MAPK) and an approximately 100-kd protein was associated with the PAF-induced aggregation, whereas phosphorylation of focal adhesion kinase (FAK) was preferably associated with cellular adherence and spreading onto immobilized Fg.

scholarly journals Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Abstract Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

scholarly journals Endothelial cells potentiate phagocytic killing by macrophages via platelet-activating factor release

2000 ◽  
Vol 278 (1) ◽  
pp. H269-H276 ◽  
Author(s):  
Tetsuhiro Owaki ◽  
Avedis Meneshian ◽  
Kosei Maemura ◽  
Sonshin Takao ◽  
Dajie Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activating Factor ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Macrophage Phagocytosis ◽  
Monocytes And Macrophages ◽  
Paf Receptor ◽  
Factor Α ◽  
Dose Dependent ◽  
Phagocytic Killing

The immunomodulatory function of endothelial cells (EC) includes the initiation of leukocyte margination, diapedesis, and activation through the upregulation of various cell surface-associated molecules. However, the effect that EC have on the phagocytic function of neighboring monocytes and macrophages is less well described. To address this issue, microvascular EC were cocultured with murine peritoneal macrophages, first in direct contact, then in a noncontact coculture system, and macrophage phagocytosis and phagocytic killing were assessed. The presence of increasing concentrations of EC resulted in a dose-dependent increase in macrophage phagocytic killing. This stimulatory effect was inhibited in a dose-dependent manner by the pretreatment of macrophage/EC cocultures with WEB-2086 or CV-6209, specific platelet-activating factor (PAF)-receptor antagonists, but not by anti-tumor necrosis factor-α, anti-interleukin (IL)-1α, or anti-IL-1β. Furthermore, the effect was reproduced in the absence of EC by the exogenous administration of nanomolar concentrations of PAF. Microvascular EC potentiate macrophage phagocytic killing via the release of a soluble signal; PAF appears to be an important component of that signal.

scholarly journals Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide

1996 ◽  
Vol 319 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Krikor TOROSSIAN ◽  
Marie AUDETTE ◽  
Richard POULIN
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Polyamine Transport ◽  
Substrate Protection

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11±4 and 96±16 µM after 30 min at 22 °C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and Km for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 °C respectively. Spermine (10 µM) almost fully protected against spermidine-transport inactivation by EDC at 22 °C, and decreased the rate of inactivation at 37 °C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 °C with EC50 values estimated at 10, 1 and less than 1 µM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89±3 compared with 44±5%), whereas the converse was true for 100 µM DCC (81±3 compared with 92±5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na+-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to cross-link a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor

2002 ◽  
Vol 282 (6) ◽  
pp. C1461-C1468 ◽  
Author(s):  
Bok Hee Choi ◽  
Jin-Sung Choi ◽  
Duck-Joo Rhie ◽  
Shin Hee Yoon ◽  
Do Sik Min ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Time Course ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Voltage Range

The action of tyrphostin AG-1478, a potent protein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels (Kv1.5) stably expressed in Chinese hamster ovary cells was investigated using the whole cell patch-clamp technique. AG-1478 rapidly and reversibly inhibited Kv1.5 currents at 50 mV in a concentration-dependent manner with an IC50 of 9.82 μM. AG-1478 accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. Pretreatment with the structurally dissimilar PTK inhibitors (genistein and lavendustin A) had no effect on the AG-1478-induced inhibition of Kv1.5 and did not modify the AG-1478-induced current kinetics. The rate constants for binding and unbinding of AG-1478 were 1.46 μM−1 · s−1 and 10.19 s−1, respectively. The AG-1478-induced inhibition of Kv1.5 channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation time course, resulting in a tail crossover phenomenon. Inhibition of Kv1.5 by AG-1478 was use dependent. The present results suggest that AG-1478 acts directly on Kv1.5 currents as an open-channel blocker and independently of the effects of AG-1478 on PTK activity.

Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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