• Journal of Life Science
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• v.13 no.6
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• pp.849-855
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• 2003

Nitric oxide (NO) plays an important role as a signaling molecule in the proliferation of placenta trophoblasts. In this study, we investigated the effect of NO on the activation of phospholipase C (PLC) in BeWo cells, choriocar-cinoma cell line. Sodium nitroprusside (SNP), an agent to produce NO spontaneously in cells, alone increased $［^3H］$ thymidine incorporation of BeWo cells, indicating NO stimulates proliferation of the cells. NO-induced proliferation of BeWo cells was blocked by U73122, an inhibitor of PLC, suggesting that NO-induced PLC activation is involved in the cell proliferation. NO also stimulated extracellular signal-regulated kinase (ERK) in BeWo cells, indicated by increased phosphorylation of ERK1/2 in Western blotting using anti-phospho-ERK1/2 antibody. NO-induced phos-phorylation of ERK1/2 was not abrogated by U73122. $PLC\gamma_1$l but not$PLC\gamma_2$ was tyrosine phosphorylated by SNP in immunoprecipitation assay using anti-$PLC\gamma_1$/$PLC\gamma_2$ antibodies, and SNP-induced phosphorylation of $PLC\gamma_1$ was abrogated by pre-treatment of cells with genistein and PD98059, indicating that NO induced-phosphorylation of $PLC\gamma_1$ is mediated by ERK. These results suggest that NO stimulates the proliferation of BeWo cells through ERK and $PLC\gamma_1$.

• BMB Reports
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• v.32 no.2
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• pp.154-160
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• 1999

A calcium-independent phospholipase $A_2$ ($iPLA_2$) was identified from the cytosolic fraction of rat liver cells. On gel filtration chromatography, the $iPLA_2$ activity was eluted as broad peaks of 150 to 500 kDa. The enzyme was maximally active at pH 7.5, retained 75% of its original activity after heating at $50^{\circ}C$ for 5 h, and was inhibited by $Ca^{2+}$, $Mg^{2+}$, and $Zn^{2+}$ ions, but was not affected by $Na^+$ and $K^+$ ions. The enzymatic activity was increased up to 150% by 1 to 4 mM DTT and was inhibited up to 25% by 0.1 to 1 mM PMSF. The $iPLA_2$ activity had preference for the head group of phospholipids, where phosphatidylethanolamine was preferred to phosphatidylcholine. The results suggest that the $iPLA_2$ may be a novel enzyme distinct from the previously reported $iPLA_2s$.

• Biomedical Science Letters
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• v.14 no.3
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• pp.199-201
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• 2008

Eosinophil is an improtant leukocyte in the development of various inflammatory diseases. Monocyte chemoattractant protein-1 (MCP-1) acts as a key regulator on monocyte movement, and activation of T cells and NK cells. However, the role of MCP-1 in eosinophils remains to be solved. In the present study, we examined the effect of MCP-1 on eosinophil migration, using human eosinophilic EoL-1 cells as an in vitro model of eosinophils. The surface expression of CCR2 in EoL-1 cells was little detected but MCP-1 strongly induced EoL-1 cell migration in a dose-dependent manner. Increased chemotactic activity due to MCP-1 was blocked by pertussis toxin, a $G_i/G_o$ protein inhibitor and U73122, a phospholipase C (PLC) inhibitor. These results suggest that MCP-1 activates $G_i/G_o$ protein and PLC and this signal pathway is involved in eosinphil movement. This finding supports the elucidation of pathogenic mechanism of eosinophilic inflammation such as asthma and atopic dermatitis.

• Natural Product Sciences
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• v.6 no.4
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• pp.170-178
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• 2000

Flavonoids are natural polyphenolic compounds widely distributed in plant kingdom. Although many flavonoids were found to show anti-inflammatory activity in vitro and in vivo, the potency of anti-inflammatory activity was not enough for a clinical trial. Thus, a search for finding potential flavonoid molecules is continuing. In this review, in vivo anti-inflammatory activity of various flavonoid derivatives is summarized mainly based on the results obtained in authors' laboratories. Among them, several biflavonoids such as amentoflavone and ginkgetin were found to possess anti-inflammatory activity on animal models of acute/chronic inflammation comparable to nonsteroidal and steroidal anti-inflammatory drugs currently used. In respect of their action mechanisms, the effects on arachidonic acid metabolism and nitric oxide production were described. Some flavonoids directly inhibit cyclooxygenase and/or lipoxygenase. Biflavones such as ochnaflavone and ginkgetin are inhibitors of phospholipase $A_2$. In recent studies, certain flavonoids were also found to suppress cyclooxygenase-2 and inducible nitric oxide synthase expression induced by inflammatory stimuli. Therefore, it is suggested that anti-inflammatory activity of the certain flavonoids (mainly flavones, flavonols and biflavonoids) may be mediated by direct inhibition of arachidonic acid metabolizing enzymes as well as suppression of the enzyme expression involved in inflammatory responses.

• Journal of Physiology & Pathology in Korean Medicine
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• v.27 no.1
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• pp.112-117
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• 2013

We studied the modulation of pacemaker activities by Samchulkunbi-tang (SCKB) in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied SCKB produced membrane depolarization in the current-clamp mode. The pretreatment with $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the SCKB-induced action. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by SCKB. However, the application of niflumic acid (a chloride channel blocker) did not inhibit the generation of pacemaker potentials by SCKB. In addition, the membrane depolarizations were inhibited by not only GDP-${\beta}$-S, which permanently binds G-binding proteins, but also U-73122, an active phospholipase C inhibitor. These results suggest that SCKB modulates the pacemaker activities by nonselective cation channels and external $Ca^{2+}$ influx and internal $Ca^{2+}$ release via G-protein and phospholipase C-dependent mechanism. Therefore, the ICC are targets for SCKB and their interaction can affect intestinal motility.

• Korean Journal of Pharmacognosy
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• v.34 no.1 s.132
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• pp.100-108
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• 2003

The regulation of plasma lipid level, particularly LDL cholesterol, represents the focus of current therapy for atherosclerosis. And $Lp-PLA_2$ is able to hydrolyse oxidized phosphatidylcholine within LDL into lyso-PC and oxidized fatty acids. $Lp-PLA_2$ is a potential biomarker of coronary heart disease and plays an important proinflammatory role in the progression of atherosclerosis. We investigated the inhibitory effects of methanol extracts of 224 natural plants on $Lp-PLA_2$ activity. Seven kinds of methanol extracts of tested plants showed above 50% inhibitory effect with the concentration of $100\;{\mu}g/ml$. The concentrated aqueous suspensions of each methanol extract were partitioned with n-hexane, $CHCl_3$, and EtOAc. Among them, EtOAc extracts of Astilbe chinensis var. davidii (root) and Pourthiaea villosa var. brunnea (leaf) significantly inhibited $Lp-PLA_2$ activity at the same concentration.

• Archives of Pharmacal Research
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• v.28 no.1
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• pp.73-80
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• 2005

The effect of capsaicin on apoptotic cell death was investigated in HepG2 human hepatoma cells. Capsaicin induced apoptosis in time- and dose-dependent manners. Capsaicin induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration, and BAPTA, an intracellular $Ca^{2+}$ chelator, significantly inhibited capsaicin-induced apoptosis. The capsaicin-induced increase in the intracellular $Ca^{2+}$ and apoptosis were not significantly affected by the extracellular $Ca^{2+}$ chelation with EGTA, whereas blockers of intracellular $Ca^{2+}$ release (dantrolene) and phospholipase C inhibitors, U-73122 and manoalide, profoundly reduced the capsaicin effects. Interestingly, treatment with the vanilloid receptor antagonist, capsazepine, did not inhibit either the increased capsaicin-induced $Ca^{2+}$ or apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the HepG2 cells may result from the activation of a PLC-dependent intracellular $Ca^{2+}$ release pathway, and it is further suggested that capsaicin may be valuable for the therapeutic intervention of human hepatomas.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2000.04a
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• pp.17-19
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• 2000

Heterotrimeric G Proteins transduce ligand binding to a wide variety of seven transmembrane cell surface receptors into intracellular signals. The currently accepted model for the activation of G protein suggests that ligand-activated receptor accelerates GDP-GTP exchange reactions on the ${\alpha}$ subunit of the heterotrimeric G protein. At least seventeen distinct isoforms of the G${\alpha}$ subunit protein have been identified in mammalian organisms. Among them, the G${\alpha}$q family consists of five members whose ${\alpha}$ subunits show different expression patterns. G${\alpha}$q and G${\alpha}$11 seem to be almost ubiquitously expressed, whereas G${\alpha}$14 is predominantly expressed in spleen, lung, kidney and testis. G${\alpha}$16 and its murine counterpart G${\alpha}$15 are expressed in hematopoietic cells and has been shown to couple a wide variety of receptors to phosphoinositide-specific phospholipase C activity. Beta-isoforms of phospholipase C were shown to be activated by all members of G${\alpha}$q family, i.e., G${\alpha}$q, G${\alpha}$11, G${\alpha}$l4 and G${\alpha}$16 subunits either in reconstitution system. or in experiments using cDNA transfection with intact Cos-7 cells.

• BMB Reports
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• v.43 no.9
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• pp.604-608
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• 2010

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) mediates proinflammatory responses in primary human umbilical vein endothelial cells (HUVECs), and it upregulates the expression of secretory group IIA phospholipase $A_2$ ($sPLA_2$-IIA). $sPLA_2$-IIA plays a pivotal role in inflammation, and antithrombin (AT) possesses properties that are beneficial to endothelial cells. Therefore, we investigated the effects of AT on the expression of $sPLA_2$-IIA in TNF-$\alpha$-stimulated HUVECs. TNF-$\alpha$ potently upregulated the expression of $sPLA_2$-IIA, and prior treatment of cells with AT inhibited the expression of $sPLA_2$-IIA in HUVECs. Also, antibodies or siRNA for syndecan-4 blocked the protective effect of AT. Furthermore, PI3-kinase and the AKT pathway are significantly involved in the AT-mediated inhibition of the expression of $sPLA_2$-IIA. These results show that AT effectively suppresses the upregulated $sPLA_2$-IIA expression, which might contribute to the cytoprotective effects of AT in the treatment of severe inflammatory diseases.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.492-498
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• 2005

The mechanism of activation of phospholipase D2 (PLD2) remains undefined although mechanisms have been described for the activation of PLDI. By expression of mutated forms of haemaglutinnin-tagged PLD2 in a mast cell (RBL-2H3) line, we show that PLD2 is phosphorylated at tyrosines -11, -14, and -470 and that tyrosine-470 is critical for activation of PLD2 by antigen. Studies were performed with mutated-DNA constructs for haemaglutinnin-tagged PLD2 in which codons for tyrosine -11, -14, -165, and -470 were mutated to phenylalanine either individually or collectively. Transient expression of these constructs showed that mutation of tyrosine -11, -14, -470, or all tyrosines (all-mutated PLD2) suppressed antigen-induced tyrosine phosphorylation of PLD2 but only the tyrosine-470 mutant failed to be activated by antigen as assessed by in vitro assay of immunoprepitated PLD2 or by assay of PLD in intact cells. The critical role of tyrosine-470 was confirmed in studies with add-back mutants (phenylalanine back to tyrosine) of the all-mutated PLD. The findings provide the first description of a mechanism of activation of PLD2 in a physiological setting.