• Archives of Pharmacal Research
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• v.26 no.6
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• pp.478-481
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• 2003

Phospholipase D is a ubiquitous enzyme that plays an important role in various lipid mediated cellular signaling pathways and produces rare phospholipids, phosphatidylethanol or phosphatidylbutanol, instead of phosphatidic acid with unique catalytic activity transphosphatidylation in the presence of primary alcohols. The reaction products, phosphatidylethanol or phosphatidylbutanol are used as markers of in vitro phospholipase D activity in many studies. For the sensitive detection of the phospholipase D products, we developed an advanced lipid extraction method that facilitates recovery of the compounds. With the new method, the activity change of phosaholipase D by agonists could be detected more easily and the recovery rate was also increased. The increase of detected enzyme activity change was about double fold compared to the conventional lipid extraction method. This method provides selective force for the phospholipase D products in the extraction procedure.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.4
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• pp.445-456
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• 1997

We previously reported that some components of ginsenosides decreased mediator releases evoked by the activation of mast cells with specific antigen-antibody reactions. This study aimed to assess the effects of ginsenosides ($Rb_2$, Re) on the mechanism of histamine release in the mast cell activation. We partially purified guinea pig lung mast cells by using enzyme digestion, the rough and the discontinuous percoll density gradient method. Mast cells were sensitized with $IgG_1$ and challenged with ovalbumin (OA). Histamine was assayed by fluorometric analyzer, leukotrienes by radioimmunoassay. Phospholipase D (PLD) activity was assessed more directly by the production of $[^3H]phosphatidylbutanol$ (PBut) which was produced by PLD-mediated transphosphatidylation in the presence of butanol. The amount of 1,2- diacylglycerol (DAG) were measured by the $[^3H]DAG$ labeled with $[^3H]palmitic$ acid or $[^3H]myristic$ acid. Pretreatment of $Rb_2$ ($300\;{\mu}g$) significantly decreased histamine release by 60%, but Re ($300\;{\mu}g$) increased histamine release by 34%. Leukotrienes release in $Rb_2$ was decreased by 40%, Re was not affected in the leukotrienes release during mast cell activations. An increasing PLD activity during mast cell activation was decreased by the dose-dependent manner in the pretreatment of $Rb_2$, but Re pretreatment facilitated the increased PLD activity during mast cell activation. The amount of DAG produced by phospholipase C (PLC) activity was decreased by $Rb_2$ pretreatment, but Re pretreatment was not affected. The amount of mass DAG was decreased by $Rb_2$ and Re pretreatment during mast cell activation. The data suggest that $Rb_2$ purified from Korean Red Ginseng Radix inhibits the DAG which is produced by the activation of mast cells with antigen-antibody reactions via both phosphatidylinositide-PLC and phosphatidylcholine-PLD systems, and then followed by the inhibition of histamine release. However, Re increases histamine release by stimulation of DAG production, which is mediated by phosphatidylcholine-PLD system rather than by phosphatidylinositide-PLC system, but inhibits the mass DAG production. Thus, it could be inferred that other mechanisms play a role in the increase of histamine release during mast cell activation.

• Proceedings of the Korean Society of Toxicology Conference
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• 1997.05a
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• pp.65-74
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• 1997

By using guinea pig lung mast cells, this study aimed to examine the effects of Aloe component(NY945) on the mediator releases caused by mast cell activation, and also aimed to assess the effects of NY945 on the mechanism of mediator releases in the mast cell activation. We partially purified mast cells from guinea pig lung tissues by using the enzyme digestion, the rough and the discontinuous density percoll gradient method. Mast cells were sensitized with $IgG_1$ (anti-OA) and challenged with ovalbumin. Histamine was assayed by fluorometric analyzer, leukotrienes by radioimmunoassay The phospholipase D activity was assessed more directly by the production of labeled phosphatidylethanol or phosphatidylbutanol which was produced by phospholipase D-mediated transphosphatidylation in the presence of ethanol or butanol. The amount of mass 1,2-diacylglycerol was measured by the [$^3H$]1,2-diacylgycerol produced when prelabeled with [$^3H$]myristic acid. In the mast cells prelabeled with L-[$^3H$]methyl methionine the phospholipid methylation was assessed by measuring the incorporation of the [$^3H$]methyl moiety into phospholipids. Pretreatment of NY945(10$\mu$g) significantly decreased histamine and leukotrienes releases during mast cell activation. The decrease of histamine release was stronger than that of leukotrienes during mast cell activation. The phospholipase D activity increased by the mast cell activation was decreased by the dose-dependent manner in the pretreatment of NY945. The amount of mass 1,2-diacylglycerol produced by activation of mast cells were decreased in the pretreatment of NY945. NY945 pretreatment strongly inhibited the incorporation of the [$^3H$]methyl moiety into phospholipids. The data suggest that NY945 purified from Aloe inhibits in part an increase of 1,2-diacylglycerol which is produced by activating mast cells with antigen-antibody complexes which is mediated via phosphatidylcholine-phospholipise D and phosphatidylinositole-phospholipise C systems, and then followed by the inhibition of histamine release. Furthermore, NY945 reduces the phosphatidylcholine production by inhibiting the methyltransfsrase I and II, which decrease the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrines.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2004.10a
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• pp.74-79
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• 2004

Little is known about the effect of epigallocatechin-3 gallate (ESCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2protein and its product, prostaglandin $E_2$ ($PGE_2$). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and $PGE_2$ synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression w3s provided by the observations that COX-2 expression was stimulated by over-expression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid(PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2expression Induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38MAPK), and specific Inhibition of p38 MAPK dramatically abolished EGCG-Induced PLD activation, COX-2 expression, and $PGE_2$ formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and $PGE_2$ accumulation. The same pathways as those obtained in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.

• Journal of Life Science
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• v.16 no.4
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• pp.691-698
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• 2006

Phospholipase D (PLD) is known to play an important role in a variety of cells. However, little is known about $CoCl_2-mediated$ PLD signaling. In this study we demonstrated for the first time that $CoCl_2$ stimulates PLD activity and increases expression of cyclooxygenase-2 (COX-2), which is known to mediate inflammatory reaction. $CoCl_2-induced$ PLD activity was assessed by measuring the formation of $[^3H]$ phosphatidylbutanol (PtdBut), the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol. To study mechanism of PLD signaling induced by $CoCl_2$, U87 human glioblastoma cells were stimulated by $CoCl_2$ and regulators of PLD activity induced by $CoCl_2$ were investigated using several inhibitors of signaling proteins. Moreover, PLD activation by $CoCl_2$ increased not only expression of COX-2 protein but also COX-2 promoter activity. In summary, these results suggest that $CoCl_2$ increases expression of COX-2 protein via PLD in human U87 glioblastoma cells.