• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.495-501
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• 2001

We have cloned the mouse neurotensin/neuromedin N (NT/N) gene from the murine mast cell line Cl.MC/C57.1 for the first time. The murine NT/N cDNA clone consisted of 765 nucleotides and coded for 169 peptide residues with an N-terminal signal peptide, and the C-terminal region contained of one copy of neurotensin (NT) and one copy of neuromedin N (NN). Total of four Lys-Arg dibasic motifs were present; one each at the middle of the open reading frame, at the N-terminal of NN, at the C-terminal of NT, and between NN and NT. Amino acid sequence analysis of the mouse NT/N revealed 90% homology to that of the rat NT/N gene. NT/N is expressed in murine mast cell lines (Cl.MC/C57.1 and P815), but not in murine bone marrow-derived mast cells (BMMCs), murine macrophage cell line (RAW 264.7), nor in murine T cell line (EL-4). NT/N mRNA in C1.MC/C57.1 is highly inducible by IgE cross-linking, phorbol myristate acetate, neurotensin, and substance P. Following the treatment of demethylating agent, 5-azacytidine (5-azaC), the NT/N gene was induced in BMMCs in response to IgE cross-linking. 5-azaC-treated BMMCs did not express the NT/N gene without additional stimuli. These findings suggested that the regulation of NT/N gene expression was dependent on the effects of not only gene methylation but also enhancer and/or repressor proteins acting on the NT/N promoter.

• IMMUNE NETWORK
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• v.2 no.4
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• pp.195-201
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• 2002

Background: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. Methods: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the ${\beta}$-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semiquantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. Results: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at $56^{\circ}C$ illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. Conclusion: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.

• The Korean Journal of Pharmacology
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• v.30 no.2
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• pp.243-254
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• 1994

Inflammatory diseases, allergic and asthmatic disorders are caused by the mediator release from the activation of the phospholipase C (PLC), phospholipase D (PLD), methyltransferase or adenylate cyclase etc. during IgG or IgE cross-linking of high affinity receptors on mast cells or basophil surface. One important enzyme activated after IgG or IgE receptor cross-linking is PLD, the enzyme which converts phosphatidylcholine (PC) to phosphatidic acid (PA). Under the hypothesis that these may be some differences in mediator release according to the difference in PLD activity, we attempted to confirm the ginseng saponin effects on the PLD activity. We examined the PLD activity during the passively sensitized mast cell activation in the presence of single component of ginsenosides $(Rc,\;Rg_1,\;Rg_2,\;Rg_3)$. We also measured the amount of mediators (histamine and leukotrienes) released by stimulating with ovalbumin (OA) or calcium ionophore (CaI), Guinea Pig lung mast cells were purified using enzyme digestion, count current elutriation, and discontinuous Percoll density gradient. In purified mast cells prelabeled with $[^3H]$ arachidonic acid or $[^3H]$ palmitic acid, PLD activity was assessed more directly by the production of labeled PEt by PLD-mediated transphosphatidylation in the presence of ethanol. Histanine release was determined by Spectrophotofluorometry, and leukotrienes by radioimmunoassay. The PLD activity during the passively sensitized mast cell activation is increased up to $3{\sim}5times$. The PLD activity during the passively sensitized mast cell activation in the presence of all ginsenosides is decreased up to $4{\sim}11$ times. $Rg_l\;and\;Rg_2$ ginsenoside pretreatment decreased histamine and leukotrienes by 50% in the OA-induced or by 40% in the Cal-induced mast cell after passively sensitization. Rc pretreatment poorly decreased histamine but leukotrienes decreased by 70% in the OA-induced or by 35% in the Cal-induced mast cell. $Rg_3$ ginsenoside pretreatment increased histamine release without challenging OA or Cal but leukotrienes decreased. These observations indicate that single unit of ginsenosldes may be an important contributor to inhibit the release of histamine and leukotrienes in the guinea pig lung mast cells, that inhibits the PLD-mediated formation of DAG evoked by mast cell activation.