Antigen-induced Ca2+ mobilization in RBL-2H3 cells:: Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production
Antigen-induced Ca2+ mobilization in RBL-2H3 cells:: Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production
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DOI：
10.1016/j.ceca.2005.08.002
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发表时间：
2005-12-01
期刊：
影响因子：
4
通讯作者：
Choi, OH
中科院分区：
文献类型：
--
作者：
Lee, HS;Park, CS;Choi, OH
肌醇1，4，5-三磷酸（IP 3）是细胞内钙离子动员的第二信使。最近，1-磷酸鞘氨醇（S1 P）已被证明参与Ca 2+从内质网（ER）释放。在此，我们研究了SlP和IP 3在RBL-2 H3肥大细胞中抗原（Ag）诱导的细胞内Ca 2+动员中的作用。鞘氨醇激酶抑制剂（DL）-苏型-二氢鞘氨醇（DHS）或IP 3受体抑制剂2-氨基乙氧基二苯基硼酸酯（2-APB）仅部分抑制抗原诱导的细胞内Ca 2+动员，而预孵育与两种抑制剂导致完全抑制。相反，用N-5-乙基羧酰胺腺苷（NECA）刺激A（3）腺苷受体引起细胞内Ca 2+动员，2-APB完全消除了这种动员，但DHS没有，这表明NECA只需要IP 3途径，而抗原同时使用IP 3和S1 P途径。然而，有趣的是，用磷脂酶C抑制剂U 73122抑制IP 3的产生完全废除了由任一刺激剂诱导的ER的Ca 2+释放。这表明，单独的S1 P，没有IP 3的伴随生产，不会导致细胞内Ca 2+动员。这在过度过表达II类磷脂酰肌醇3-激酶（P13 KC 2 β）的β同种型的RBL-2 H3细胞的一些克隆中得到进一步证实。在包括克隆5A 4C在内的这些克隆中，P13 KC 2 β在整个细胞中过表达，尽管内源性P13 KC 2 β通常仅在ER中表达。胞质溶胶和PM中P13 KC 2 β的过表达导致磷脂酰肌醇4，5-二磷酸（PI（4，5）P-2）的耗尽，导致IP 3产生的显著减少。这可以解释在克隆5A 4C中细胞内Ca 2+动员的消除。通过在这些细胞中加入外源性PI（4，5）P-2来重建Ca 2+动员，从而支持这一假设。我们的结果表明，IP 3和S1 P都有助于Fc γ R1诱导的Ca 2+从ER释放，并且IP 3的产生对于S1 P引起Ca 2+从ER动员是必需的。(c)2005爱思唯尔有限公司保留所有权利。
Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S I P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor (DL)-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A(3) adenosine receptors with N-5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (Pl3KC2 beta). In such clones including clone 5A4C, P13KC2 beta was overexpressed throughout the cell, although endogenous P13KC2 beta was normally expressed only in the ER. Overexpression of P13KC2 beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P-2 in these cells. Our results suggest that both IP3 and S1P contribute to Fc epsilon Rl-induced Ca2+ release from the ER and production Of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER. (c) 2005 Elsevier Ltd. All rights reserved.