Noh S J, Kim M J, Shim S, Han J K
Department of Life Science, Pohang University of Science and Technology, South Korea.
J Cell Physiol. 1998 Aug;176(2):412-23. doi: 10.1002/(SICI)1097-4652(199808)176:2<412::AID-JCP20>3.0.CO;2-3.
In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes.
在非洲爪蟾卵母细胞中，鞘氨醇 -1- 磷酸（S1P）和溶血磷脂酸（LPA）均通过与膜结合受体相互作用来激活钙离子依赖的振荡性氯离子电流。外部施加50微摩尔/升的S1P会引发一种持续较长时间的振荡电流，从振荡开始持续超过30分钟，通常最大电流峰值为300纳安（n = 11），而1微摩尔/升的LPA处理仅显示短暂振荡但更强烈的电流反应，最大峰值幅度为2800纳安（n = 18）。两种磷脂诱导的钙离子依赖的氯离子电流在细胞外无钙离子的情况下均可观察到，被细胞内注射钙离子螯合剂乙二醇双四乙酸（EGTA）阻断，并且不能由内质网（ER）钙离子ATP酶抑制剂毒胡萝卜素处理引发。细胞内钙离子释放似乎来自肌醇1,4,5 - 三磷酸（IP3）敏感的钙离子储存库，因为注射肝素可阻断氯离子电流。用氨基类固醇U - 73122（一种G蛋白介导的磷脂酶C（PLC）激活抑制剂）预处理卵母细胞，可抑制S1P和LPA引发的电流反应。然而，当向卵母细胞注射10纳克针对非洲爪蟾磷脂酶C（PLC - xβ）的反义寡核苷酸（AS - ODN）时，卵母细胞对S1P刺激无反应，而对LPA刺激反应正常，这表明S1P信号通路通过PLC - xβ，而LPA信号通路通过另一种未知的PLC。为了确定所涉及的G蛋白类型，我们引入了针对在非洲爪蟾中鉴定出的四种G蛋白α亚基的AS - ODN；G(q)α、G11α、G0α和G(i1)α。在所测试的针对Gα的AS - ODN中，针对S1P的AS - G(q)α和AS - G(i1)α以及针对LPA的AS - G(q)α和AS - G11α分别将电流反应特异性降低至对照的约20 - 30%。这些结果表明，LPA和S1P虽然具有相似的结构特征，但在非洲爪蟾卵母细胞中，它们从IP3敏感池中释放细胞内钙离子，并且在信号转导途径中使用不同的组分。
