cPLA2α-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic β-cells
cPLA2α-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic β-cells
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DOI：
10.1152/ajpendo.00086.2003
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发表时间：
2003-07-01
影响因子：
5.1
通讯作者：
Gromada, J
中科院分区：
文献类型：
--
作者：
Juhl, K;Hoy, M;Gromada, J
利用电容测量，我们研究了细胞内应用重组人胞浆磷脂酶A（2）（cPLA（2 α））及其脂解产物花生四烯酸和溶血磷脂酰胆碱对单个小鼠胰腺β细胞中Ca 2+依赖性胞吐作用的影响。cPLA（2 α）剂量依赖性（EC 50 = 86 nM）刺激去极化诱发的胞吐作用达450%，而不影响全细胞Ca 2+电流或胞质Ca 2+水平。刺激作用涉及分泌颗粒的引发，如通过颗粒的易释放池的大小从70-80增加到280-300所反映的。cPLA（2 α）刺激的胞吐作用可被cPLA（2）特异性抑制剂AACOCF（3）拮抗。在用AACOCF（3）或针对cPLA（2 alpha）的反义寡核苷酸处理的细胞中，Ca 2+诱发的胞吐减少了40%。cPLA（2 α）的作用通过花生四烯酸和溶血磷脂酰胆碱（470%刺激）的组合来模拟，其中每种化合物单独使胞吐反应加倍。据报道，含胰岛素分泌颗粒的启动涉及通过ClC-3 Cl通道的Cl-摄取。因此，cPLA（2 α）的刺激作用被Cl-通道抑制剂DIDS和用CIC-3 Cl-通道反义寡核苷酸预处理的细胞抑制。我们认为cPLA（2 α）在控制β细胞胞吐速率方面具有重要作用。cPLA（2 α）的这种作用反映了增强的跨颗粒Cl-通量，导致可用于释放的颗粒数量增加，并且需要花生四烯酸和溶血磷脂酰胆碱的联合作用。
Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A(2) (cPLA(2alpha)) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA(2alpha) dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA(2alpha)-stimulated exocytosis was antagonized by the specific cPLA(2) inhibitor AACOCF(3). Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF(3) or an antisense oligonucleotide against cPLA(2alpha). The action of cPLA(2alpha) was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl channels. Accordingly, the stimulatory action of cPLA(2alpha) was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with CIC-3 Cl- channel antisense oligonucleotides. We propose that cPLA(2alpha) has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA(2alpha) reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.