Juhl Kirstine, Høy Marianne, Olsen Hervør L, Bokvist Krister, Efanov Alexander M, Hoffmann Else K, Gromada Jesper
Laboratory of Islet Cell Physiology, Novo Nordisk, Bagsvaerd, Denmark.
Am J Physiol Endocrinol Metab. 2003 Jul;285(1):E73-81. doi: 10.1152/ajpendo.00086.2003. Epub 2003 Mar 18.
Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha 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. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha 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 cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha 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.
我们通过电容测量，研究了细胞内应用重组人胞质型磷脂酶A2（cPLA2α）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Ca2+依赖性胞吐作用的影响。cPLA2α以剂量依赖性方式（EC50 = 86 nM）将去极化诱发的胞吐作用刺激了450%，而不影响全细胞Ca2+电流或细胞质Ca2+水平。这种刺激作用涉及分泌颗粒的启动，表现为可快速释放颗粒池的大小从70 - 80增加到280 - 300。cPLA2α刺激的胞吐作用被特异性cPLA2抑制剂AACOCF3所拮抗。在用AACOCF3或针对cPLA2α的反义寡核苷酸处理的细胞中，Ca2+诱发的胞吐作用降低了40%。花生四烯酸和溶血磷脂酰胆碱的组合（刺激470%）模拟了cPLA2α的作用，其中每种化合物单独作用时可使胞吐反应增加一倍。据报道，含胰岛素分泌颗粒的启动涉及通过ClC - 3 Cl-通道摄取Cl-。因此，cPLA2α的刺激作用被Cl-通道抑制剂DIDS以及用ClC - 3 Cl-通道反义寡核苷酸预处理的细胞所抑制。我们提出，cPLA2α在控制β细胞的胞吐速率中起重要作用。cPLA2α的这种作用反映了跨颗粒Cl-通量的增强，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共同作用。
