Pedersen Stine F, Poulsen Kristian A, Lambert Ian H
Department of Biochemistry, Institute for Molecular Biology and Physiology, Copenhagen, Denmark.
Am J Physiol Cell Physiol. 2006 Dec;291(6):C1286-96. doi: 10.1152/ajpcell.00325.2005. Epub 2006 Jul 19.
Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca(2+)-independent phospholipase A(2) (iPLA(2)) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA(2)-IVA, cPLA(2)-IVB, cPLA(2)-IVC, iPLA(2)-VIA, iPLA(2)-VIB, and secretory sPLA(2)-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA(2)-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA(2)-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA(2) substrate availability, potentiated arachidonic acid release and osmolyte efflux in a volume-sensitive, 5-lipoxygenase-dependent, cyclooxygenase-independent manner. Melittin-induced arachidonic acid release was inhibited by manoalide and slightly but significantly by BEL. A BEL-sensitive, melittin-induced PLA(2) activity was also detected in lysates devoid of sPLA(2), indicating that both sPLA(2) and iPLA(2) contribute to arachidonic acid release in vivo. Swelling-induced taurine efflux was inhibited potently by BEL and partially by manoalide, whereas the reverse was true for melittin-induced taurine efflux. It is suggested that in NIH3T3 cells, swelling-induced taurine efflux is dependent at least in part on arachidonic acid release by iPLA(2) and possibly also by sPLA(2), whereas melittin-induced taurine efflux is dependent on arachidonic acid release by sPLA(2) and, to a lesser extent, iPLA(2).
NIH3T3小鼠成纤维细胞的渗透性肿胀激活了一种对溴苯乙酮内酯(BEL)敏感的牛磺酸外流,这表明一种不依赖钙离子的磷脂酶A2(iPLA2)参与其中(兰伯特·IH。《膜生物学杂志》192:19 - 32,2003年)。我们报告,NIH3T3细胞的牛磺酸外流不仅因细胞肿胀而增加,还因细胞收缩而减少。花生四烯酸向细胞外的释放同样因收缩而减少,但未因肿胀而显著增加。发现NIH3T3细胞表达胞质钙依赖性cPLA2 - IVA、cPLA2 - IVB、cPLA2 - IVC、iPLA2 - VIA、iPLA2 - VIB和分泌性sPLA2 - V。BEL和sPLA2抑制剂 manoalide可部分抑制肿胀细胞中花生四烯酸的释放。细胞肿胀引发了从细胞核中释放出对BEL敏感的花生四烯酸,iPLA2 - VIA定位于细胞核。暴露于蜂毒肽蜂毒素以增加PLA2底物的可用性,以一种体积敏感、5 - 脂氧合酶依赖性、环氧化酶非依赖性的方式增强了花生四烯酸的释放和渗透溶质的外流。蜂毒素诱导的花生四烯酸释放被manoalide抑制,被BEL轻微但显著地抑制。在不含sPLA2的裂解物中也检测到了对BEL敏感的、蜂毒素诱导的PLA2活性,这表明sPLA2和iPLA2都在体内花生四烯酸的释放中起作用。肿胀诱导的牛磺酸外流被BEL强烈抑制,被manoalide部分抑制,而蜂毒素诱导的牛磺酸外流则相反。这表明在NIH3T3细胞中,肿胀诱导的牛磺酸外流至少部分依赖于iPLA2以及可能还有sPLA2释放的花生四烯酸,而蜂毒素诱导的牛磺酸外流则依赖于sPLA2释放的花生四烯酸,且在较小程度上依赖于iPLA2释放的花生四烯酸。