Jan C R, Wu S N, Tseng C J
Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan, Republic of China.
Br J Pharmacol. 1999 Jul;127(6):1502-10. doi: 10.1038/sj.bjp.0702691.
The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.
以fura - 2作为钙离子探针，研究了醚脂1 - O - 十八烷基 - 2 - O - 甲基 - sn - 甘油 - 3 - 磷酸胆碱（ET - 18 - OCH3）对犬肾Madin Darby（MDCK）细胞内游离钙离子浓度（[Ca2 + ]i）的影响。在含钙培养基中，ET - 18 - OCH3在10 - 100微摩尔浓度范围内可使[Ca2 + ]i显著升高，浓度依赖性延迟为45 - 175秒。[Ca2 + ]i信号由逐渐升高和持续平台期组成。在无钙培养基中，ET - 18 - OCH3（10 - 100微摩尔）可诱导细胞内钙库释放钙离子，浓度依赖性延迟为45 - 175秒。这种细胞内钙离子的释放以浓度依赖性方式触发了容量性钙离子内流。这种容量性钙离子内流不受酮康唑（25微摩尔）、1 - [β - [3 - （4 - 甲氧基苯基）丙氧基] - 4 - 甲氧基苯乙基] - 1H - 咪唑盐酸盐（SKF96365；50微摩尔）、硝苯地平（10微摩尔）、维拉帕米（10微摩尔）、地尔硫卓（10微摩尔）和镉（0.5微摩尔）的抑制。血小板活化因子（PAF）受体拮抗剂2 - （苯硫基）乙基 - 1,4 - 二氢 - 2,4,6 - 三甲基吡啶 - 3,5 - 二羧酸甲酯（PCA - 4248）在1 - 20微摩尔浓度范围内以浓度依赖性方式抑制25微摩尔ET - 18 - OCH3诱导的[Ca2 + ]i升高，20微摩尔时可完全阻断。当磷脂酶C抑制剂U73122（2微摩尔）抑制肌醇1,4,5 - 三磷酸（IP3）的产生时，ET - 18 - OCH3（25微摩尔）诱导的[Ca2 + ]i升高未改变，但部分受到磷脂酶D抑制剂普萘洛尔（0.1毫摩尔）或磷脂酶A2抑制剂马兜铃酸（二十 - 四十微摩尔）的抑制。在无钙培养基中，用25微摩尔ET - 18 - OCH3预处理可完全耗尽内质网钙泵抑制剂毒胡萝卜素敏感的钙库。相反，用毒胡萝卜素预处理可消除0.1毫摩尔ATP诱导的[Ca2 + ]i升高，而不改变ET - 18 - OCH3诱导的[Ca2 + ]i升高。这表明ET - 18 - OCH3耗尽了毒胡萝卜素敏感的钙库，并且还从毒胡萝卜素不敏感的钙库中释放了钙离子。毒胡萝卜素不敏感的钙库涉及线粒体，因为线粒体解偶联剂羰基氰化物间氯苯腙（CCCP；2微摩尔）诱导的线粒体钙离子释放可被25微摩尔ET - 18 - OCH3预处理所消除。ET - 18 - OCH3（25微摩尔）在360纳米激发波长下可诱导fura - 2荧光显著的锰淬灭，证实ET - 18 - OCH3诱导了容量性钙离子内流。镧离子（0.1毫摩尔）或钆离子（50微摩尔）可消除ET - 18 - OCH3诱导的锰淬灭和[Ca2 + ]i升高。我们的数据表明，ET - 18 - OCH3通过激活PAF受体导致细胞内钙离子释放，随后引起容量性钙离子内流，从而诱导MDCK细胞内[Ca2 + ]i升高。磷脂酶D和磷脂酶A2，而非磷脂酶C，可能参与介导容量性钙离子内流。镧离子可能通过抑制PAF受体消除ET - 18 - OCH3诱导的[Ca2 + ]i升高。
