The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells

Abstract

1. 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 μM with a concentration-dependent delay of 45-175 s. The [Ca2+](i) signal was composed of a gradual rise and a sustained plateau. 2. In Ca2+-free medium, ET-18-OCH3 (10-100 μM) 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 μM). 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 μM), nifedipine (10 μM), verapamil (10 μM), diltiazem (10 μM) and cadmium (0.5 μM). 3. Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylate (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 μM ET-18-OCH3-induced [Ca2+](i) rise in a concentration-dependent manner between 1-20 μM, with 20 μM exerting a complete block. 4. The [Ca2+](i) rise induced by ET-18-OCH3 (25 μM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 μM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 μM) or the phospholipase A2 inhibitor aristolochic acid (20-40 μM). 5. In Ca2+-free medium, pretreatment with 25 μM 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 μM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 μM ET-18-OCH3. 6. ET-18-OCH3 (25 μM) 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 μM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+](i) rise. 7. 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.