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

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.