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

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.

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.

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).

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.

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 mM) or the phospholipase A₂ inhibitor aristolochic acid (20–40 μM).

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.

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.

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.

British Journal of Pharmacology (1999) 127, 1502–1510; doi:10.1038/sj.bjp.0702691

DOI: 10.1038/sj.bjp.0702691

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