Article date: July 1999
By: Chung‐Ren Jan, Sheng‐Nan Wu, Ching‐Jiunn Tseng, in Volume 127, Issue 6, pages 1502-1510
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.
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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