Thapsigargin selectively stimulates synthesis of phosphatidylglycerol in N1E-115 neuroblastoma cells and phosphatidylinositol in C6 glioma cells.
Phospholipid metabolism was studied in N1E-115 neuroblastoma and C6 glioma cells exposed to thapsigargin, a selective inhibitor of endoplasmic reticulum Ca(2+)-ATPase that raises the cytosolic free Ca2+ concentration [Ca2+]i. Thapsigargin caused only a transient increase of [Ca2+]i (< 1 min) in N1E-115 cells similar in magnitude and duration to agonist-induced calcium release mediated by inositol trisphosphate. Sustained elevation of [Ca2+]i due to influx of extracellular calcium, as occurs in most other cell lines including C6 cells, did not occur in N1E-115 cells. Increased uptake of inorganic phosphate (Pi) associated calcium influx was observed in C6 but not in N1E-115 cells. Thapsigargin affected phospholipid synthesis in both cell lines, most likely by inhibiting phosphatidic acid phosphohydrolase as indicated by diversion of [3H]oleic acid incorporation from triacylglycerol to phospholipid synthesis and stimulation of [32P]Pi incorporation into anionic phospholipids at the expense of phosphatidylcholine synthesis. The response to increased phosphatidate/phosphatidyl-CMP availability was cell specific. Thapsigargin (> 100 nM) selectively stimulated phosphatidylglycerol synthesis 20-30-fold in N1E-115 neuroblastoma cells while phosphatidylinositol synthesis was increased < 2-fold. In contrast, phosphatidylglycerol was not affected in C6 glioma cells and phosphatidylinositol synthesis was stimulated 8-fold by thapsigargin (> 1 microM). Agonist-stimulated calcium release did not increase phosphatidylglycerol synthesis in N1E-115 cells. Thapsigargin-stimulated phosphatidylglycerol synthesis and agonist-stimulated phosphatidylinositol synthesis could occur at the same time. Similar results were obtained with TMB-8, an inhibitor of intracellular Ca2+ release that decreases diacylglycerol utilization by blocking choline uptake and phosphatidylcholine synthesis without affecting resting [Ca2+]i. Thus [Ca2+]i does not directly mediate the effects of thapsigargin, TMB-8 or agonist stimulation on anionic phospholipid metabolism. These additional effects may limit the use of thapsigargin to assess Ca(2+)-dependence of phospholipid metabolism associated with Ca(2+)-mediated signal transduction.
Palmer
F B
FB
Atlantic Research Centre, Halifax, Nova Scotia, Canada.
Cook
H W
HW
Byers
D M
DM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D009829:Oleic Acids; D010710:Phosphates; D010715:Phosphatidylglycerols; D010716:Phosphatidylinositols; D013729:Terpenes; D019301:Oleic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D019284:Thapsigargin; D002118:Calcium