Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.
1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.
D000255:Adenosine Triphosphate / Q000032:analysis; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D005990:Glycerol / Q000031:analogs & derivatives* / Q000378:metabolism; D005991:Glycerol Kinase / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D010712:Phosphatidic Acids / Q000032:analysis / Q000096:biosynthesis*; D010743:Phospholipids / Q000096:biosynthesis*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D005227:Fatty Acids; D010712:Phosphatidic Acids; D010743:Phospholipids; D000001:Calcimycin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D005991:Glycerol Kinase; D005990:Glycerol; D002118:Calcium