Phosphatidic acid that accumulates in platelet-derived growth factor-stimulated Balb/c 3T3 cells is a potential mitogenic signal.
We developed a monoclonal antibody specific to phosphatidic acid (PA). Using this antibody, a novel method to quantify trace amounts of PA was achieved. With the method, PA can be measured in the range of 20-500 pmol. We applied this method to quantify changes in PA levels in Balb/c 3T3 cells stimulated by platelet-derived growth factor. PA contents were very low in quiescent cells and dramatically increased with time up to 15 min. On the other hand, a biphasic diacylglycerol (DG) increase was found. The early phase showed a transient small peak of DG at 30 s followed by a decrease to 1 min. In the second phase, DG accumulated gradually but very markedly up to 15 min. Treatment with propranolol, a PA phosphohydrolase inhibitor, enhanced the accumulation of PA and inhibited the formation of DG in the second phase. However, R59022, a DG kinase inhibitor, did not influence the accumulation of DG or PA, suggesting that platelet-derived growth factor stimulates mainly phospholipase D-catalyzed hydrolysis of phospholipids rather than phospholipase C-catalyzed hydrolysis in the second phase. PA, even after contaminating lyso-PA was removed, could stimulate DNA synthesis, although lyso-PA was 25 times more potent. Moreover, phospholipase D was found to be a much stronger mitogen than phospholipase C. Phospholipase D treatment caused a biphasic accumulation of PA. PA levels reached a maximum at 1 h, and then decreased between 1 and 2 h; finally, there was a gradual elevation up to 10 h. In this case, there was no significant DG accumulation. On the other hand, phospholipase C treatment induced only DG accumulation without any significant change in PA. These results indicate that PA accumulation, rather than an increase in DG, correlates well with mitogenesis.
Fukami
K
K
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
Takenawa
T
T
D016475:3T3 Cells; D000818:Animals; D000918:Antibody Specificity; D002855:Chromatography, Thin Layer; D004075:Diglycerides / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens; D010712:Phosphatidic Acids / Q000276:immunology / Q000378:metabolism*; D010739:Phospholipase D / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011433:Propranolol / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology
D004075:Diglycerides; D008934:Mitogens; D010712:Phosphatidic Acids; D010982:Platelet-Derived Growth Factor; D011744:Pyrimidinones; D013844:Thiazoles; C047505:R 59022; D011433:Propranolol; D010738:Type C Phospholipases; D010739:Phospholipase D