ATP stimulates the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells. Potentiating effects of guanosine triphosphates and sphingosine.
Recently, phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) was shown to be stimulated by activators of protein kinase C (Kiss, Z., and Anderson, W. B. (1989) J. Biol. Chem. 264, 1483-1487), suggesting that PtdEtn metabolism may play a role in signal transduction. Here we have studied the possible regulation of PtdEtn hydrolysis by adenine and guanine nucleotides, as well as by sphingosine, both in membranes isolated from [14C]ethanolamine- or [32P]PtdEtn-prelabeled NIH 3T3 cells and in intact cells. In isolated membranes both ATP and ADP stimulated the hydrolysis of PtdEtn. Both nucleotides had maximal (approximately 2-fold) effects at about 0.5 mM concentration. The main water-soluble product of [14C]PtdEtn hydrolysis was [14C]ethanolamine, while in [32P] PtdEtn-prelabeled membranes the nucleotides stimulated the formation of [32P]phosphatidic acid, suggesting the involvement of a phospholipase D-type enzyme. The hydrolysis-resistant analogs of GTP, such as guanosine 5'-3-O-(thio)triphosphate and guanyl-5'-yl imidodiphosphate, greatly potentiated the stimulatory effects of ATP and ADP on PtdEtn hydrolysis. On the other hand, the nonphosphorylating analogs of ATP, adenyl-5'-yl beta,gamma-imidodiphosphate and beta,gamma-methyl-eneadenosine 5'-triphosphate, failed to stimulate PtdEtn hydrolysis both in the absence and presence of guanosine triphosphates. Sphingosine, while exhibiting no effect alone, had a relatively modest (1.2-1.3-fold) potentiating effect on ATP-stimulated PtdEtn hydrolysis in isolated membranes. The effect of sphingosine was mimicked by threo- and erythrosphinganines, while N-acetylsphingosine was without effect. In studies with [14C]ethanolamine-prelabeled intact NIH 3T3 cells, externally added ATP did not stimulate PtdEtn hydrolysis. In contrast, sphingosine and sphinganines had much greater stimulatory effects on PtdEtn hydrolysis in intact cells than with isolated membranes. These data indicate that PtdEtn hydrolysis may be regulated by adenine and guanine nucleotides in addition to, or in cooperation with, the activators of protein kinase C, and that sphingosine may be an additional regulator of PtdEtn hydrolysis.
Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
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D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology
D000227:Adenine Nucleotides; D008563:Membrane Lipids; D010714:Phosphatidylethanolamines; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013110:Sphingosine