Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor.
Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.
Orlati
S
S
Dipart. di Biologia Ev.Sp., Università di Bologna, Bologna, Italy.
Porcelli
A M
AM
Hrelia
S
S
Van Brocklyn
J R
JR
Spiegel
S
S
Rugolo
M
M
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008246:Lysophospholipids; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D012137:Respiratory System / Q000166:cytology / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004075:Diglycerides; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins; D008246:Lysophospholipids; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D010712:Phosphatidic Acids; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D010566:Virulence Factors, Bordetella; D000072000:NF-KappaB Inhibitor alpha; D005576:Colforsin; C060506:sphingosine 1-phosphate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D013110:Sphingosine; D002118:Calcium