Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay.
In Chinese hamster ovary cells overexpressing Edg-1, one of the sphingosine 1-phosphate (S1P) receptor subtypes, [(3)H]S1P binding was displaced by unlabeled S1P with IC(50), a half-maximal concentration to inhibit the binding, of about 20 nM. This radioreceptor binding was used for quantitative measurement of S1P. Among the various lipids employed, only sphingosylphosphorylcholine (SPC), other than S1P, practically displaced the binding; however, the potency of SPC was about 100 to 1000 times less than that of S1P. Thus, SPC bound to the S1P receptors inefficiently. Furthermore, before the application of test samples to this assay, S1P was partially purified: the lipid was extracted first into the aqueous phase and separated from other lipids under alkaline conditions, and then reextracted into the chloroform phase under acidic conditions. With this assay, we could specifically and quantitatively measure S1P from 2 to 40 pmol per assay well in biological samples including serum samples and various tissues. This assay also allowed us to measure the change in cellular S1P content in U937 cells after treatment with exogenous sphingosine.
Murata
N
N
Laboratory of Signal Transduction, Gunma University, Maebashi, 371-8512, Japan.
Sato
K
K
Kon
J
J
Tomura
H
H
Okajima
F
F
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008246:Lysophospholipids; D008297:Male; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000302:isolation & purification / Q000494:pharmacology; D014018:Tissue Distribution; D020298:U937 Cells
D017874:Immediate-Early Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; C060506:sphingosine 1-phosphate; D013110:Sphingosine