Quantification of extracellular UDP-galactose.
The human P2Y(14) receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y(14) receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with subnanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1-4-beta-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [gamma-(32)P]ATP and the formation of [gamma-(32)P]UTP is monitored by high-performance liquid chromatography. The overall conversion of UDP-Gal to [gamma-(32)P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. The occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule.
Lazarowski
Eduardo R
ER
Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, 7017 Thurston-Bowles Building, CB 7248, University of North Carolina, Chapel Hill, NC 27599-7248, USA. eduardo_lazarowski@med.unc.edu
D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002417:Cattle; D002460:Cell Line; D004285:Dogs; D005110:Extracellular Space / Q000737:chemistry* / Q000187:drug effects; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D007785:Lactose / Q000096:biosynthesis; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D014531:Uridine Diphosphate Galactose / Q000032:analysis* / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D009701:Nucleoside-Diphosphate Kinase; D013917:Thrombin; D007785:Lactose; D014532:Uridine Diphosphate Glucose
Annu Rev Biochem. 1998;67:49-69
9759482
Brain Res Mol Brain Res. 2003 Oct 21;118(1-2):10-23
14559350
Trends Pharmacol Sci. 2003 Feb;24(2):52-5
12559763
J Physiol. 2007 Oct 1;584(Pt 1):245-59
17656429
J Biol Chem. 2003 Jun 27;278(26):23331-42
12684505
Br J Pharmacol. 2008 Apr;153(7):1528-37
18204471
Br J Pharmacol. 1996 Jan;117(1):203-9
8825364
J Biol Chem. 1975 Jan 25;250(2):739-46
1112787
Anal Biochem. 1994 Oct;222(1):49-54
7856870
J Biol Chem. 2009 May 1;284(18):12572-83
19276090
J Biol Chem. 2000 Apr 14;275(15):10767-71
10753868
J Biol Chem. 2000 Oct 6;275(40):31061-8
10913128
Br J Pharmacol. 2005 Oct;146(3):435-44
15997228
J Immunol. 2003 Aug 15;171(4):1941-9
12902497
Am J Respir Cell Mol Biol. 2005 Dec;33(6):601-9
16109883
Eur J Pharmacol. 2006 Aug 14;543(1-3):166-73
16820147
Purinergic Signal. 2008 Jun;4(2):139-46
18368524
Br J Pharmacol. 2006 Jan;147 Suppl 1:S172-81
16402102
Genes Dev. 2003 Jul 1;17(13):1592-604
12842911
J Biol Chem. 2001 Nov 9;276(45):41527-42
11553646
J Cell Biol. 1971 Nov;51(21):536-47
5165268
J Pharmacol Exp Ther. 2009 Jul;330(1):162-8
19339661
Am J Physiol Cell Physiol. 2008 Jul;295(1):C231-41
18495810
Mol Pharmacol. 2003 Oct;64(4):785-95
14500734
Mol Pharmacol. 2003 May;63(5):1190-7
12695547
Br J Pharmacol. 2004 Jul;142(6):1002-14
15210578
Anal Biochem. 2001 Jun 1;293(1):129-37
11373089
Curr Drug Targets. 2008 Apr;9(4):292-309
18393823
Anal Biochem. 2006 Jan 15;348(2):243-51
16325757
Brain Res Brain Res Rev. 2005 Apr;48(2):144-56
15850653
Blood. 2008 Mar 15;111(6):3062-9
17993619
J Biol Chem. 1970 Apr 25;245(8):2103-8
5440844
Science. 2003 Sep 12;301(5639):1531-4
12970565
Glia. 2003 Sep;43(3):218-03
12898701
J Pharmacol Exp Ther. 2000 Dec;295(3):862-9
11082418
Biochemistry. 2008 Sep 2;47(35):9269-78
18693752