Abstract
The nucleotide P2Y1 receptor (P2Y1R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y1R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y1R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y1R and obliterated the P2Y1R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y1R agonist, not only displaced within 4 min the P2Y1R localization out of membrane rafts but also induced its subsequent internalization. 2′-Deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y1R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y1R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y1R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y1R to membrane rafts, highlighting the role of this microdomain in P2Y1R signaling.
Footnotes
-
A.N. is a postdoctoral fellow funded by the Centre for Cell Regulation and Pathology; C.S.E. is an undergraduate biochemistry student.
-
This work was partially funded by grant 13980001 to the Centre for Cell Regulation and Pathology. The Millennium Institute MIFAB also contributed with funds to the Centre.
-
ABBREVIATIONS: PM, plasma membrane; P2Y1R, P2Y1 receptor; P2Y2R, P2Y2 receptor; MβCD, methyl β-cyclodextrin; GPCR, G protein-coupled receptor; HCA, human chorionic artery; Tfn-R, transferrin receptor; MRS 2365, [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt; MRS 2179, 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt; ARC 69931MX, N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5′-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid); 2-MeSADP, 2-methylthio ADP; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; ADO, adenosine; 5-HT, 5-hydroxytryptamine.
-
↵1 Current affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia.
- Received May 1, 2008.
- Accepted September 16, 2008.
- The American Society for Pharmacology and Experimental Therapeutics