Browsing by Author "Poblete, M. Ines"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    P2Y(1) Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling
    (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2008) Norambuena, Andres; Poblete, M. Ines; Donoso, M. Veronica; Espinoza, C. Sofia; Gonzalez, Alfonso; Huidobro Toro, J. Pablo
    The nucleotide P2Y(1) receptor ( P2Y(1) R) 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 P2Y(1) R 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 P2Y(1) R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1) R and obliterated the P2Y(1) R-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 P2Y(1) R agonist, not only displaced within 4 min the P2Y(1) R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N-6-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1) R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1) R 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 P2Y(1) R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1) R to membrane rafts, highlighting the role of this microdomain in P2Y(1) R signaling.