title: Internalization and down-regulation of the prostacyclin receptor in human platelets.
abstract: The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 degrees C, addition of unlabelled ligand at either 37 degrees C or 4 degrees C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 degrees C or 4 degrees C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 degrees C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 degrees C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 degrees C, two classes of binding sites were present, whereas after preincubation at 37 degrees C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Giovanazzi
authors.forename: M R
authors.initials: MR
authors.lastname: Accomazzo
authors.forename: O
authors.initials: O
authors.lastname: Letari
authors.forename: D
authors.initials: D
authors.lastname: Oliva
authors.forename: S
authors.initials: S
authors.lastname: Nicosia
mesh_terms: D000262:Adenylyl Cyclases / Q000097:blood; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D011464:Epoprostenol / Q000097:blood; D006801:Humans; D016285:Iloprost / Q000097:blood / Q000494:pharmacology; D066298:In Vitro Techniques; D007231:Infant, Newborn; D007700:Kinetics; D010974:Platelet Aggregation; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000096:biosynthesis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011464:Epoprostenol; D000262:Adenylyl Cyclases; D016285:Iloprost