INTERNALIZATION AND DOWN-REGULATION OF THE PROSTACYCLIN RECEPTOR IN HUMAN PLATELETS

The internalization of [H-3]iloprost, a prostacyclin analogue, was stu died in human platelets by binding studies. After incubation with [H-3 ]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 bou nd ligand respectively, suggesting that a portion had been internalize d. The percentage of [H-3]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 degrees C or 4 degrees C w as markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting a gents (dinitrophenol and 2-deoxyglucose) completely inhibited internal ization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated t hat, 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), where as, when platelets were preincubated at 37 degrees C with iloprost, th e receptor was found mainly in the cytosolic fraction. In platelets pr eincubated with iloprost at 4 degrees C: two classes of binding sites were present, whereas after preincubation at 37 degrees C only the low er-affinity sites were detected. After exposure to the agonist, ilopro st-induced inhibition of platelet aggregation and activation of adenyl ate cyclase and cAMP production were significantly lower. Taken togeth er, these data demonstrate that human platelets can internalize a high -affinity binding site for iloprost, presumably the prostacyclin recep tor.