Human platelet β2‐adrenoceptors: agonist‐induced internalisation and down‐regulation in intact cells

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Abstract
1 The effect of isoprenaline (10 μm at 37°C for 30 min) pretreatment on [125I]-(—)-pindolol ([125I]-(—)-Pin) binding to β2-adrenoceptors on intact human platelets has been examined. 2 By use of saturation analysis, maximal binding capacity (Bmax) of [125I]-(—)-Pin binding in control and treated cells was assessed in the presence of 1 μm (—)-propranolol or 1 μm (±)-CGP 12177 which were taken to represent total or cell surface β-adrenoceptors respectively. Assay incubations were performed at 37°C and 4°C, the latter to prevent recycling of internalised receptors. 3 Isoprenaline treatment resulted in an identical, highly significant, loss of binding sites (⋍ 25%) defined by (—)-propranolol at both assay temperatures as compared to control cells. Binding sites identified in the presence of (±)-CGP 12177 were reduced to a much greater extent (⋍ 70%), but this was only seen when assays were performed at 4°C. 4 Agonist-induced changes in receptor numbers were concentration-dependent with half maximal receptor loss occurring at an isoprenaline concentration of approximately 2 × 10−8 m. These effects were inhibited by the presence of a β-adrenoceptor antagonist and absent if agonist pretreatment was performed at 4°C. 5 Recovery experiments showed that the isoprenaline-induced reduction in total receptor number defined by (—)-propranolol was irreversible whereas the reduction in cell surface receptors defined by (±)-CGP 12177 was rapidly reversible (< 40 min). 6 These data suggest that isoprenaline treatment of intact human platelets causes redistribution of β2-adrenoceptors. A proportion are sequestered away from the cell surface (internalised), becoming inaccessible to the hydrophilic ligand (±)-CGP 12177. A smaller proportion defined by (—)-propranolol are apparently totally lost from the cell (down regulated).