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

Article date: November 1987

By: N. Cook, S.R. Nahorski, D.B. Barnett in Volume 92, Issue 3, pages 587-596

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.

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.

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.

Agonist‐induced changes in receptor numbers were concentration‐dependent with half maximal receptor loss occurring at an isoprenaline concentration of approximately 2 × 10−8m. These effects were inhibited by the presence of a β‐adrenoceptor antagonist and absent if agonist pretreatment was performed at 4°C.

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).

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).

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.

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.

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.

Agonist‐induced changes in receptor numbers were concentration‐dependent with half maximal receptor loss occurring at an isoprenaline concentration of approximately 2 × 10−8m. These effects were inhibited by the presence of a β‐adrenoceptor antagonist and absent if agonist pretreatment was performed at 4°C.

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).

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).

DOI: 10.1111/j.1476-5381.1987.tb11360.x

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