Non-specific uptake of the radioligand 125I-IHYP by intact human lymphocytes

Reversal of the uptake process

Elisabeth M. Paietta, D. Hoyer, G. Engel, J. D. Schwarzmeier

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Binding of (±)[125I]iodohydroxybenzylpindolol (IHYP) to β-adrenoceptors on intact human peripheral blood lymphocytes was found to be associated with a high degree of non-specific intracellular uptake of the radioligand. Non-specific binding on cellular surfaces was identified by displacing IHYP from its specific binding sites with the cold competing antagonist (±)-propranolol. Another methodological approach, however, was necessary to eliminate the uptake problem: release of intracellularly accumulated IHYP was achieved by sedimentation of the IHYP-loaded cells and resuspension in hypotonie buffer (HME buffer) for 10 min at + 4°C. The true value of maximal IHYP binding measured after IHYP release was 970 binding sites/cell as compared with 2300 receptors/cell found under standard binding conditions. This difference in maximal IHYP binding obtained by the 2 methods is attributed to the strong interference of the uptake process with the measurement of specific IHYP binding. Further evidence for the efficiency of the procedure to reverse the uptake process was obtained with the very slowly dissociable β-adrenoceptor antagonist FM 24. Pre-incubation of the cells with 5 × 10-5 M FM 24 totally prevented specific IHYP binding. Any IHYP measured on cells in the presence of FM 24, therefore, must have been due to mere non-specific binding and cellular uptake of the radioligand. Even though the uptake of IHYP was enhanced in FM-24-treated cells, this influx could be completely reversed by exposing the IHYP-loaded cells to HME buffer for 10 min at +4°C. Our findings indicate that our experimental conditions are suitable for β-receptor binding studies on intact cells with IHYP, because the uptake of the labeled ligand is eliminated.

Original languageEnglish (US)
Pages (from-to)267-276
Number of pages10
JournalMolecular and Cellular Endocrinology
Volume25
Issue number3
DOIs
StatePublished - 1982
Externally publishedYes

Fingerprint

Lymphocytes
Buffers
iodohydroxybenzylpindolol
Adrenergic Receptors
Binding Sites
Cells
Sedimentation
Propranolol
Blood

Keywords

  • radioligand binding
  • β-adrenoceptor
  • β-receptor/adenylate cyclase system

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Non-specific uptake of the radioligand 125I-IHYP by intact human lymphocytes : Reversal of the uptake process. / Paietta, Elisabeth M.; Hoyer, D.; Engel, G.; Schwarzmeier, J. D.

In: Molecular and Cellular Endocrinology, Vol. 25, No. 3, 1982, p. 267-276.

Research output: Contribution to journalArticle

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abstract = "Binding of (±)[125I]iodohydroxybenzylpindolol (IHYP) to β-adrenoceptors on intact human peripheral blood lymphocytes was found to be associated with a high degree of non-specific intracellular uptake of the radioligand. Non-specific binding on cellular surfaces was identified by displacing IHYP from its specific binding sites with the cold competing antagonist (±)-propranolol. Another methodological approach, however, was necessary to eliminate the uptake problem: release of intracellularly accumulated IHYP was achieved by sedimentation of the IHYP-loaded cells and resuspension in hypotonie buffer (HME buffer) for 10 min at + 4°C. The true value of maximal IHYP binding measured after IHYP release was 970 binding sites/cell as compared with 2300 receptors/cell found under standard binding conditions. This difference in maximal IHYP binding obtained by the 2 methods is attributed to the strong interference of the uptake process with the measurement of specific IHYP binding. Further evidence for the efficiency of the procedure to reverse the uptake process was obtained with the very slowly dissociable β-adrenoceptor antagonist FM 24. Pre-incubation of the cells with 5 × 10-5 M FM 24 totally prevented specific IHYP binding. Any IHYP measured on cells in the presence of FM 24, therefore, must have been due to mere non-specific binding and cellular uptake of the radioligand. Even though the uptake of IHYP was enhanced in FM-24-treated cells, this influx could be completely reversed by exposing the IHYP-loaded cells to HME buffer for 10 min at +4°C. Our findings indicate that our experimental conditions are suitable for β-receptor binding studies on intact cells with IHYP, because the uptake of the labeled ligand is eliminated.",
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