Evidence for an agonist-induced, ATP-dependent change in muscarinic receptors of intact 1321N1 cells

R. K. Hoover, Myron Lee Toews

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Abstract

The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate ([3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine ([3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cells competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H]NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.

Original languageEnglish (US)
Pages (from-to)63-70
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume251
Issue number1
StatePublished - Jan 1 1989
Externally publishedYes

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Muscarinic Receptors
Adenosine Triphosphate
Adrenergic beta-1 Receptors
Adrenergic alpha-1 Receptors
Quinuclidinyl Benzilate
Antimycin A
Muscarinic Agonists
Scopolamine Hydrobromide
Astrocytoma
Carbachol
Atropine
Sucrose

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Evidence for an agonist-induced, ATP-dependent change in muscarinic receptors of intact 1321N1 cells",
abstract = "The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate ([3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine ([3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cells competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H]NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.",
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AU - Hoover, R. K.

AU - Toews, Myron Lee

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N2 - The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate ([3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine ([3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cells competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H]NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.

AB - The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate ([3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine ([3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cells competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H]NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.

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