Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Mediation of separate responses by high affinity and low affinity agonist-receptor conformations

M. McKinney, S. Stenstrom, E. Richelson

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Abstract

Murine neuroblastoma cells (clone N1E-115) possess muscarinic receptors that mediate multiple responses, including the elevation of cyclic GMP levels and the inhibition of receptor-mediated increases in cyclic AMP. Evidence is presented showing that two muscarinic agonist-receptor conformations in N1E-115 cells each separately mediate a cyclic nucleotide response. Pirenzepine inhibited the [3H]cyclic GMP response to carbachol with a KD value of aproximately 6 nM, whereas it inhibited the ability of carbachol to reduce prostaglandin E1-mediated elevations in [3H]cyclic AMP levels with a KD value of 93 nM, thus differentiating between two classes of receptors involved in these responses. Ten muscarinic agonists were studied for their ability to mediate the two cyclic nucleotide responses. Six were as effective as acetylcholine in the reduction of [3H]cyclic AMP levels, but only two were as effective as acetylcholine in elevating [3H]cyclic GMP levels. Four agonists (arecoline, pilocarpine, oxotremorine, and McN-A343) were ineffective in increasing [3H]cyclic GMP levels. These four agonists and bethanecol, which could increase [3H]cyclic GMP levels only 18% as well as acetylcholine, behaved as competitive antagonists in this response to carbachol. These partial agonists, in contrast to carbachol, bound to only one class of muscarinic sites in N1E-115 cells with equilibrium dissociation constants determined by competition binding assay which agreed well with their respective EC50 values for their effect on [3H]cyclic AMP levels. The equilibrium dissociation constants for the partial agonists determined by their inhibition of carbachol in the [3H]cyclic GMP response also agreed well with their respective EC50 values for mediating the [3H]cyclic AMP response. Thus, the partial agonists bound to the same receptors at which carbachol mediated [3H]cyclic GMP formation, but with KD values about the same as their respective EC50 values for inhibition of prostaglandin E1-mediated [3H]cyclic AMP increases. The full agonists acetylcholine and methacholine, like carbachol, bound to two sites in N1E-115 cells. For the six agonists able to stimulate both responses at least to some degree, the ratio of their potencies at each response correlated with their respective efficacies at each response but with much more dependence in the [3H]cyclic GMP response. It is proposed that the low affinity agonist-receptor conformation, inducible by full agonists, activates the effector for the cyclic GMP response and its formation is inhibited by pirenzepine with high potency; and the high affinity agonist-receptor conformation, inducible by both full and partial agonists, activates the effector for the cyclic AMP reduction and is inhibited by pirenzepine with lower potency.

Original languageEnglish (US)
Pages (from-to)223-235
Number of pages13
JournalMolecular Pharmacology
Volume27
Issue number2
StatePublished - 1985

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Cyclic GMP
Neuroblastoma
Cholinergic Agents
Carbachol
Clone Cells
Cyclic AMP
Pirenzepine
Acetylcholine
Muscarinic Agonists
Alprostadil
Cyclic Nucleotides
Muscarinic Receptors
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride
Arecoline
Bethanechol
Oxotremorine
Cholinergic Agonists
Pilocarpine
Methacholine Chloride

ASJC Scopus subject areas

  • Pharmacology

Cite this

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Mediation of separate responses by high affinity and low affinity agonist-receptor conformations. / McKinney, M.; Stenstrom, S.; Richelson, E.

In: Molecular Pharmacology, Vol. 27, No. 2, 1985, p. 223-235.

Research output: Contribution to journalArticle

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abstract = "Murine neuroblastoma cells (clone N1E-115) possess muscarinic receptors that mediate multiple responses, including the elevation of cyclic GMP levels and the inhibition of receptor-mediated increases in cyclic AMP. Evidence is presented showing that two muscarinic agonist-receptor conformations in N1E-115 cells each separately mediate a cyclic nucleotide response. Pirenzepine inhibited the [3H]cyclic GMP response to carbachol with a KD value of aproximately 6 nM, whereas it inhibited the ability of carbachol to reduce prostaglandin E1-mediated elevations in [3H]cyclic AMP levels with a KD value of 93 nM, thus differentiating between two classes of receptors involved in these responses. Ten muscarinic agonists were studied for their ability to mediate the two cyclic nucleotide responses. Six were as effective as acetylcholine in the reduction of [3H]cyclic AMP levels, but only two were as effective as acetylcholine in elevating [3H]cyclic GMP levels. Four agonists (arecoline, pilocarpine, oxotremorine, and McN-A343) were ineffective in increasing [3H]cyclic GMP levels. These four agonists and bethanecol, which could increase [3H]cyclic GMP levels only 18{\%} as well as acetylcholine, behaved as competitive antagonists in this response to carbachol. These partial agonists, in contrast to carbachol, bound to only one class of muscarinic sites in N1E-115 cells with equilibrium dissociation constants determined by competition binding assay which agreed well with their respective EC50 values for their effect on [3H]cyclic AMP levels. The equilibrium dissociation constants for the partial agonists determined by their inhibition of carbachol in the [3H]cyclic GMP response also agreed well with their respective EC50 values for mediating the [3H]cyclic AMP response. Thus, the partial agonists bound to the same receptors at which carbachol mediated [3H]cyclic GMP formation, but with KD values about the same as their respective EC50 values for inhibition of prostaglandin E1-mediated [3H]cyclic AMP increases. The full agonists acetylcholine and methacholine, like carbachol, bound to two sites in N1E-115 cells. For the six agonists able to stimulate both responses at least to some degree, the ratio of their potencies at each response correlated with their respective efficacies at each response but with much more dependence in the [3H]cyclic GMP response. It is proposed that the low affinity agonist-receptor conformation, inducible by full agonists, activates the effector for the cyclic GMP response and its formation is inhibited by pirenzepine with high potency; and the high affinity agonist-receptor conformation, inducible by both full and partial agonists, activates the effector for the cyclic AMP reduction and is inhibited by pirenzepine with lower potency.",
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