Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: Adenylate cyclase and phosphatidylinositol turnover

K. Akiyama, T. W. Vickroy, M. Watson, William R Roeske, T. D. Reisine, T. L. Smith, H. I. Yamamura

Research output: Contribution to journalArticle

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Abstract

(-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mose pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 x 108 M-1 min-1 and 6.8 x 10-3 min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (K(d)) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 μM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or K(i) = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (K(i) = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a K(i) value of 72 nM. In contrast, atropine was nearly equipotent (K(i) = 0.3-0.5 nM) in binding studies and in both effector systems.

Original languageEnglish (US)
Pages (from-to)653-661
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume236
Issue number3
StatePublished - 1986
Externally publishedYes

Fingerprint

Pituitary Neoplasms
Carbachol
Quinuclidinyl Benzilate
Phosphatidylinositols
Adenylyl Cyclases
Cholinergic Agents
Dexetimide
Ligands
Pirenzepine
Cyclic AMP
Muscarinic Antagonists
Time and Motion Studies
Muscarinic Receptors
Atropine
Isoproterenol
Population
Inhibition (Psychology)

ASJC Scopus subject areas

  • Pharmacology

Cite this

Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors : Adenylate cyclase and phosphatidylinositol turnover. / Akiyama, K.; Vickroy, T. W.; Watson, M.; Roeske, William R; Reisine, T. D.; Smith, T. L.; Yamamura, H. I.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 236, No. 3, 1986, p. 653-661.

Research output: Contribution to journalArticle

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abstract = "(-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mose pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85{\%}). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 x 108 M-1 min-1 and 6.8 x 10-3 min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (K(d)) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 μM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or K(i) = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (K(i) = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a K(i) value of 72 nM. In contrast, atropine was nearly equipotent (K(i) = 0.3-0.5 nM) in binding studies and in both effector systems.",
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AU - Vickroy, T. W.

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AU - Roeske, William R

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AU - Smith, T. L.

AU - Yamamura, H. I.

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N2 - (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mose pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 x 108 M-1 min-1 and 6.8 x 10-3 min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (K(d)) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 μM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or K(i) = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (K(i) = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a K(i) value of 72 nM. In contrast, atropine was nearly equipotent (K(i) = 0.3-0.5 nM) in binding studies and in both effector systems.

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