Molecular Pharmacology, Vol 14, 768-780, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Biochemical Characterization of the Muscarinic Cholinergic Receptor in Human Brain: Alterations in Huntington’s Disease

¹ Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724

[³H]3-Quinuclidinyl benzilate was used to characterize the muscarinic cholinergic receptor binding in three regions of the normal human brain and in rat brain. In each of these tissues, specific ligand binding was linear between 0.05 and 0.7 mg tissue protein/assay at [³H]QNB concentrations between 20 and 200 pM. For the human brain regions, the bimolecular rate constant of association was 6.9 x 10⁸ M^-1 min^-1 and the dissociation rate constant was 14.3 x 10^-3 min^-1. The K_D calculated from the ratio of these two values was 20.1 pM. The kinetic K_D determined for rat brain, under these same conditions, was 25 pM. The dissociation rate constant and the kinetic K_D for both the human brain regions and the rat brain were similar whether dissociation was initiated before or after the ligand-receptor interaction had achieved equilibrium.

Scatchard analysis of saturation isotherms, using similar receptor concentrations for each of the human brain regions, gave K_D values of 60-70 pM with binding capacities between 600 and 900 fmol/mg protein. Analysis of saturation data from rat brain using similar receptor concentrations gave a K_D of 70 pM and a binding capacity of 900 fmol/mg protein.

[³H]Quinuclidinyl benzilate binding was inhibited by muscarinic agonists and antagonists; the antagonists were 1000-fold more potent than were the agonists. Under the specified conditions the antagonists and agonists had IC₅₀ values between 0.1-2 nM and 0.8-10 µM, respectively. The muscarinic antagonists had Hill coefficients of 1.0 and the agonists had Hill coefficients between 0.70 and 0.85. Nicotinic and noncholinergic drugs were ineffective in inhibiting [³H]quinuclidinyl benzilate binding at concentrations up to 10 µM.

Huntington’s diseased brains had significant decreases in muscarinic receptor binding in the caudate nucleus and putamen. Choline acetyltransferase activity was decreased significantly in the caudate nucleus, putamen and globus pallidus of Huntington’s diseased brains.

Saturation isotherms for [³H]quinuclidinyl benzilate binding were determined in each of the three control brain regions and in Huntington’s diseased putamen at different receptor concentrations. Each of these brain regions had a "true" K_D between 20 and 40 pM. These studies, plus inhibition studies using dexetimide, the biologically-active stereoisomer of benzetimide, revealed that the decreases in ligand binding were due to receptor loss rather than to changes in receptor affinity.

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				W. Eckelman, R. Reba, W. Rzeszotarski, R. Gibson, T Hill, B. Holman, T Budinger, J. Conklin, R Eng, and M. Grissom External imaging of cerebral muscarinic acetylcholine receptors Science, January 20, 1984; 223(4633): 291 - 293. [Abstract] [PDF]