The specific muscarinic ligand [3H]quinuclidinyl benzilate ([3H]QNB) was used to label acetylcholine receptors in the submandibular gland of the rat. Specific binding of [3H]QNB increased linearly with tissue concentration in the range of 0.02-0.3 mg of protein/ml. Kinetic analysis of [3H]QNB binding revealed the presence of a single population of high affinity binding sites, with a dissociation constant of 87.2 pM and a Hill coefficient of 0.95. The binding was saturable and the receptor density was 214 fmol/mg of protein. The rate constants at 37 degrees C for association and dissociation of the [3H]QNB-receptor complex were 5.98 X 10(-8) M-1 X min-1 and 6.6 X 10(-3) X min-1, respectively. The ratio k-1/k+1 gave a Kd value of 11.1 pM, similar to the Kd value (13.1 pM) determined by kinetic parameters when extrapolated at infinitely low receptor concentration. Muscarinic antagonists displaced [3H]QNB from muscarinic receptors with a Hill coefficient near to 1.0. Displacement curves for muscarinic agonists and for the atypical antagonist pirenzepine had Hill values significantly less than one. In the presence of 0.1 mM GPP(NH)P, the potency of agonists but not antagonists in displacing [3H]QNB binding decreased 2 to 3-fold. The [3H]QNB binding site was sensitive to the inhibitory effect of various sulfhydryl reagents. Repeated treatments of rats with an acetylcholinesterase inhibitor led to a decreased density of muscarinic receptors in the submandibular gland. This alteration was specific for the muscarinic recognition site and was paralleled by a reduced sensitivity to carbachol.
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