Muscarinic receptors in porcine caudate nucleus. I. Enhancement by nickel and other cations of [3H]cis-methyldioxolane binding to guanyl nucleotide-sensitive sites

T Nukada, T Haga and A Ichiyama

Muscarinic acetylcholine receptors in the synaptic membrane fraction of porcine caudate nucleus were characterized by using a radiolabeled agonist, [3H]cis-methyldioxolane [( 3H]CD) and an antagonist, [3H]quinuclidinyl benzilate [( 3H]QNB). Scatchard analysis of the specific binding of [3H]CD gave a single equilibrium dissociation constant of 8.1 nM when a concentration of less than 80 nM [3H]CD was used. The binding capacity was 390 fmoles/mg of protein and corresponded to about 10% of the binding sites of [3H]QNB. Agonist/[3H]CD competition binding experiments indicated that [3H]CD was selectively bound to the sites with a high affinity for agonists. [3H]CD binding was inhibited by Na+, K+, Mg2+, and Ca2+ with the half- maximal effect at 10-50 mM. Nickel ion showed biphasic effects on [3H]CD binding: a 2- to 3-fold enhancement of binding at 0.1-10 mM and inhibition above 10 mM. Other cations, including Co2+, Mn2+, and Zn2+, at 1 mM also increased [3H]CD binding by a factor of 1.5-1.8. Among 18 cations examined, only Cd2+, Hg2+, and Cu2+ caused significant inhibition of [3H]CD binding at 1 mM. [3H]CD binding was decreased to about 20% of the control value in the presence of guanylyl-5'- imidodiphosphate (GppNHp), GTP, and GDP with the half-maximal effect at 1.3, 32, and 45 microM, respectively. [3H]CD binding in the presence of Ni2+ was decreased by GppNHp to a level obtained in the presence of GppNHp alone. The increase caused by Ni2+ in [3H]CD binding was due to the increase in the maximal binding capacity (Bmax) without changes in the affinity for [3H]CD. We conclude that Ni2+ increases the proportion of a muscarinic receptor subclass (or state) that is sensitive to guanyl nucleotide.

Volume 24, Issue 3, pp. 366-373, 11/01/1983
Copyright © 1983 by American Society for Pharmacology and Experimental Therapeutics

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