Muscarinic acetylcholine receptor in chick limb bud during morphogeneis

In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate ((3H)QNB), a muscarinic antagonist, to homogenates of chicken limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of (3H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 microM). Specific binding of (3H)QNB reflected a muscarinic receptor. The Kd in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol (3H)QNB/mg protein and 12.0 fmol (3H)QNB/mg protein, respectively. Data on displacement of specific bound (3H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the (3H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis.