Muscarinic Acetylcholine Receptors in Developing Chick Heart*

The chronotropic response of embryonic chick hearts to cholinergic agents is first clearly detectable on day 5 in ouo and full responsiveness develops by day 7. Muscarinic acetylcholine receptors in fully responsive 9day-old hearts specifically bind the muscarinic antagonist quinuclidinyl benzilate. Binding is blocked effectively by muscarinic agonists and antagonists but is poorly inhibited by the nicotinic antagonist o-tubocurarine. Hill coefficients for the three antagonists studied were approximately 1.0, but for the two agonists studied ranged from 0.4 to 0.7. Kinetic analysis of the binding of quinuclidinyl benzilate by muscarinic receptors showed that the reaction proceeds by a two-step mechanism. Initially, a rapidly reversible complex with K, = 0.7 to 1.0 nM is formed. After additional incubation, this complex is transformed to a slowly reversible complex. Despite the fact that hearts from embryos younger than 7 days in ouo have diminished physiologic response to cholinergic drugs, hearts from 3to 1%day-old embryos contain similar numbers of receptors per mg of protein (165 to 230 fmol/mg of protein). Thus, there are physiologically inactive receptors in young embryonic hearts. Studies of ligand binding by muscarinic receptors in unresponsive 3-day-old hearts revealed extensive similarity to physiologically responsive g-day-old hearts. The only difference detected was an increase in the initial inhibition of quinuclidinyl benzilate binding by agonists in g-day-old hearts. This small difference is insufficient to account for the inactivity of receptors. Other events in the physiologic response occurring subsequent to ligand binding may be more dramatically altered in unresponsive hearts, or young embryonic hearts may contain hidden or intracellular forms of the muscarinic receptor that are accessible to muscarinic agents in homogenates but not in intact tissue.