The biphasic response of muscarinic cholinergic receptors in cultured heart cells to agonists. Effects on receptor number and affinity in intact cells and homogenates.

A biphasic time course of the agonist-mediated loss of muscarinic cholinergic receptors has been demonstrated in cultured chick embryo heart cells by radioligand binding studies using the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB). This agonist-mediated receptor loss was associated with decreased affinity of the receptor for agonist as judged by competitive binding of the agonist carbamylcholine with [3H]QNB to cell homogenates (Galper, J. B., and Smith, T. W. (1980) J. Biol. Chem. 255, 9571-9579). In the current studies the concentration dependence of agonist-mediated receptor loss was also found to be biphasic. The apparent shift of affinity following brief (15 min) agonist exposure coincided with the agonist-mediated loss of a subclass of high affinity receptors with an IC50 for carbamylcholine inhibition of [3H]QNB binding of 3.9 x 10(-7) M. Those receptors remaining constituted a subclass of low affinity receptors with IC50 = 8.2 x 10(-5) M. The data further suggest that an apparent decrease in agonist affinity after guanine nucleotide exposure represents conversion of high affinity receptors to a similar low affinity state, IC50 = 8.6 x 10(-5) M. The rapid loss of [3H]QNB binding sites in the presence of agonist did not require interaction of agonist with intact cells, but also occurred if cells were homogenized and then subjected to a brief (15 min) exposure to agonist. The slow loss over 3 h of [3H]QNB binding sites could only be demonstrated in intact cells incubated with agonist prior to homogenization. To probe further the later phase of agonist-mediated receptor loss, we developed new assay methods for determining muscarinic antagonist binding to intact cells. In control cells, binding of the hydrophobic antagonist [3H]QNB was quite similar in extent to binding of the more hydrophilic antagonist [3H]methylscopolamine ([3H]MS), with Kd values of 0.11 and 0.47 nM, respectively. Kinetic analysis of the binding of these two ligands was performed to determine whether they might distinguish between two states of the receptor. Both ligands bound to the receptor by a two step mechanism consistent with the formation of a low affinity complex followed by conversion to a high affinity complex. However, the ratio of reverse to forward rate constants of the second step of [3H]MS binding was roughly 100-fold greater than that for the more hydrophobic ligand [3H]QNB. Comparison of the time course of agonist-induced receptor loss as measured by binding of [3H]MS or [3H]QNB was consistent with muscarinic agonist mediation of a stepwise alteration in receptor configuration from a form that bound both [3H]MS and [3H]QNB to a form that bound only [3H]QNB and thence to a form that bound neither [3H]MS nor [3H]QNB. The relationship of such a sequential mechanism to agonist-induced changes in the relationship of the receptor to the cell membrane and agonist-induced endocytosis of the receptor is discussed.