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The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic antagonist [3H]methylscopolamine ([3H]MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 d in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist carbamylcholine were the responses studied. Exposure to 10(-3) M carbamylcholine caused a 15% decrease in beating rate and a 33% increase in the rate of 42K+ efflux from cells labeled to equilibrium. An assay for binding of [3H]MS to intact cells was developed. [3H]MS bound specifically to intact heart cells (185 fmol/mg protein) with a Kd of 0.48 nM. Exposure of cells for various times to 10(-3) M carbamylcholine followed by binding of [3H]MS to intact cells demonstrated that a gradual loss of 70% of [3H]MS binding sites took place over the next 6 h with a T 1/2 of 30 min. A decrease in the ability of carbamylcholine to stimulate K+ efflux and to decrease beating rate was observed after pre-exposure of cells to muscarinic agonists. A close correlation was found between the loss of the subclass of muscarinic receptors subject to agonist control and the loss of physiologic responsiveness after agonist exposure. The data suggest the absence of significant numbers of "spare" receptors within this group.

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.

Prior to ingrowth of the vagus nerve (4-5 days in ovo), embryonic chick hearts are relatively unresponsive to muscarinic stimulation ( Pappano , A. J. (1977) Pharmacol. Rev. 29, 3-33). We studied the correlation between the development of physiologic responsiveness in the embryonic chick heart and changes in the properties of muscarinic receptors. In cultures from hearts 10 days in ovo, muscarinic agonists decreased beating rate by 15% and increased the rate of K+ efflux by 35%. In cultures of embryonic hearts 3 1/2 days in ovo, muscarinic receptors had no effect on beating rate and mediated only an 11% increase in the rate of K+ efflux. We previously demonstrated that in cells cultured from hearts 10 days in ovo, 26% of receptors bound agonist with a high affinity (RH) and that incubation with guanine nucleotides mediated the conversion of RH to a low affinity form (RL ( Galper , J. B., Dziekan , L. C., O' Hara , D. S., and Smith, T. W. (1982) J. Biol. Chem. 237, 10344-10356]. In cultures of hearts 3 1/2 days in ovo, RH constituted 52% of total [3H] quinuclidinyl benzilate-binding sites, and guanine nucleotides had no effect on the conversion of RH to RL. Growth of cells cultured from hearts 3 1/2 days in ovo in medium supplemented with specific lots of serum resulted in a 3-fold increase (from 11 to 30%) in the ability of muscarinic agonists to increase K+ permeability. This increased sensitivity to muscarinic stimulation was accompanied by a 20% increase in RH and sensitivity of 75% of RH to guanine nucleotides. Thus, enhanced number of RH and development of guanine nucleotide responsiveness are associated with the development of a physiologic response. The relationship of these developmental changes to the appearance of a guanine nucleotide regulatory protein is discussed.

Na+ channels in chick brain and heart have been directly compared by measuring binding of tritium-labeled saxitoxin ([3H]STX) to the two tissues under identical conditions. Maximum saturable uptake and toxin affinity were considerably less in chick heart than in chick brain, requiring the development of an assay method to resolve specific [3H]STX uptake in heart. With this method, binding to both preparations consisted of a specific saturable component and a linear nonspecific component. The equilibrium dissociation constant for [3H]STX measured in chick heart (6.2-8.8 nM) was 20-30 times higher than that measured in chick brain (0.3 nM). The dissociation rate for [3H]STX was only about twice as fast in heart as it was in brain, indicating that the decrease in toxin affinity in heart results predominantly from a slowed toxin association rate. The decreased affinity for [3H]STX found at the chick heart Na+ channel is compared with toxin-resistant Na+ channels in other preparations. The existence of two Na+ channel subtypes is proposed, with high affinity and low affinity for saxitoxin and tetrodotoxin; the significance of this classification is discussed.