Agonist and Guanine Nucleotide Modulation of Muscarinic Cholinergic Receptors in Cultured Heart Cells*

Agonist modulation of muscarinic cholinergic recep- tor properties was studied in cultured chick embryo heart cells. Exposure of cultured heart cells to musca- rinic agonists caused a biphasic decrease in the number of muscarinic receptors as measured by binding of the potent muscarinic antagonist ['H]quinuclidinyl benzil-ate (['HJQNB) to homogenates of these cultures. A rapid loss of 26% of receptors occurred during the first minute of exposure to agonist, foliowed by a gradual loss of another 44% of receptors by 3 h. Changes in the extent of [3H]QNB binding after a 3-h exposure to agonist were not the result of agonist-induced changes in affinity or rate of ['HJQNB binding to the receptor since ['HIQNB bound with a kd of 0.21 x lo-' M and a half-time to equilibrium of 13.0 min to homogenates of control cells compared to a kd of 0.18 X lo-' M and half-time to equilibrium of 13.0 min in homogenates of agonist- treated cells. However, studies of the rapid phase of agonist-induced receptor loss indicated that: 1) this phase was accompanied by a shift in the affinity of the remaining receptors for agonist from an IC50 for car- bamylcholine inhibition of ['H]QNB binding of 1.5 X M to 9.2 X M; 2) exposure of cell homogenates to M guanosine 5'-@,y-imino)triphosphate caused a similar shift in affinity unaccompanied by alteration in receptor number; 3) incubation with guanine nucleo- tides caused the reappearance of [3H]QNB-binding sites lost during the first minute of agonist exposure; and 4) incubation with colchicine had no effect on the rapid phase of receptor loss. Studies of the slow phase of agonist-induced receptor loss indicated that: 1) inhibitors of microtubule function including colchicine and vinblastine inhibited up to two-thirds of agonist-in-duced receptor loss with half-maximal effects at 1.4 x lo-' M and 8.0 X 10" M, respectively; 2) recovery of receptors following washout of agonist was preceded by a 3-h lag period after which receptor number re- turned to 95% of control levels over 9 h; and 3) the protein synthesis inhibitor cycloheximide (2 pg/ml) prevented recovery. These data indicate a complex set of interactions of muscarinic agonists, guanine nucleo- tides, and cytokinetic events in the modulation of muscarinic receptor activity. comparison at various times after initiation of UV exposure. The conversion to /? and known spectra (19). y lumicolchicines was nearly complete after 90 min as compared to

Agonist modulation of muscarinic cholinergic receptor properties was studied in cultured chick embryo heart cells. Exposure of cultured heart cells to muscarinic agonists caused a biphasic decrease in the number of muscarinic receptors as measured by binding of the potent muscarinic antagonist ['H]quinuclidinyl benzilate (['HJQNB) to homogenates of these cultures. A rapid loss of 26% of receptors occurred during the first minute of exposure to agonist, foliowed by a gradual loss of incubation with colchicine had no effect on the rapid phase of receptor loss. Studies of the slow phase of agonist-induced receptor loss indicated that: 1) inhibitors of microtubule function including colchicine and vinblastine inhibited up to two-thirds of agonist-induced receptor loss with half-maximal effects at 1.4 x lo-' M and 8.0 X 10" M, respectively; 2) recovery of receptors following washout of agonist was preceded by a 3-h lag period after which receptor number returned to 95% of control levels over 9 h; and 3) the protein synthesis inhibitor cycloheximide (2 pg/ml) prevented recovery. These data indicate a complex set of interactions of muscarinic agonists, guanine nucleotides, and cytokinetic events in the modulation of muscarinic receptor activity.
Heart, Lung and Blood Institute, National Institutes of Health Grants * This study was supported by research grants from the National HL-22775 and HL-18003, American Heart Association Grant 79875, and an award from the William F. Milton Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Recipient of Young Investigator Award, National Heart, Lung and Blood Institute, National Institutes of Health.
The role of agonists in modulating the number and function of cell surface receptors and the relationship of this modulation to the dynamic nature of the cell membrane has been described for several systems (1). Incubation of cultured human lymphocytes with insulin has been shown to cause up to a 70% decrease in the binding of 1251-labeled insulin to the cells, while the affinity of the remaining receptors for insulin was unchanged (2). The number of sites for binding of Iz5Ilabeled human epidermal growth factor in human fibroblasts has been shown to decrease after treatment of cells with epidermal growth factor. The data suggest that endocytosis of the receptor.growth factor complex, with subsequent lysosomal degradation of both the hormone and receptor within the cell, may be responsible for this process (3). The activity of P-adrenergic receptors in avian and frog erythrocytes (4)(5)(6) and astrocytoma cells (7,8) is modulated by P-agonists as well as guanine nucleotides. In astrocytoma cells, short term incubation with /3-agonists is followed by uncoupling of hormone binding from the adenylate cyclase response, while more prolonged incubation is followed by an actual decrease in receptor number (5). Studies utilizing radiolabeled P-adrenergic agonists suggest that persistent binding of agonist and agonist-induced modification of the receptor might be responsible for these effects (9,10).
Our previous studies of the effects of carbamylcholine in modulating the responsiveness of the explanted embryonic chicken heart to this muscarinic agonist demonstrated a rapid but transient decrease in the frequency (11) and force' of contraction with return to base-line within 3 min. Earlier studies have been interpreted as suggesting that muscarinic cholinergic effects are mediated by increased intracellular cGMP levels and that rapid desensitization occurs because of the transient nature of the cGMP elevation (12). Other possible mechanisms can now be probed because of the availability of the potent radiolabeled muscarinic antagonist [3H]quinuclidinyl benzilate, allowing more direct studies of the interaction of agonists and antagonists with the muscarinic receptor. Studies utilizing [3H]QNB' (13) have defined the properties of ['HIQNB binding to muscarinic receptors in embryonic chicken heart (ll), rabbit, rat, and guinea pig heart (14), and in monolayer cultures of embryonic chicken heart cells (15).
Using embryonic chicken heart cell cultures, we have demonstrated that prolonged exposure to muscarinic agonists (up to 3 h) was accompanied by marked decreases (65 to 70%) in both the number of muscarinic receptors and in the physiological responsiveness of these cells to muscarinic agonists (15). The present investigation deals with the effect of both short and long term exposure of muscarinic receptors of cultured heart cells to muscarinic agonists. The time course of changes in receptor number and receptor properties was studied, together with the effect of guanine nucleotides and inhibitors of endocytosis on these agonist-mediated events.

EXPERIMENTAL PROCEDURES
MateriaZs-Chemicals were obtained from the following sources: carbamylcholine chloride, vinblastine sulfate, 8 Br-adenosine 3':5'cyclic monophosphoric acid, N2,02-dibutyryl guanosine 3':5"cyclic monophosphoric acid, cycloheximide, guanosine 3':5"cyclic monophosphoric acid, GMP, and GTP from Sigma; podophyllotoxin, cytochalasin B, colchicine, and oxotremorine from Aldrich Chemical Co., guanosine 5"(/?,y-imino)triphosphate tetrasodium salt from ICN Pharmaceuticals; medium "199 from Microbiological Associates; 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer from Calbiochem; Heart Cell CuZtures-Heart cell cultures were prepared by a modification of the method of DeHaan (16) as described (17) except that Ca2+-Mg2'-free Hanks' balanced salt solution was used in place of Pucks saline G. Hearts were removed, minced, and incubated with 0.02576 (w/v) trypsin in Ca2'-Mg2'-free Hanks' balanced salt solution at 37°C for 8 min. The trypsin solution was removed and diluted into medium "199 containing 50% heat-inactivated horse serum at room temperature. After successive trypsinizations, suspensions of trypsinized cells were sedimented at IO00 rpm in a desk-top centrifuge, resuspended in growth medium, and incubated in a 100-mm Petri dish (Falcon) for 45 min at 37°C in a humidifed atmosphere of 5% C02/95% air. During this incubation, nearly 95% of the fibroblasts in the suspension adhered to the dish. The heart cells then were plated at a density of 2.0 X lo4 cells per cm2 on collagen-coated 100-mm Petri dishes. On the third day of incubation, the medium was changed. Cells were harvested on culture day 4 unless otherwise indicated.
Measurements of r3H]QNB Binding to Homogenates-The assay procedure was a modification of the method of Yamamura and Snyder (11,13). After three rinses with ice-cold "199 Hepes, cells were harvested in a small volume of M-I99/Hepes. After freezing and thawing twice at -70°C, the cells were homogenized in a glass on glass homogenizer and allowed to warm to 22°C prior to assay.
The final assay mixture was 0.5 ml of Hepes-buffered "199 containing [3H]QNB (28 Ci/mmol, 1 nM in [3H]QNB unless otherwise specified), drugs as indicated in the figure legends, and 0.5 ml of homogenate. At the appropriate time (1 h at 22°C unless otherwise stated), 5 ml of wash medium (120 nm NaC1, 5.4 mM KCl, 0.8 mM MgSO4, 1.8 m CaC12, 50 m Hepes, 1.0 nm NaH2PO4, adjusted to pH 7.4 with NaOH) at 22°C was added to terminate the incubation. The reaction mixture was passed through a Whatman glass fiber (GF/C) fiter, the assay tube washed three times, and filters were dried and assayed for radioactivity in a liquid scintillation counter with 10 ml of Instagel (Packard). Protein was determined by a modification of the procedure of Lowry et al. (18) 16,700) and 244 nm ( E 30,000) (19). For conversion of colchicine to lumicolchicine, a 33 pM solution of colchicine in 9576 ethanol, which gave an optical density reading of 1.0 at 244 n m , was exposed to a 366-nm source in a Chromata-Vu black box, Blacklight Eastern Corporation, Port Washington, NY, in order to avoid extraneous light. Conversion to lumicolchicine was followed by comparison of the ultraviolet absorption spectrum of an aliquot determined on a Carey recording spectrophotometer at various times after initiation of UV exposure. The conversion to /? and known spectra (19). y lumicolchicines was nearly complete after 90 min as compared to

Effects of Agonist Exposure on Binding of [3H/QNB to
Muscarinic Receptors-We have demonstrated previously that exposure of intact cultured chick heart cells for 3 h to M carbamylcholine caused a 65 to 70% decrease in muscarinic receptor number as measured by the binding of the potent muscarinic antagonist ['HIQNB to homogenates of these cells (15). Such an agonist-induced decrease in ["HIQNB binding might reflect a decrease in the number of receptors available for binding. Alternatively, either a decreased affinity of the receptor for [3H]QNB or a substantial decrease in the rate of approach of [3H]QNB binding to equilibrium could explain an apparent decrease in [3H]QNB binding.
In order to distinguish among these mechanisms, an experiment comparing the binding of [3H]QNB to homogenates of intact cells which had been exposed to agonists and to homogenates of control cells is illustrated in Fig. 1. The specific binding was saturable. Scatchard analysis of the data gave two nearly parallel straight lines for carbamylcholine-treated and control cells corresponding to K d values of 0.18 k 0.03 (S.E.) and 0.21 k 0.04 (n = 3) nM, respectively, intersecting the x axis at 78 and 194 fmol/mg of protein (Fig. 1). Hence the data are consistent with a 60% carbamylcholine-mediated decrease in r3H]QNB binding with no significant alteration in affinity for 13H]QNB.
In order to determine whether carbamylcholine treatment affected the time course of [3H]QNB binding, a kinetic analysis of QNB binding to homogenates of control cells and homogenates of intact cells treated with carbamylcholine was performed. The rate of binding of [3H]QNB at a concentration of 1 nM is illustrated in Fig. 2. In homogenates of both control and carbamylcholine-pretreated cells, binding proceeded without a lag and reached 50% saturation at 13.2 -+-0.7 (S.E) and 12.0 k 0.8 min (n = 3), respectively, with saturation levels  reflecting a 63% decrease in receptor number after agonist exposure. Previous studies have demonstrated that the binding of [3H]QNB to whole heart homogenates and homogenates of cultured heart cells is a biphasic process (11,15). When the data in Fig

Relationship of Relative Pharmacologic Potency of Muscarinic Agonists to Effectiveness in Mediating a Decrease in
Receptor Number-In order to test the hypothesis that agonist modulation of receptor number involves specific binding of agonists to muscarinic receptors, the relative potency of muscarinic agonists in mediating a decrease in muscarinic receptor number was compared to their relative pharmacologic potency and to their relative ability to compete with [3H]QNB for receptor binding. Intact cells were exposed for 3 h to various concentrations of the muscarinic agonists carbamylcholine and oxotremorine. As shown in Fig. 3, left panel, both agonists caused a maximum decrease of 70% in specific [3H]QNB binding to homogenates of the intact cells. Oxotremorine and carbamylcholine exerted half-maximal effects of 35% at 1.0 X M and 0.8 X M, respectively. Analysis of these curves by the procedure of Brown and Hill (20) showed that oxotremorine and carbamylcholine have Hill coefficients of less than 1.0 (0.53 and 0.48, respectively; Fig. 3

, rightpanel).
These results suggest that the agonist binding reaction that mediates the apparent decrease in receptor number occurs either a t multiple classes of sites or a t a single class of sites exhibiting negatively cooperative site-site interactions. Similarly, the ICm'' values for oxotremorine and carbamylcholine ' Although studies of antagonist competition with [3H]QNB binding allow calculation of K, for antagonists assuming a simple competitive interaction between ['HIQNB and the competing ligand, the Hill coefficients for agonists are less than one, and a simple competitive inhibition of ['HIQNB binding by agonists cannot be assumed. Hence we refer to an IC, for the agonist which is specific for the experimental conditions described. with Hill coefficients of 0.58 and 0.55 (15). The marked similarities among the data for the relative ability of these agonists to compete with [3H]QNB for receptor binding, their relative potencies in inducing decreases in receptor number (Fig. 3), and their relative pharmacologic potencies indicate that agonist modulation of receptor number involves specific binding to muscarinic receptors.

Effect of Nucleotides on Agonist-mediated Decreases in Antagonist Binding to Muscarinic Receptors-It has been
demonstrated in a number of systems including the fl-adrenergic receptors of frog erythrocyte membranes that following an agonist-mediated decrease in receptor-binding sites, the number of receptors can be restored to near-control levels by incubation with GTP or with Gpp(NH)p, a nonhydrolyzable GTP analogue (21). To determine the effects of nucleotides on agonist modulation of muscarinic receptor number after prolonged agonist exposure, we exposed cultured heart cells to

Effect of Prior Exposure to Agonist on the Properties of
Agonist Binding to Muscarinic Receptors-Although the affinity of the receptor for antagonist and the kinetics of binding of antagonist appear to be unaltered by agonist exposure, the binding properties of agonists might reflect more subtle changes in the receptor than the less specific binding of antagonists. Therefore, the affinity of receptor for agonist in control cells and in cells exposed to lo-' M carbamylcholine for 3 h was estimated by determining the ability of agonists to compete with ['HIQNB binding to homogenates of control and agonist-treated cells.
The experiment summarized in Fig. 4   Having shown (Fig. 4) that the apparent affinity of receptors for agonist was decreased 6-fold after a 3-h exposure of cells to agonist, we studied the time course over which agonistinduced changes in affinity took place. As summarized in (data not shown). No change in K d for antagonist could be detected after such a brief exposure to agonist. This shift in affinity for agonist was not reversible after washout of agonist and incubation for 3 h in fresh medium (Fig. 6). Thus, both the loss of 26% of [3H]QNB-binding sites and the decrease in apparent affinity of the remaining receptors occur during the first minute of agonist exposure and are not reversible with incubation in fresh medium without agonist for 3 h.
The Effect of Guanine Nucleotides on Binding of Agonists to Muscarinic Receptors-Guanine nucleotides have been shown to decrease the affinity of agonists for the &adrenergic receptor and in homogenates to modulate the release of bound agonist from the receptor (10,22). It has also been shown that GTP and Gpp(NH)p are capable of lowering the apparent affinity of carbamylcholine for the muscarinic receptor in homogenates of rat heart (23,24). The ICra for carbamylcholine displacement of ["HIQNB binding in homogenates of heart cell cultures increased from 1.5 f 0.2 X M (n = 15) to 8.7 & 1.5 X M (n = 4) with the addition of 0.1 mM Gpp(NH)p (Fig. 6). This shift was similar to that seen in homogenates of intact cells exposed for 1 to 15 min to carbamylcholine ( Fig. 6 and Table I). However, unlike the effect of exposure to carbamylcholine, no effect of Gpp(NH)p on total number of [3H]QNB-binding sites could be demonstrated. Incubation with GTP and GDP also increased the concentration of carbamylcholine required to inhibit 50% of ["HIQNB binding while GMP and cGMP had no significant effect on carbamylcholine binding (Table I).
If the loss of 26% of ['HIQNB-binding sites after brief exposure of cultured heart cells to muscarinic agonists represents persistent binding of agonist to the receptor, and if muscarinic receptors are subject to regulation by guanine nucleotides, treatment with guanine nucleotides of homoge-    (Fig. 6). However, in contrast to results obtained after 3 h of agonist exposure (see above), r3H]QNB-binding sites lost during brief agonist exposure recovered fully from 26.3 f 2.1% (n = 9) below base-line levels to a mean only 3.2 f 2.5% (n = 6) below control in homogenates treated with Gpp(NH)p. These data are consistent with the hypothesis that muscarinic receptors are subject to regulation at a guanine nucleotide regulatory site and suggest release of persistently bound agonist during guanine nucleotide-mediated conversion of a high affinity receptor to a low affinity form.
As described previously in the case of homogenates of intact cells exposed to agonist for 3 h, incubation with guanine nucleotides had no effect on recovery of even a fraction of the 70% of r3H)QNB-binding sites lost. It would appear that after a 3-h exposure to agonist the subclass of receptors lost after a 15-min exposure of intact cells to agonist will no longer recover if homogenized and incubated in the presence of guanine nucleotides. These data may be explained by a loss of guanine nucleotide sensitivity during prolonged agonist exposure. Alternatively, if prolonged exposure to agonist results in inclusion of these receptors into closed vesicles during their removal from the cell surface membrane, such receptors might become inaccessible to guanine nucleotides.
Time Course of Recovery of Receptors After Agonist Exposure-As discussed previously, the decrease in muscarinic receptor number after exposure to agonist is a biphasic process which may involve more than a single class of receptors with a rapid early phase reversible by guanine nucleotides followed by a more gradual later phase. To determine whether the recovery of these receptors after removal of agonist followed the same or separate pathways, we examined the recovery of receptors after a 15-min or 3-h agonist exposure. In the experiments shown in Fig. 7, cells incubated for 3 h with agonist were washed and incubated with fresh medium. A 3-h lag period followed during which no recovery of receptors could be detected. Recovery then proceeded to 95% of initial levels, with 50% recovery at 7.3 h (Fig. 7). In some experiments recovery was greater than 100%, reaching 120 to 130% of initial levels. Fig. 7 also shows that in the presence of cycloheximide (2 pg/ml), no recovery of [3H]QNB binding occurred for up to 16 h. This concentration of cycloheximide was capable of inhibiting up to 98% of protein synthesis in these cultures as measured by inhibition of [3H]leucine incorporation into trichloroacetic acid-precipitable protein. However, cycloheximide had no effect on the time course of agonist-induced disappearance of r3H]QNB-binding sites, which proceeded with a time course indistinguishable from that shown in Fig.  5 in the presence of cycloheximide. In all of these recovery experiments, cells were used on the fourth day of culture at which time there is little cell division as judged by [3H]thymidine incorporation (25) and repeated cell count^.^ We conclude that the rate of recovery of receptors is too rapid to be explained merely by cell division with appearance of new receptor-containing membrane.
In order to study the recovery of that subgroup of 26% of receptors lost rapidly during the early phase of agonist exposure, cells were incubated for 15 min in M carbamylcholine, washed, and incubated in fresh medium. As shown in Fig.  6, these [3H]QNB-binding sites did not recover during a 3-h incubation in fresh medium. Unlike the recovery of receptors lost after a 3-h incubation with agonist, however, further incubation for up to 12 h demonstrated no significant recovery. Thus, recovery of this class of rapidly lost receptors was significantly slower than recovery of receptors lost after a 3-h incubation with agonist. However, data in Fig. 7 indicate that after a 3-h incubation with agonist, receptors recover to control levels after 12 h in fresh medium, suggesting that all classes of receptors recover during this period. One possible explanation of these findings is that a 3-h exposure to agonist alters the subgroup of receptors lost during the first 15 min of exposure to agonist, rendering these receptors unresponsive to guanine nucleotides and shortening their prolonged recovery period to approximate the recovery kinetics shown in Fig.  7.
Effect of Inhibitors of Endocytosis on Agonist-induced Loss of Receptor Number-The preceding data are consistent with loss of ["HIQNB-binding sites either due to irreversible alteration of the properties of the receptor by agonist exposure or actual agonist-induced removal of the receptor from the cell surface membrane. Both of these mechanisms are consistent with a requirement for synthesis of protein for recovery of [3H]QNB-binding capacity. Endocytosis and subsequent lysosomal degradation of the receptor has been demonstrated for turnover of other cell surface receptors (26) and could account for these findings. In order to determine whether inhibitors of cytokinesis interfered with agonist-mediated changes in receptor number, cells were incubated for varying periods of time with colchicine after which incubation was continued for 3 h in fresh medium containing colchicine plus carbamylcholine (Fig. 8). In this experiment, incubation of cells for 3 h with carbamylcholine alone produced a 60% decrease in the number of [3H]QNB-binding sites. The presence of colchicine alone for 3 h had no significant effect on [3H]QNB binding (data not shown). However, the presence of colchicine during a 3-h incubation with carbamylcholine reduced the receptor loss due to carbamylcholine from the control value in this experiment of 60% to 53% at 1O"j M colchicine, to 48% at M colchicine, and to 40% at M colchicine (zero time points, Fig. 8). Treatment of cells with colchicine alone for varying times prior to a 3-h incubation with carbamylcholine plus colchicine produced progressively more marked inhibition of the effects of carbamylcholine on [3H]QNB binding with a maximum reduction in receptor loss to 20% after a 45-min preincubation with M colchicine (Fig. 8).
Morphological changes observed by phase contact microscopy during incubation with colchicine ranged from ruffling of the cell membrane at to M to loss of well defined cell boundaries at higher concentrations. Cells continued to contract normally at rates of 120 k 15 beats per min through- out the experiment at M colchicine, but after 2 h at M colchicine no further contractions could be discerned.
To determine the concentration dependence and specificity of this effect of colchicine on the carbamylcholine-mediated decrease in receptor number, several agents known to interact with microtubules or microfilaments were studied. These included vinblastine, a vinca alkaloid that binds to tubulin causing precipitation of an alkaloid. tubulin complex (27), podophyllotoxin, a competitive inhibitor of colchicine with an affinity for tubulin twice that of colchicine (27), cytochalasin B, an inhibitor of microfilament function (28), and lumicolchicine, a photochemical product of colchicine that does not interact with microtubules (27). Results of these experiments are summarized in Fig. 9. Like colchicine, agents known to interact with microtubules decreased the carbamylcholineinduced loss of receptor number in a concentration-dependent manner. Colchicine exerted a half-maximal effect at 1.4 X M (Fig. 9), vinblastine at 2.0 X M, and podophyllotoxin at 8.0 x M. Cytochalasin B and lumicholchicine had negligible effects even at M (Fig. 9). The effect of colchicine reached a maximum at a concentra-   tion of M, at which point exposure to carbamylcholine was still capable of effecting a loss of 24% of r3H]QNB-binding sites. One possible explanation of this finding is that a subset of receptor sites is subject to modulation by agonist by a mechanism not involving microtubule function. The 26% of receptor sites lost rapidly after exposure of cells to carbamylcholine might represent such a class of receptors. As shown in Table 11, a 15-min exposure to carbamylcholine decreased receptor number by about 30%. In the presence of colchicine, a 15-min exposure to carbamylcholine still induced a 27% loss of receptors. Hence two classes of receptors could be demonstrated based on the ability of colchicine to inhibit their regulation by a g~n i s t .~

DISCUSSION
The studies reported here address mechanisms by which muscarinic agonists, guanine nucleotides, and cytokinetic mechanisms interact with the muscarinic receptor to mediate and control muscarinic cholinergic activity. Our findings indicate that both pharmacologic action and agonist modulation of receptor number are mediated through specific binding of agonist to muscarinic receptors, suggesting that activation and regulation of receptors may share common steps. Since the It is important to note that the effect of colchicine and vinblastine varied somewhat in our experiments depending on the flock of hens from which eggs were taken for culture of embryonic heart cells. Flock MR 56 used in experiments prior to those reported here exhibited a maximum colchicine inhibition of agonist effect on receptor number of only 40 to 50% rather than the 66% value found with cultures from flock MR 58 reported here. One possible explanation for these findings is that the relative number of receptors subject to rapid, colchicine-insensitive agonist control varies with the age of the laying hen and/or with the genotype of the flock. affinity of receptors for r3H]QNB and the kinetics of r3H]QNB binding were the same in control and agonist-treated cells, the agonist-induced decrease in the number of r3H]QNB-binding sites presumably reflects a decrease in the number of receptor sites available to interact with this potent antagonist. Although exposure of cells to muscarinic agonists did not affect a f f i t y or kinetics of antagonist binding, a more subtle and potentially important agonist-induced effect was the significant decrease in the apparent affinity of the receptor for agonist (Fig. 6).
The regulation of receptor number and receptor affinity by agonist was separable temporally into two distinct phases, shown schematically in the upper portion of Fig. 10. As early as 1 min after addition of agonist, a 26% decrease in [3H]QNBbinding sites occurred, together with a concomitant decrease in the apparent affinity of all remaining receptors for agonist.
With more prolonged agonist exposure, another 4 4 % of r3H]QNB-binding sites were lost over 2% h. We also found that guanine nucleotides decreased the affinity of muscarinic receptors for agonists in homogenates of control cells to the same extent as a brief exposure to agonist. Furthermore, guanine nucleotides were able to facilitate the recovery of the [3H]QNB-binding sites lost during the early phase of exposure to agonists.
These findings suggest that muscarinic receptors in cultured heart cells may exist in three states: 1) a high affinity guanine nucleotide-sensitive state characterizing a subset of receptors (26% shown schematically in the upper portion of Fig. 10) capable of binding agonist in a quasi-irreversible fashion, subsequently unavailable for [3H]QNB binding; 2) a resting state that characterizes the majority of receptors (74%; lower portion of Fig. 10) prior to exposure of intact cells to agonist or prior to exposure of homogenates to elevated levels of guanine nucleotides, with an IC50 for carbamylcholine inhibition of [3H]QNB binding of 1.4 X M (Fig. 6); and 3) a low affinity state assumed by receptors either after treatment of homogenates with guanine nucleotides or immediately after exposure of cells to agonist, with an IC% for carbamylcholine displacement of r3H]QNB of 8.9 X M (Fig. 6). In this scheme, rapid quasi-irreversible binding of agonist to a group of high affinity receptors would cause a loss of [3H]QNB-binding sites and be accompanied by conversion of additional receptors in the resting state to the low affinity activated state. The concept of partial occupancy by agonist of a group of receptor sites with an associated decrease in the affinity for agonist of the remaining unbound sites is quite analagous to the concept of negative cooperativity, which is well demonstrated for muscarinic agonist binding (Fig. 3). We postulate that binding of guanine nucleotides to a nucleotide regulatory site associated with the high aftinity receptor may convert these high affinity receptors to a low affinity form and, in addition, convert the remaining receptor population to the low affinity form. Any agonist persistently bound to the high affinity form would be released during this process with recovery of ["HIQNB-binding sites. Alternatively, all receptors may be associated with a guanine nucleotide regulatory site and be converted independently to the low affinity state.
Under physiologic conditions, muscarinic cholinergic agonists and guanine nucleotides may act in concert at their respective sites to mediate rapid conversion of the receptor to the low affinity activated state. Whether or not the low affinity state of the receptor is actually the physiologically active form of the receptor cannot be determined from the data presented.
The model outlined in Fig. 10 depends on the presence of a high aftinity receptor and persistent binding of agonist to this receptor associated with regulation of receptor affinity. Traditional analysis of hormone-receptor interactions has been

Control of Muscarinic Receptors in Cultured
Heart Cells based on the assumption that the hormone. receptor complex is readily reversible. However, studies of the P-adrenergic receptor (9) and the glucagon receptor (29) have demonstrated persistent binding of agonists and a role of guanine nucleotides in mediating the release of agonist from receptor in homogenates. Furthermore, exposure of astrocytoma cells (7) to either guanine nucleotides or /3-adrenergic agonists has been reported to decrease the affinity of the receptor for agonist as well as to mediate a decrease in adenylate cyclase activation, suggesting that such agonist-induced affinity changes are also involved in regulation of P-adrenergic receptor activity. Although we have not adduced direct evidence in the present studies for the existence of the high affinity form of the receptor, Birdsall et al. have presented a computer analysis of data from studies of direct binding of tritiated muscarinic agonists to synaptosomal preparations from rat cerebral cortex suggesting that 25 to 30% of receptors may exist in a high a f f i t y form (30).
Of the 60 to 70% of E3H]QNB-binding sites lost during agonist exposure, 26% are lost during the first minute of exposure while 43% of sites are lost between 30 min and 3.0 h during a slow second phase (Fig. 5). Agents known to interfere with microtubule function (Fig. 9) inhibit 40 to 45% of the total agonist-induced receptor loss. Hence a subclass of receptors (25 to 30%) is subject to agonist control by a mechanism independent of microtubule function. The absence of an effect of colchicine on the rapid loss of receptor sites during brief exposure of cells to agonist (Table 11) suggests that these sites may represent the 25 to 30% subset insensitive to colchicine. The total receptor population, then, may be divided into subgroups as shown schematically in Fig. 10.
Studies of recovery of receptors lost during brief exposure to agonist revealed that the receptor sites did not recover significantly over periods greater than 12 h after removal of agonist. However, if cells were incubated for 3 h with agonist, guanine nucleotides had no effect on recovery of receptors, and recovery to base-line levels occurred over a 12-h period and required protein synthesis (Fig. 7). One possible explanation of these data is that after a 3-h exposure to agonist, receptors in the 26% subgroup undergoing rapid loss are also subject to endocytosis.
The role of microtubules in the process of agonist-induced loss of muscarinic receptors is further supported by the finding that the half-maximal inhibitory effect of colchicine on agonist-mediated receptor loss occurs at a concentration of 1.4 X lop6 M (Fig. 9), comparable to the value of 2.3 X lo-" M observed for half-maximal binding of colchicine to the tubulin of sea urchin eggs (31). However, it should be noted that colchicine exerts an inhibitory effect on nucleotide transport (28) which could also interfere with agonist modulation of receptors. However, lumicolchicine, which had no effect on agonist-induced changes in receptor number (Fig. 9), also has a potent inhibitory effect on nucleoside transport. Polymerization of tubulin involves GTP (27) which was found (Table  I) to decrease apparent receptor affinity for carbamylcholine. Shifts in GTP concentrations during depolymerization of microtubules by colchicine could secondarily alter receptor affinities and perhaps also interfere with agonist-induced changes in receptor number. The most likely explanation is that disruption of microtubular structure interferes with the agonistinduced disappearance of [3H]QNB-binding sites, presumably by inhibition of endocytosis. Agonist stimulation of endocytosis of cell surface receptors has been demonstrated following epidermal growth factor interaction with human fibroblasts (3), antigen interaction with antigen receptor sites on B lymphocytes (32,33), and low density lipoprotein interaction with fibroblasts (26). Recently Siman and Klein have demonstrated that agonist-mediated decreases in muscarinic receptors in neuroblastoma hybrids were inhibited by cytochalasin B, which interacts with microfilaments (34). The absence of an effect of cytochalasin B in our system is presumably related to differences in species and cell type.
Further studies to explore the presence of a high affinity subclass of receptors, the role of endocytosis of receptor. ligand complexes, and persistent binding of agonist to high affinity receptors after short agonist exposure will be necessary to elucidate more fully the mechanism of agonist control of muscarinic receptor activity and the interaction of agonists, guanine nucleotides, and the cell membrane in this process.