JOURNAL OF Vol. No. 12, of June 25, pp. 7382-7390,1984 of ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 12,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 12, Ieaue of June 25, pp. 7382-7390,1984 Printed in U.S.A.

The Development of Physiologic Responsiveness to Muscarinic Agonists in Chick Embryo Heart Cell Cultures ROLE OF HIGH AFFINITY RECEPTORS AND SENSITIVITY TO GUANINE NUCLEOTIDES*

(Received for publication, October 3, 1983)

Jonas B. Galper$, Louise C. Dziekan, and Thomas W. Smith From the Cardiovascular Division, Brigham and Women’s Hospital and the Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Prior to ingrowth of the vagus nerve (4-6 days in ouo), 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% days in ouo, 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 ouo, 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% days in ouo, 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% days in ovo in medium supplemented with specific lots of serum resulted in a %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.

Parasympathetic stimulation of the heart causes a decrease in the rate and force of contraction. These changes have been associated with an increase in K’ permeability (1) and in some experimental preparations with a decrease in Ca2+ movement into the cell (2, 3). The vagus nerve, which carries parasympathetic stimuli to the heart, synapses on ganglion cells within the myocardial tissue located principally in the

* This study was supported by research grants from the National Heart, Lung, and Blood Institute, National Institute of Health Grants HL-22775 and 18003, and American Heart Association Grant 79825. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Established Investigator of the American Heart Association.

atria (4). Short neurons originating in these ganglia release acetylcholine in the region of muscarinic cholinergic receptors which mediate the physiologic response. Studies of the embryonic development of the chicken heart have demonstrated that prior to ingrowth of the vagus nerve (day 4-5 in ouo), a physiologic response to muscarinic agonists was either absent or markedly diminished (5-7). The object of the current studies was to determine the factor(s) responsible for the absence of a physiologic response in these young hearts. Specifically, we determined whether differences in the binding of agonists to muscarinic receptors in these hearts or the absence of factors that couple muscarinic receptors to a physiologic response might explain the lack of response to muscarinic stimulation.

Previously, no difference in the number of muscarinic receptors, as demonstrated by the binding of the tritium-labeled muscarinic antagonist [3H]QNB,’ could be detected in heart homogenates studied either prior to or following ingrowth of the vagus nerve. However, differences in muscarinic receptor number as measured by antagonist binding do not necessarily reflect differences in agonist binding or in coupling of muscarinic receptors to a physiologic response.

Recently, we and others have described at least two subsets of muscarinic receptors using competitive binding of muscarinic agonists with (3H]QNB to homogenates of hearts 10 days in ouo as a measure of receptor affinity for agonists (8, 9). These subsets comprise a high affinity subtype constituting 26% of total [3H]QNB-binding sites with an IC, for carbamylcholine inhibition of [3H]QNB binding of 3.9 f 1.0 x M and a low affinity subset with an ICso of 4.5 f 0.7 X lo-’ M (9). The functional role of these receptor subsets is of consid- erable interest. If high affinity muscarinic receptors play a critical role in the mediation of a physiologic response, then the absence of high affinity receptors prior to vagal innervation might be responsible for the absence of a response to muscarinic agonists in these hearts. Such a finding would parallel the observation that high affinity @-adrenergic receptors appear to be involved in 0-adrenergic agonist stimulation of adenylate cyclase activity. An agonist-high affinity 8-adrenergic receptor complex has been shown to interact with guanine nucleotides followed by dissociation of bound agonist and concomitant stimulation of adenylate cyclase activity (13, 14). We and others have previously reported data suggesting that an agonist-high affinity muscarinic receptor complex may interact with guanine nucleotides in a manner analogous to that demonstrated for the &adrenergic receptor. Specifi-

‘The abbreviations used are: QNB, quinuclidinyl benzilate; Gpp(NH)p, guanosine 5’-(B,yimino)-triphosphate; Hepes, 4 4 2 - h ~ - droxyethy1)-1-piperazineethanesulfonic acid; RH, high affinity receptors; RL, low affinity receptors.

7382

Development of Muscarinic Response in Cultured Heart Cells 7383

cally: 1) guanine nucleotides facilitate a decrease in muscarinic receptor affinity for agonist, probably by mediating the conversion of high affinity muscarinic receptors to a low affinity form (9-11); 2) prior incubation of cultured embryonic chick heart cells with muscarinic agonist resulted in the loss of high affinity receptors in homogenates of these cells; and 3) incubation of these homogenates with guanine nucleotides resulted in the reappearance of receptors in a low affinity form (9, 12). These data are consistent with the hypothesis that following agonist exposure, agonist remains persistently bound to a subclass of high affinity receptors rendering them unavailable for binding of [3H]QNB. Recovery of receptor number could then occur after release of agonist during the guanine nucleotide-mediated conversion of the high affinity receptor to a low affinity form.

These findings suggest that the interaction between agonist, high affinity receptor, and guanine nucleotides, which paral- lels that for @-adrenergic stimulation of adenylate cyclase, may be critical for the mediation of a muscarinic response in the heart. The developing embryonic chick heart offers the opportunity to address this question by determining whether the appearance of a physiologic response to muscarinic stimulation, as measured by the ability of muscarinic agonists to decrease beating rate and/or increase the rate of K+ efflux from heart cells, is correlated with changes in receptor affinity state and sensitivity to guanine nucleotides. Specifically, the studies here take advantage of two findings: 1) that cells cultured from embryonic chick hearts reflect the sensitivity to muscarinic agonists present in the hearts from which cultures are derived; and 2) that growth of heart cell cultures from hearts 3% days in ouo in media supplemented with certain lots of horse serum resulted in the conversion to a state responsive to muscarinic stimulation. This induction of a muscarinic response was associated with the development of increased numbers of high affinity receptors and also of responsiveness to guanine nucleotides. Since the ability of guanine nucleotides to interact with receptors has been shown to require the presence of a guanine nucleotide regulatory protein (13), the possibility must be considered that the "defect" in the response to muscarinic agonists prior to vagal innervation of the heart could be a qualitative or quantitative lack of a guanine nucleotide regulatory protein.

EXPERIMENTAL PROCEDURES

Materials-Chemicals were obtained from the following sources: carbamylcholine chloride, cycloheximide, and GTP from Sigma; guanosine-(&y-imino)-triphosphate tetrasodium salt from ICN Phar- maceuticals (Cleveland, OH); oxotremorine from Aldrich; medium "199 from Microbiological Associates (Bethesda, MD); Hepes buffer from Calbiochem; I-['HIQNB (specific activity, 43 Ci/mmol) and 'zK+ from New England Nuclear; horse serum and fetal calf serum from North American Biological, Miami, FL; and embryonated chicken eggs (Flock MR62) from Spafas, Inc. (Norwich, CT).

Heart CeU Cultures-Heart cell cultures were prepared by a modification of the method of DeHaan (15). Embryos were removed from embryonated Leghorn chicken eggs on day 3% or day 10 in ouo, minced, and incubated with 0.25% (w/v) trypsin in Ca"-M$+-free Hanks' balanced salt solution at 37 "C for 8 min. Embryonic age was determined by the method of Hamberger and Hamilton (16). 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 sed- imented at lo00 rpm in a benchtop centrifuge, resuspended in growth medium, and incubated in a 100-mm Petri dish (Falcon, Oxnard, CA) for 45 min at 37 "C in a humidified atmosphere of 5% COZ/95% air. During this incubation, nearly 95% of the fibroblasts in the suspension adhered to the dish. Heart cells were then plated at a density of 1.3 X 106 cells/cm2 on collagen-coated 100-mm Petri dishes. On the third culture day, the medium was changed. Unless otherwise indicated, cells were used for experiments on culture day 4.

Media-Cells were grown in a modification of medium "199 consisting of 20-21% (v/v) "199 and 79-80% (v/v) of a buffered salt solution containing 117 mM NaCl, 4.4 mM KCI, 1.8 mM CaC12, 0.8 mM MgSO,, 25 mM Hepes (adjusted to pH 7.4 with NaOH), 5 mM glucose, and 0.001% (v/v) phenol red.

Measurement of [BHIQNB Binding to Homogenates-The assay procedure was a modification of the method of Yamamura and Snyder as described (17, 18). After three rinses with ice-cold "199 Hepes, cells were harvested in a small volume of Hepes-buffered medium M- 199. After freezing at -70 "C and thawing twice, the cells were homogenized in a glass on glass homogenizer and allowed to warm to 22 "C prior to assay.

The final assay mixture consisted of 0.5 ml of Hepes-buffered M- 199 containing Z-['HIQNB (43 Ci/mmol, 1 nM in ['HIQNB 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 mM NaCl, 5.4 mM KC1, 0.8 mM MgS04, 1.8 mM CaC12, 50 mM Hepes, 1.0 mM NaHzPO., 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) filter, the assay tube washed 3 times, and filters dried and assayed for radioactivity in a liquid scintillation counter with 10 ml of Insta-Gel (Packard). Protein was determined by the procedure of Lowry et nl. (19) after precipitation of the homogenate protein with trichloroacetic acid, using bovine serum albumin as standard.

Specific binding is defined as binding inhibited by saturating concentrations (0.1 mM) of oxotremorine. ['HIQNB binding to homogenates was 90% specific by this criterion. All data were corrected for nonspecific binding in the presence of 0.1 mM oxotremorine. A typical assay tube containing 0.2 mg of protein/0.5 ml of homogenate suspension bound 900 to lo00 cpm of f3H]QNB at a counting effi- ciency of 33%. '%+ Efflrwl-Cells grown on circular glass coverslips were incu-

bated for 24 h with [4,5-'H]leucine, labeled to equilibrium (3 h) with "K' (5 mCi/ml, total K' = 4.5 mM) as described (18). Cells were then rinsed once in fresh growth medium containing unlabeled K+, transferred to a perfusion chamber, and perfused with unlabeled growth medium at a flow rate of 0.98 ml/min at 37 "C. The effluent was collected at 30-s intervals for 5 min, 10 ml of scintillation fluid added, and "K+ determined. The protein from each coverslip was solubilized by soaking the coverslip in 1 ml of 0.5 N NaOH, and protein content was determined as described previously (18). An aliquot of each sample was taken and neutralized with 0.5 ml of Tris- HC1 (pH 7.4), 10 ml of scintillation fluid added, and "K' and 'H determined by double label counting in a liquid scintillation spectrom- eter. Aliquots were taken from half the samples and protein measured by the method of Lowry et al. (19), allowing the determination of milligrams of protein/1000 'H counts and the calculation of milligrams of protein/coverslip preparation. After correction for decay of "K+, K+ uptake was normalized to uptake in nanomoles/mg of cell protein.

Measurement of Changes in Beating Rate-Cells grown on glass coverslips were placed in a Sykes-Moore chamber (Bellco Glass, Inc., Vineland, NJ) and continuously perfused via inlet and outlet porta. The chamber was placed on the stage of an inverted phase contrast microscope enclosed in a Lucite box maintained at 37 "C. The inlet side of the chamber was connected by polyethelene tubing to two syringe pumps to allow the cultures to be sequentially perfused by separate solutions. Perfusion at 0.98 ml/min did not disturb cell adhesion to the coverslip. Beating was determined visually or by monitoring the movement of the border of a single cardiac cell with a video motion detector and recording the output with a physiologic recorder (Hewlett-Packard Co., Palo Alto, CA) as described (18,20).

Computer Analysi-The data for competitive binding of carbamylcholine with ['HIQNB were fit by a nonlinear least square analysis to the binding equation

for a single site model or the sum of two such binding equations for a two-site model. Data were weighted by the inverse of the square of the variance of individual values. B, = total number of receptor- binding sites; F = the concentration of free ligand; KI = R . Z/RI where R = free receptor, Z = concentration of free inhibitor; RZ = bound inhibitor; and B = the binding at a given concentration of free ligand

7384 Development of Muscarinic Response in Cultured Heart Cells and inhibitor. Using an F test as described by Munson and Rodbard (21) to determine whether the data gave the best fit to a single population of receptors or two populations with differing affinities, we found that in all cases presented here the two-site analysis gave F values greater than the critical level. Hence, although under certain conditions carbamylcholine and GppNHp markedly decreased the number of high affinity receptors, a fraction of the high affinity sites still remained as judged by this method.

RESULTS

The Physiologic Response to Muscarinic Agonists in Heart Cells Cultured from Embryonic Chick Hearts Prior to Ingrowth of the Vagus Nerue-Embryonic chick hearts studied prior to ingrowth of the vagus nerve (day 4-5 in ouo) demonstrated a markedly diminished response to muscarinic agonists (5-7). In contrast, hearts studied following vagal innervation (e.g. 10 days in ouo) demonstrate readily apparent decreases in the rate and force of contraction on exposure to muscarinic agonists. An initial goal of the present experiments was to study the development of physiologic responsiveness to muscarinic stimulation in cultures of hearts 3% days in ouo. Since it was not clear whether cells cultured from embryonic chick hearts demonstrated sensitivity to muscarinic stimulation similar to that of the hearts from which they were derived, we first compared the effect of the muscarinic agonist carbamylcholine on beating rate and K+ permeability in cells cultured from hearts of embryos 3% days in ouo to the effect on cultures from hearts 10 days in ouo. The experiment summarized in Table I demonstrates that beating rate decreased 16% from 140 -t- 5 to 118 k 7 (&LE., n = 20) beats/min in the presence of M carbamylcholine in cultures from hearts 10 days in ouo. This change, although small, was highly reproducible and statistically significant (p < 0.001). In cultures of hearts from embryos 3% days in ouo, carbamylcholine had no discernible effect on beating rate.

The explanation usually offered for the effect of acetylcholine on the mechanical and electrical activity of atrial muscle has been based on the finding that acetylcholine selectively increases the membrane permeability to K+ ions (1). To compare the effect of muscarinic agonists on K+ permeability in cultures of heart cells from hearts 3% days in ouo with cultures from hearts 10 days in ouo, we compared the ability of carbamylcholine to increase the 42K+ efflux rate from cultures from hearts of these two ages. To study K+ efflux, the intracellular K+ pools of cultures from embryonic hearts 3% and 10 days in ouo were first labeled to equilibrium with

the medium was complete for both sets of cultures at 3 h at 975 -t- 10 (n = 14) nmol/mg of protein for cultures of hearts 10 days in ouo and 1065 k 52 (n = 14) nmol/mg of protein for

TABLE I Effect of carbamylchline on beating rate in heart cell cultures from

chick embryos 3% and 10 days in ouo Cells grown on glass coverslips were perfused in Sykes Moore

chambers, and the beating rate was determined as described under “Experimental Procedures.” For each determination, cells were first perfused with growth medium. After establishment of a stable base- line, perfusion was switched to medium M in cholinergic agonist for 5 min and the effect on beating rate determined. All observations in a given series were performed on the same group of cells in the microscoDic field.

42 K + (data not shown). Equilibration of intracellular 4ZK+ with

Beating rate/min (&.E.) Days in ouo‘ control Carbamylcholine

110-3 M) ~~. .~ ,

10 (n = 20) 140 f 5 118 & 7 3% ( n = 6) 130 F 8 137 & 6

Age of the embryo from which hearts were taken.

cultures of hearts 3% days in ouo. These data indicated that intracellular K+ content/mg of protein was significantly greater in cells cultured prior to vagal innervation of the heart ( p < 0.001).

As described previously (18), efflux of 42K+ from cells cultured from hearts 10 days in ouo was found to follow an exponential time course for up to 30 min during which “K+ remaining in the cells decreased from 975 +- 15 to 280 f 14 nmol/mg of protein. In the experiment described in Fig. 1, the rate of efflux of 42K+ from heart cell cultures of embryos 3% and 10 days in ouo was compared. Each plot represents the least squares fit of a semilogarithmic plot of the data to a straight line. These data indicate that under control conditions during the time studied, the efflux of 42K+ from both sets of cultures follows an exponential time course. The half- time of K+ efflux derived from the slope of the curve in Fig. l a was 13.8 k 0.3 min (n = 14) for cultures from hearts 10 days in ouo and (Fig. l b ) 9.8 f 0.4 (n = 14) min for cultures from hearts 3% days in ouo. Hence under control conditions the rate of efflux of “K+ from heart cells cultured from hearts of embryos 3% days in ouo was 26% faster than that in cultures from hearts 10 days in ouo.

The effect of the muscarinic cholinergic agonist carbamylcholine on the rate of efflux of 42K+ from cultured cells is also shown in Fig. 1. Incubation with M carbamylcholine increased the T% of efflux of 42K+ by 33% from 13.2 & 0.2 min to 8.8 -C 0.3 min in cultures of hearts 10 days in ouo (Fig. la) compared to an increase in 42K+ efflux of only 11% from 9.8 t 0.4 (n = 14) min to 8.7 +- 0.3 min (n = 14) in cultures from hearts of embryos 3% days in ouo (Fig. lb). This small change in response to carbamylcholine in the rate of efflux from cultures from embryos 3% days in ouo was significant (p < 0.002) but represented a markedly decreased response to carbamylcholine in cultures studied prior to vagal innervation of the heart. The concentration gradient of K+ across the cell membrane is a major driving force in the movement of K+ in response to changes in membrane permeability. If the intracellular K+ concentration varied with time of exposure to muscarinic agonists, the driving force for K+ efflux would vary, and the comparison of relative efflux rates could not be used as an indicator of changes in K+ permeability in cells exposed to agonists for various times. However, we have previously demonstrated that intracellular K’ content did not vary from steady state levels during exposure to carbamylcholine for up to 3 h (18).

All of these experiments were carried out on cells after 3 days in culture. However, the decreased responsiveness to muscarinic stimulation in cultures from hearts 3% days in ouo persisted in cells studied after 6 or 9 days in culture and hence was a stable property of these cultures.

We have previously demonstrated a close correlation between the ability of muscarinic agonists to increase 42K+ efflux and their ability to decrease beating rate (18). The absence of any discernible effect of carbamylcholine on beating rate (Table I) in the presence of an 11% carbamylcholine-induced increase in ‘*K+ efflux rate in cultures of hearts 3% days in ouo (Fig. l b ) may be due to the fact that the decrement in beating rate associated with such a small increase in “K+ permeability is too small to be appreciated (18). In any case, these data demonstrate that cells cultured from hearts prior to ingrowth of the vagus nerve exhibit a markedly diminished physiologic response to muscarinic stimulation compared to cultures from innervated hearts.

Binding of Muscarinic Agonists and Antagonists to Homog- enutes of Heart Cells Cultured from Embryonic Hearts Prior to Ingrowth of the Vagus Nerue-The effects of muscarinic

Development of Muscarinic Response in Cultured Heart Cells 3 ‘12

“ T 1 I I I a

I I I I 1

Time (min) 1 2 3 4 5

1000

- 900 clr E 1 0

v E

800 c),

.- 0 : a 1 Y

r U

700

IO

I I I I I

b

I I I I I 1 2 3 4 5

T i m e ( m i n )

7385

FIG. 1. Comparison of effect of carbamylcholine on raK* efflux from cells cultured from hearts of embryos 3% days and 10 days in o m . Dishes containing heart cells cultured from chicken embryos 3% days (a) or 10 days ( 6 ) in ouo and grown on glass coverslips were labeled overnight with 0.2 pCi/ml of [4,5-3H]leucine, incubated for 3 h in growth medium containing 5 pCi/ml of “K+ (total K+ = 4.4 mM), rinsed, and placed in an efflux chamber as described under “Experimental Procedures.” Cells were perfused with growth medium containing unlabeled K+ without carbamylcholine (0); with M carbamylcholine (A); with M carbamylcholine plus

M atropine (0). “K+ efflux was determined at the times indicated as described under “Experimental Procedures” and plotted as “K+ remaining in the cells a t time t , calculated by subtracting total efflux of “K+ from the cells a t time t from the initial “K+ content at time zero. “K+ at time zero was determined as the mean K+ content/mg of protein of 14 coverslips exposed to 5 pCi/ml of “K+ for 3 h. After rinsing, *‘K+ was determined as described under “Experimental Procedures.” Each curue is the mean of two sets of seven replicate determinations each. The lines are the least squares fit of the log,, of “K+ remaining at time t to a straight line. Correlation coefficients were at least 0.99 for all plots. The half-time of efflux is calculated from the relationship T.+, = ln2/kl where k , is the slope of the curve at each set of conditions in Fig. 1 and equals the rate constant for the movement of “K+ out of the cell.

agonists on K+ permeability and beating rate in cultured heart cells require the interaction of agonists with muscarinic receptors. The absence of muscarinic receptors in hearts prior to vagal innervation might explain the absence of a physiologic response in these hearts. However, we have previously shown that muscarinic receptor numbers measured by the binding of the potent muscarinic antagonist [3H]QNB were the same in homogenates of embryonic chick heart from 2% to 18 days in ouo (7). If the number of antagonist binding sites is the same at all embryonic ages, then at least two possible explanations for the absence of a physiologic response remain: 1) that although total receptor numbers are the same, the distribution of receptors among subtypes with high and low affinities for agonist binding changes with embryonic age; and/or 2) that a factor or factors such as guanine nucleotide- binding protein which couples agonist binding to a physiologic response is not present prior to vagal innervation of the heart (13). In order to test these hypotheses we studied the binding of [3H]QNB to homogenates of heart cells cultured from hearts of embryos 3% days in ouo and determined affinity of receptors for agonists by competition of various concentrations of agonist with a fixed concentration of [3H]QNB. These data were compared to similar studies of homogenates of heart cells cultured from embryo hearts 10 days in ouo.

We first determined whether the characteristics of binding of [3H]QNB to homogenates of cultures of hearts 3% days in ouo were similar to those previously determined for cultures of hearts 10 days in ouo. The binding of [3H]QNB to homogenates of cultures 3% days in ouo was saturable, and 92% of binding was specific as measured by displacement by lo“ M oxotremorine, a potent muscarinic agonist. Scatchard analysis of the binding of [3H]QNB to chick heart homogenates demonstrated that binding was saturable at 147 f 5 fmol/mg of protein (S.E., n = 8) with a K d of 0.12 f 0.1 nM (Fig. 2). We have previously determined that the binding of [3H]QNB to homogenates of cultures of hearts from embryos 10 days in ouo was saturable at 184 & 13 fmol/mg of protein (S.E., n = 6) with a K d of 0.11 nM (9). Hence, although cultures from both 3%- and 10-day hearts did not exhibit any significant difference in affinity for [3H]QNB, there were 20% fewer receptors/mg of protein in cultures of hearts 3% days in ouo. No such difference was found in homogenates of intact hearts (7).

In order to determine the specificity of [3H]QNB binding for the muscarinic receptor in homogenates of cells from hearts 3% days in ouo, we compared the ability of selected muscarinic agonists and antagonists to compete with [3H] QNB for binding to the receptor. In the experiments sum-

7386 Development of Muscarinic Response in Cultured Heart Cells

Bound I Fmol lmg)

FIG. 2. Scatchard plot of binding of [8H]QNB to homogenates of chick heart cells cultured from embryos 3% days in 000. Replicate cultures in multiwell dishes were incubated overnight in medium containing 0.01 pCi/ml of [U-’4C]leucine. Cells were washed, harvested, and cells from each group combined and homogenized as described under “Experimental Procedures.” Aliquots of homogenates were incubated for 2 h with the concentrations of [3H] QNB indicated and specific binding determined as described under “Experimental Procedures.”

0 b / k i [LIGAND] .M

FIG. 3. Inhibition of [‘HIQNB binding by muscarinic agonists and antagonists. Replicate culture dishes were labeled overnight in medium containing 0.01 pCi/ml of [U-“C]leucine. Cells were washed, harvested, combined, and homogenized as described under ”Experimental Procedures.” Aliquots were incubated for 1 h with 1 nM [3H]QNB plus the indicated concentrations of agonist or antagonist. Specific binding was determined as described under “Experi- mental Procedures.” A, unlabeled QNB; 0, scopolamine; 0, oxotremorine; 0, carbamylcholine.

marized in Fig. 3 the concentrations of the muscarinic antagonists QNB and scopolamine and the agonists oxotremorine and carbamylcholine required to inhibit 50% of [3H]QNB binding at a concentration of [3H]QNB of 1 nM were 3 and 1.3 nM, respectively, for the antagonists, and 0.22 and 22 pM, respectively, for the agonists. These relative values correspond to the relative pharmacologic potency of these ligands for a muscarinic response. The Hill coefficients for scopolamine and QNB were 0.98, indicating a single class of binding sites for antagonists, while oxotremorine and carbamylcholine exhibited Hill coefficients of 0.91 and 0.8, respectively. The Hill coefficients for agonists, although less than one, are significantly greater than the values of 0.69 and 0.56 for binding of oxotremorine and carbamylcholine, respectively, to muscarinic receptors in homogenates of cultures of hearts 10 days in ouo (12), suggesting that the degree of heterogeneity of sites or the degree of negative site-site interaction was less marked in cells cultured prior to ingrowth of the vagus nerve.

High Affinity Muscarinic Receptors in Cultures from Hearts from Chick Embryos 3% Days in Ouo-The affinity of agonist for the muscarinic receptor was studied using competitive binding of carbamylcholine with [3H]QNB to cell homogenates as a measure of affinity. Computer analysis of data from

experiments summarized in Fig. 4 (closed circles) showed that muscarinic receptors in cultures from hearts from embryos 3% days in ouo were composed of nearly equal numbers of high affinity (RH) and low affinity (RL) subtypes: 66 f 5 (S.E., n = 12) fmol/mg of protein or 52% with a Kd for carbamylcholine of 6.0 f 1.0 X M and 62 f 5 fmol/mg of protein with a Kd of 3.4 f 0.4 X M. We have previously demonstrated that muscarinic receptors in cultures of heart cells 10 days in ouo exist in high- and low-affinity subtypes, 26% or 34 fmol/mg of protein having a Kd of 3.9 X M and 95 fmol/mg of protein with a Kd of 4.5 X lo-‘ M (9). Hence the absence of a physiologic response in heart cell cultures derived from hearts 3% days in ouo is not associated with an absence of high affinity muscarinic receptors in these cells.

Effect of Guanine Nucleotides and Prior Exposure to Mus- carinic Agonists on High Affinity Muscarinic Agonist Bind- ing-Several groups have demonstrated that exposure of heart cell homogenates to guanine nucleotides results in a decrease in apparent affinity of muscarinic receptors for agonists (9- 12). We have recently demonstrated that guanine nucleotides mediate the interconversion of RH to RL and that prior exposure of heart cell cultures from embryos 10 days in ouo to muscarinic agonists leads to the loss of RH sites (18). We have suggested that this loss of RH might be due to persistent binding of agonist to RH, thus rendering them unavailable for binding of [3H]QNB in the competitive binding assay. We have also suggested that the interaction of an agonist-high affinity receptor complex with guanine nucleotides may be associated with the mediation of a physiologic response to muscarinic stimulation.

To determine whether the absence of a physiologic response in cultures from hearts 3% days in ouo is associated with the absence of a response of high affinity receptors to guanine nucleotides, we studied the effects of prior exposure to agonist on high affinity muscarinic receptor number and the effect of the GTP analogue GppNHp on receptor number and on affinity of receptors for agonist in homogenates of cultures from hearts 3% days in ouo. Data in Fig. 4 summarize the means of 12 separate experiments. Computer analysis re- vealed no significant effect of GppNHp on the relative numbers of RH and RL or on affinity of RH and RL for agonist (Table 11); RH constituted 48% or 61.5 f 4.8 fmol/mg of protein with a Kd of 6.7 f 1.5 X M ( n = 12) for carbamylcholine, and RL constituted 65.5 f 5.1 fmol/mg of protein with Kd of 2.6 f 0.4 X M. Furthermore, no effect of Gpp(NH)p on RH could be detected when cultures from hearts 3% days in ouo were studied after 6 or even 9 days in culture. When cells were preincubated for 15 min with M carbamylcholine, washed, and receptor subtypes determined by competitive binding of various concentrations of carbamylcholine with 1 nM [3H]QNB (Fig. 4), a 22% decrease in total [3H]QNB bound (from 127 to 107 fmol/mg of protein) OC- curred. This was due to a decrease of 41% in the high affinity binding sites from 66 fmol/mg in control cells to 39 k 5 fmol/ mg of protein with a Kd of 14 f 5 X lo-‘ M in cells pre- exposed to carbamylcholine (Table 11).

These data demonstrate that although prior treatment with agonist did decrease the number of RH, GppNHp had no discernible effect in converting RH to RL in cultures taken from hearts at 3% days in ouo. Furthermore, no effect of GTP on RH was found in these cultures (data not shown). If the physiologic response to muscarinic agonist stimulation of the heart involves the interaction of the muscarinic receptor with a guanine nucleotide-binding protein, followed by conversion of RH to RL, the absence of an effect of Gpp(NH)p and/or GTP on RH might be directly related to the absence of a


i 5 o t

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T L I 1 1 I I I

L o ' 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2

Carbomylcholine Concentration, M

FIG. 4. Effeet of carbamylcholine and quanine nucleotides on apparent receptor affinity for agonist. Replicate culture dishes of heart cells from embryos 3% days in ouo were labeled overnight with 0.01 pCi/ml of [U-"Clleucine. Cells were washed and incubated with (A) and without (0) M carbamylcholine for 15 min; cells were harvested and homogenized as described under "Ex- perimental Procedures" and incubated for 1 h at room temperature in medium containing 1 nM IaH]QNB at the concentrations of the agonist indicated, and [3H]QNB binding determined. In a similar manner, aliquota of homogenates from control cells not previously exposed to agonist were incubated with M Gpp(NH)p (0) and [3H]QNB binding determined in the presence of the indicated concentration of agonist. Each point represents the mean of three replicate determinations repeated 12 times for each group. The curues are drawn by eye.

TABLE I1 Summury of effects of carbamykholine and guanine nucleotides on apparent receptor affinity for agonists in homogenutes of cultures

from hearts 3% days in ouo grown in control serum Values derived from nonlinear least squares fit of the data in Fig.

4 to a two-component binding equation as described under "Experi- mental Procedures."

RH (+S.E., n = 12) RL (+S.E., n = 12)

Receptor &b Receptor receptors number number

Total

pM fmoll? pM fmollmg fmllmg protein protezn protein

Control 0.60 f 0.10 66 f 5 34 f 4 62 f 5 127 GppNHp(l0" 0.67f0.15 62 f 5 2 6 f 4 6 5 f 5 127

Carbamylcholine 1.40 f 0.50 39 f 5 27 & 2 69 & 5 107 M)

(10-3 M) The differences between & values for R H in control, GppNHp-

treated, and carbamylchoIine-treated homogenates are not statistically significant.

* The differences in I(d values for RL in control, GppNHp-treated, and carbamylcholine-treated homogenates are not statistically significant.

physiologic response to muscarinic agonists in these cells. Effect of Growth of Heart Cells from Chick Embryo Hearts

3% Days in Ouo in the Presence of Medium Supplemented with Selected Lots of Horse Serum on the Development of a Physiologic Response to Muscarinic Stimulation-As described above, heart cell cultures taken from chick embryo hearts prior to ingrowth of the vagus nerve contained RH but were deficient both in a physiologic response to muscarinic agonists and in a biochemical response to guanine nucleotides. Pro- longed growth of these cells in culture for up to 9 days had no effect on either the development of a physiologic response or the development of sensitivity to guanine nucleotides. The temporal relationship between the ingrowth of the vagus nerve and the development of a physiologic response in the intact embryonic heart suggested that hormonal and/or neurohu-

moral factors might induce the development of muscarinic responsiveness in these cultures. For this reason, we tested 5 lots of horse serum for their ability to induce a K+ permeability response to carbamylcholine in cultures of hearts 3% days in ouo. Cells were grown in medium "199 supplemented with 4% fetal calf serum and 2% of a given lot of horse serum. As described previously, cells were equilibrated with "K+ and the rate of efflux determined during superfusion with control medium or with medium containing M carbamylcholine. The half-time of efflux of 42K+ was determined from the slope of a semilogarithmic plot of "K+ remaining in the cells a t a given time as described in Fig. 1. The experiment summarized in Table I11 is typical for 2 of the 5 lots of serum tested. Cells grown for 3 days in medium containing horse serum from lot 9938 exhibited a somewhat slower rate of resting "K+ efflux, 10.5 ? 0.2 (fS.E., n = 7) min compared to 9.8 -C 0.4 min in cells grown in standard serum. This difference was statistically significant (p < 0.002). Cells grown in serum 9938 also exhibited a 30% decrease in the t% of K+ efflux on exposure to carbamylcholine, from 10.5 f 0.2 ( n = 14) min to 7.5f 0.4 min compared to an 11% decrease in t% of K+ efflux in cells grown in standard medium in which t% decreased from 9.7 to 8.7 min (Table 111). Hence growth in medium containing horse serum lot 9938 resulted in a modest but significant decrease from the control rate of efflux of "K+ and a marked increase in responsiveness of "K+ efflux rate to muscarinic agonist. Furthermore, steady state K+ levels in cells from 3%-day hearts grown in serum lot 9938 decreased to 1010 & 25 (S.E., n = 14) nmol/mg of protein. This value was not significantly different from 1065 f 52 nmol ( n = 14) seen in cells grown in control serum and also not significantly different from the 970 & 10 ( n = 14) nmol/mg of protein observed in cultures of heart cells from embryos 10 days in ouo.

High Affinity Receptors and the Response to Guanine Nu- cleotides in Heart Cells Cultured from Embryos 3% Days in Ouo and Incubated with Media Supplemented with Horse Serum Lot 9938-In order to determine the relationship between physiologic responsiveness and a guanine nucleotide

TABLE I11 Efflux rate of "K+ from cells grown in control medium and medium

supplemented with horse serum 9938 Experiments were carried out as described in Fig. 1. After labeling

to equilibrium (3 h) with "K+, cells were incubated for the indicated times in the continuing presence of 5 pCi/ml of "K+ with or without

M carbamylcholine. '*K+ efflux was measured as described in Fig. 1. Half-times for efflux were derived from slopes of regression lines obtained from the efflux data as described in Fig. 1. The correlation coefficients for these regression lines were at least 0.99. The data for each curve represent the mean of seven replicate determinations.

tln of '*K+ efflux (+S.E., n = 7)

Days in in ouo"

Control Carbamyl-

ehol ie (10" M)

lob min

3%b (control serum) 13.2 f 0.2 8.8 f 0.3

3% (serum 9938) 9.8 f 0.4' 8.7 f 0.3

10.5 f 0.2' 7.5 f 0.4

From data in Fig. 1. The differences of control values for tllz of efflux for cultures of

hearts 3% days in ouo grown in control serum or in serum 9938 are significant within 98% confidence limits as demonstrated by the method of covariance analysis (22). The difference between the control rate of efflux and the rate of efflux in the presence of M carbamylcholine is significant within 99% confidence limits by similar criteria.

"Age of the embryo from which hearts were taken for culture.


effect on agonist affinity for the receptor, we studied the effects of guanine nucleotides on agonist binding to muscarinic receptors in cultures of heart cells from embryos 3% days in ouo which had developed a K+ permeability response to muscarinic agonist following growth in medium supplemented with horse serum lot 9938. Experiments are summarized in Fig. 5 and Table IV, in which the effect of GppNHp on the competitive binding of various concentrations of carbamylcholine with [3H]QNB to homogenates of cultures of hearts from embryos 3% days in ouo grown in media supplemented with serum lot 9938 was studied. Computer fitting of these data indicated the presence of two receptor subtypes, a high affinity receptor with K d of 6.0 f 1.0 X lop7 M (S.E., n = 12) and a low affinity subtype with Kd of 3.4 f 0.4 x M. These K d values were not significantly different from those found for receptor subtypes in cultures of heart cells grown in control media (Fig. 4 and Table 11). However, compared to cells grown in control sera, total receptor number increased

L. L1 I I I 1 I “ - e , \ o ’10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2

Carbamylcholine Concant ra t ion , M

FIG. 5. Effect of carbamylcholine and guanine nucleotides on apparent affinity for agonist in cells cultured from hearts of embryos 3% days in ouo in medium supplemented with 2% horse serum, lot 9938. The procedures and data analysis were as described in Fig. 4, except that cells were grown for 3 days in culture with medium “199 supplemented with 4% fetal calf serum and 2% horse serum lot 9938. 0, control (n = 12); 0, control plus M Gpp(NH)p (n = 12); A, after carbamylcholine pretreatment for 15 min (n = 12).

TABLE IV Summary of effects of cholinergic agonist and guanine nucleotides on apparent affinity for agonists in homogenutes of cultures of hearts 3%

days in ouo grown in horse serum 9938 Values derived from nonlinear least squares fit of the data in Fig.

5 to a two-component binding equation as described under ”Experi- mental Procedures.”

RH (+S.E., n = 12) RL (&S.E., n = 12) Total

Receptor &b Receptor receptors number number

protem protetn protein llM fmollmg pM fmollmg fmollw

Control 0.42 f 0.03 93 f 2 32 f 2 66 f 2 159 GppNHp (lov4 0.43 f 0 . 1 3 2 3 f 4 11 f 5 1 3 6 f 4 159

Carbamylcholine 1.1 f 1.0 37 f 11 11 f 1 80 f 11 117 M)

(10-3 M)

“The differences between I& values for RH in control, GppNHp- treated, and carbamylcholine-treated homogenates are not statistically significant.

*The differences between & values for RL in control, GppNHp- treated, and carbamylcholine-treated homogenates are not statistically significant.

by 20% in homogenates of cells grown in serum 9938 from 127 fmol/mg of protein (Table 11) to 159 fmol/mg of protein (Table IV). Since the number of low affinity receptors remained nearly constant at 66 f 2 fmol/mg in cells grown in serum 9938 compared to 62 k 5 fmol/mg in cells grown in control sera, this increase in receptor number was due almost completely to an increase in high affinity sites from 62 k 5 (S.E., n = 12) fmol/mg of protein in cells grown in control serum to 93 f 2 fmol/mg of protein in cells grown in serum 9938.

Incubation with the GTP analogue Gpp(NH)p had no effect on total receptor numbers in cells grown in serum 9938 but resulted in a decrease of 75% in RH from 93 f 2 fmol/mg of protein in control cells to 23 .S 4 fmol/mg of protein in the presence of Gpp(NH)p and a nearly 2-fold increase in R L from 66 f 2 fmol/mg in control cells to 136 f 4 fmol/mg of protein in the presence of Gpp(NH)p (Fig. 5, Table IV). No significant difference in the K d of RH and R L in control and Gpp(NH)p- treated homogenates of cells incubated in serum 9938 could be detected. These data are consistent with a GppNHp- mediated interconversion of RH to R L in these cells.

Prior incubation of intact cells grown in serum 9938 with lob3 M carbamylcholine resulted in the disappearance of 26% of total [3H]QNB-binding sites (Fig. 5, Table IV) from 159 fmol/mg of protein in control cells to 117 fmol/mg in carbamylcholine-pretreated cells. This decrease was due to a decrease of 55.6 fmol/mg of protein in RH. No statistically significant change in the number of R L could be determined following agonist pretreatment. The K d of those high affinity receptors remaining following pre-exposure to agonist was somewhat higher than that seen in control cells. Although the computer-generated goodness of fit did not improve for a 3- component system, the analysis used here does not rule out the presence of three receptor subtypes, a superhigh affinity, high affinity, and low affinity subtype as proposed by Birdsall et al. (8). In the presence of 3 subtypes the loss of superhigh affinity receptors due to agonist pre-exposure might leave a high affinity receptor subtype with intermediate affinity and the low affinity form.

Our findings indicate that the development of a physiologic response to muscarinic agonists in cultures grown in serum 9938 is associated with at least 3 changes in the cell: 1) the development of increased numbers of R H ; 2) an increase in the number of RH which are lost following exposure to muscarinic agonists; and 3) the development of coupling of binding of guanine nucleotides to the conversion of high affinity receptors to a low affinity form. These findings suggest that both high affinity receptors and a guanine nucleotide-mediated interconversion of RH to R L may be necessary for, or closely associated with, a physiologic response of the heart to muscarinic stimulation.

DISCUSSION

The response of the heart to muscarinic stimulation is presumed to be mediated by a complex series of interactions set in motion at the receptor site by agonist binding. Among the events reported to be associated with the muscarinic response in various experimental systems are inhibition of adenylate cyclase (24), changes in cyclic GMP levels (25), decreased phosphorylation of specific proteins due to decreased activation of cyclic AMP-dependent protein kinases (26), and changes in the turnover of phosphatidylinositol (27). The absence of a physiologic response to muscarinic agonists could be due to a defect in any one or more of these or other processes.

It is well established that prior to ingrowth of the vagus


nerve, embryonic chick hearts are relatively unresponsive to muscarinic stimulation (6, 7). The studies described here demonstrate that cells cultured from embryonic chick hearts prior to ingrowth of the vagus nerve continue to maintain this unresponsiveness to muscarinic stimulation. Furthermore, a response to carbamylcholine did not develop during growth of these cells in culture for up to 9 days. Hence these cultures constitute a stable system in which to study not only the biochemical "defect" responsible for the absence of a muscarinic response, but also to determine what hormonal, neuro- humoral, or other factors might be responsible for inducing the development of a physiologic response in these cells. Finally, by defining the "defect" responsible for the absence of a physiologic response in the developing heart, new insight may be obtained into those factors that mediate parasympathetic control over myocardial function in the mature adult heart.

Studies of "K+ efflux from cells cultured from hearts 3% and 10 days in ouo reported here indicate 1) that the rate of base-line K+ efflux (efflux in the absence of carbamylcholine) decreases significantly (40%) during the course of embryonic development, 2) that the ability of muscarinic agonists to increase K' efflux increases at least %fold during embryonic development, and 3) that growth of heart cells from embryos 3% days in ouo in selected lots of horse serum resulted in both a decrease in base-line K' efflux and an increase in responsiveness of K' efflux rate to muscarinic stimulation.

The validity of comparisons of efflux data and intracellular [K+] data from cultures of hearts 3% days in ouo to those from cultures of hearts 10 days in ouo is supported by the findings that 1) beating rates in cultures of hearts of both ages were not significantly different (Table I), 2 ) total cell number per culture disc was essentially the same for cultures of hearts of both ages, and 3) total protein per disc was the same at both ages.

The changes in rate of K' efflux during embryonic development reported here are in agreement with studies of Car- meliet et d. (35), who determined that the rate coefficients for 4'K+ efflux were higher in ventricular strips of chick hearts 3-5 days in ouo compared to strips from chick hearts 6-8 days in ouo, but increased again prior to hatching. These findings could be explained, at least in part, by the increased intracellular K' reported here in cultures of hearts 3% days in ouo. An increase in intracellular K' at early embryonic ages was demonstrated by McDonald and DeHaan (33) in ventricular strips of embryonic chick heart. They showed that intracellular K+ decreased from 167 mM at day 2 in ouo to 117 mM at day 14 in ouo. However, Fozzard and Sheu (34) using K'- sensitive electrodes in the embryonic chick ventricle reported an increase in intracellular K' concentration from 71.3 to 89.9 mM from day 4 to day 8 in ouo.

It has also been reported that during early embryonic development chick heart cells are relatively depolarized (33, 34). Membrane potential increased from -61.8 mV at day 3 in 000 to -80 mV at days 14-18 in ouo (33). The relative depolarization of heart cells at early embryonic ages and an increased intracellular [K+] might explain the increased rate of K' efflux in the cells reported here. The K' concentration gradient across the cell membrane is the driving force for the passive movement of K' out of the cells. The negative charge at the internal surface of the cell membrane favors the movement of K+ into the cell (30). EK is the reversal potential for K+ at which the diffusional forces due to the K+ gradient are exactly balanced by the membrane potential (Em). In depolarized cells where E,,, is significantly less negative than Ex, K' will move down its concentration gradient at a more rapid

rate. Hence both an elevated [K']i and the relative depolarization of the membrane in cells cultured from hearts 3% days in ouo could contribute to an increased base-line K' efflux.

The markedly reduced response of K+ permeability to muscarinic stimulation in cultures from hearts of embryos 3lh days in ouo compared to that in cultures from hearts 10 days in ouo might be secondary to uncoupling of muscarinic receptors from control of K' permeability at this stage of development. Alternatively, one must consider the possibility that elevation in the base-line 4'K+ efflux rate in cultures from hearts of embryos 3% days in ouo may reflect the fact that potassium permeability (PK) is already maximal in these cells and cannot be elevated further by muscarinic agonists.

However, other muscarinic functions not dependent on K' permeability are also attenuated prior to ingrowth of the vagus nerve. Although the response of adenylate cyclase to padre- nergic stimulation was fully expressed as early as 21h-3% days in ouo (29), we recently demonstrated that prior to vagal innervation of the heart carbamylcholine inhibition of isoproterenol-stimulated adenylate cyclase activity is decreased 10- fold compared to inhibition in hatched chick. In addition, the sensitivity of isoproterenol-stimulated adenylate cyclase activity to carbamylcholine prior to vagal innervation is decreased 35-fold compared to that in post-hatched chick hearts.' Hence, the finding that muscarinic inhibition of adenylate cyclase activity is also markedly decreased prior to vagal innervation argues that perhaps muscarinic preceptors may be uncoupled from various physiologic functions in the cell prior to vagal innervation, including the ability to increase resting K' permeability.

The apparent inability of muscarinic receptors to interact with guanine nucleotides in hearts studied prior to ingrowth of the vagus nerve may have important physiologic implica- tions. The interaction of guanine nucleotides with a guanine nucleotide regulatory protein has been shown to play a critical part in mediating a number of hormonal responses (13). In the stimulation of adenylate cyclase, guanine nucleotides have been shown to mediate three separate but related processes: 1) direct stimulation of adenylate cyclase activity, 2) conversion of high affinity /%adrenergic receptors to a low affinity form (28), and 3) coupling of the @-adrenergic agonist-receptor complex to the stimulation of the catalytic unit of adenylate cyclase (13). Data have been presented that support a model for hormonal stimulation of adenylate cyclase activity in which all three of these processes play an important part (14). Hence the stimulatory guanine nucleotide-binding protein (N,) plays a central role in regulating activity of adenylate cyclase and receptor affinity for agonist.

The function of guanine nucleotides in the modulation of muscarinic cholinergic activity in the heart is less well established. In parallel with findings in the P-adrenergic system, guanine nucleotides mediate the conversion of a high affinity muscarinic receptor to a low affinity form (9-11). The relationship of this guanine nucleotide effect to a physiologic response to muscarinic stimulation is not yet clear. The studies reported here demonstrate that an association exists between physiologic responsiveness of the heart to muscarinic stimulation and the ability of guanine nucleotides to mediate the conversion of RH to RL. Specifically, while high affinity receptors were unaffected by guanine nucleotides in homogenates of cultures from hearts 3% days in ouo, induction of responsiveness to muscarinic stimulation either during embryonic development (between day 3% and day 10 in ouo) or following growth of cells cultured from hearts 3% days in ouo

* B. T. Liang, E. J. Neer, and J. 3. Galper, submitted for publication.


in the presence of specific lots of horse serum was associated with development of the ability of guanine nucleotides to mediate the conversion of RH to RL. Growth in culture in the presence of these horse sera also resulted in an increase in the fraction of RH. One possible interpretation of these findings is that induction of a physiologic response to muscarinic stimulation involves the appearance of a specific guanine nucleotide regulatory protein in the cell that couples the muscarinic receptor-agonist complex to a physiologic response.

The existence of a guanine nucleotide-binding protein responsible for coupling of the muscarinic receptor-agonist complex to a physiologic response in the adult heart cell is suggested by studies of muscarinic inhibition of adenylate cyclase activity (23). Muscarinic inhibition of P-adrenergically stimulated adenylate cyclase activity is dependent on GTP and Na' ions and is associated with a GTP-mediated decrease in the affinity of the muscarinic receptor for agonist. GTP- dependent coupling of muscarinic inhibition of adenylate cyclase activity could in principle be associated either with the same guanine nucleotide regulatory protein (Ns) responsible for GTP-dependent @-adrenergic stimulation of adenylate cyclase or an independent inhibitory guanine nucleotide regulatory protein (Ni). Available evidence suggests that such an independent inhibitory guanine nucleotide regulatory protein is present in mature cardiac tissue. In rat heart membrane preparations, N-ethylmaleimide has been shown to interfere with a GTP-mediated decrease in agonist affinity for the muscarinic receptor, while having no effect on the GTP- mediated decrease in agonist affinity for the @-adrenergic receptor (31). Islet-activating protein, which interferes only with muscarinic inhibition of adenylate cyclase, has been shown to catalyze the ADP-ribosylation of a 41,000-dalton protein that is presumed to be an inhibitory guanine nucleotide protein (32). Furthermore, we have previously demonstrated that as early as 3 days in ovo, @-adrenergic agonists are capable of stimulating adenylate cyclase activity (29), and more recently we have shown that muscarinic inhibition of adenylate cyclase activity is markedly attenuated in hearts 3 days in O V O . ~ Hence the stimulatory guanine nucleotide regulatory protein must be present in cells in which muscarinic receptors are not yet coupled to the effects of guanine nucleotides.

These findings suggest the following conditions prior to vagal innervation of the heart: 1) a factor that couples the muscarinic receptor to guanine nucleotide responsiveness is either absent or present in an inactive form; 2) a muscarinic coupling factor is probably independent of the stimulatory guanine nucleotide regulatory protein; and 3) the factor that couples muscarinic receptors to the effects of guanine nucleotides, quite possibly an inhibitory guanine nucleotide regulatory protein, may be necessary for the coupling of the muscarinic agonist-receptor complex to physiologic responses such as alterations in K+ permeability and beating rate. If this were the case, heart cell cultures from hearts 3% days in ovo might offer an analog of the cyc- mutant of the S49 lymphoma cell line (13) for the study of the inhibitory guanine nucleotide regulatory protein.

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