DEVELOPMENTAL BIOLOGY 57, 254-269 (1977)

Cholinergic

and Adrenergic

Receptors

on Mouse Cardiocytes

MARY-ANNE LANE,’ ANTONIO SASTRE,' MARGARET AND MIRIAM M. SALPETER~ Section of Neurobiology

and Behavior

and School of Applied and Engineering Ithaca, New York 14853

Received July 21,1976;

accepted in revised form January

in Vitro

LAW,

Physics,

Cornell

University,

10, 1977

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2-14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via a- and padrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [3HlAlprenolol, a potent p-adrenergic receptor antagonist, and [3H]quinuclidinyl benzilate ([3H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize padrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave -800 sites/pm*, a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells. INTRODUCTION

level. Realization of these expectations has been hampered by the reported insensitivity of cardiocytes in vitro to a large number of such agents. Thus, one finds major studies which have shown heart cells in vitro to be insensitive to epinephrine, norepinephrine, acetylcholine, etc. (Sperelakis and Lehmkuhl, 1965; Sperelakis, 1972). Other authors have found sensitivity to catecholamines, but not to cholinergic agents (Noda and Yugari, 19731, or paradoxical responses that are not dosedependent (Ertel et al., 1971; Clarke et al., 1971). The present study re-examines this question in a mammalian system using spontaneously beating mouse cardiocytes. We were particularly interested in examining the response characteristics of isolated single cardiocytes and observing whether the same cell can have more than one pharmacologically specific receptor. We found that single cardiocytes tested can have both cholinergic (muscarinic) and ad-

The adult heart is innervated by the sympathetic and parasympathetic branches of the autonomic nervous system and is fully responsive to both adrenergic and cholinergic agents (e.g., Trautwein, 1963; Cooper, 1965: Higgins et al., 1973). Numerous investigators have shown that isolated beating heart cells can be readily obtained from embryonic material grown in vitro (e.g., Cedergren and Harary, 1964; DeHaan, 1967; Sperelakis, 1972). The availability of such systems, which share a number of properties with adult tissue, offers the possibility of studying molecular events underlying the action of autonomic and cardioactive agents at the single-cell 1 On leave at Department of Bacteriology and Immunology, University of California, Berkeley, Calif. 94720. 2 Present address: Department of Pharmacology, Cornell Medical College, 1300 York Avenue, New York, N. Y. 10021. 3 To whom correspondence should be addressed. 254 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

LANE

ET AL.

Cholinergic

renergic (a and p) receptors and that they can respond to their respective agonists in a dose-dependent manner and at physiological concentrations. Clusters of cardiocytes were more sensitive to both adrenergic and cholinergic agonists than were the single cells. Fine-structural and electrophysiological studies further confirmed the myocardial nature of these cells. We conclude that such mammalian cardiocytes do provide a fruitful system for studying cardiocyte development and the action of cardioactive drugs at a single-cell level. MATERIALS

AND

METHODS

Culturing. Cultures were prepared by the method of DeHaan (1967). Briefly, hearts from 9-, 14-, or 17-day in utero or 1 day postpartum mice (NIH Swiss, randombred) were aseptically removed from the donors, minced, washed free of red blood cells in Hanks’ balanced salt solution, and subjected to stepwise 7-min trypsinizations (0.05%; Flow Laboratories). The resultant cell suspensions were pooled, washed, resuspended in complete medium, and plated in 35- or 60-mm tissue culture (Falcon or Corning) dishes. Cells were grown in Ham’s F12 medium (GIBCO) freshly supplemented with 2% horse serum and 4% fetal calf serum (Flow Laboratories; both heat-inactivated for 30 min at 56”C),2 mM glutamine, 25 III/ml of penicillin, 25 pglml of streptomycin, in an atmosphere of 5% COZ, 95% air, 99% humidity at 37°C. Approximately 5 x lo3 tells/35-mm dish or 5 x lo4 cells/go-mm were plated. The cultures were left undisturbed for the first 48 h, after which they were washed and fed daily. Care was taken in the dissection to trim off visible arterial and venous tissue. Hearts from an entire litter constituted a single culture. The stages of development of the mouse embryos are given by Theiler (1972). Pharmacology. Additions of cardioactive reagents were made by preparing a

and Adrenergic

Receptors

255

final dilution in a 2-ml volume of medium and adding this to drained cultures. Where several active ingredients were added sequentially, lo- ~1 quantities were added with a microliter pipet directly to the dish into a 2-ml volume of medium. All reagents were allowed to equilibrate in cultures for 10 min at 37°C. Beat rates were monitored using a Wild inverted microscope at 450 x for 2-min periods at room temperature or on a 37°C temperature controlled stage (McCall 1976). a-Bungarotoxin was purified from crude venom by the method of Lee et al. (1972). m-Norepinephrine HCl (DL-NEpi), m-epinephrine (DL-Epi), m-propranolol HCl, m-isoproterenol HCl, L-phenylephrine HCl, and Bungarus multicinctus crude venom were obtained from Sigma; phentolamine methanesulfonate from CIBAGEIGY; carbamylcholine chloride, d-tubocurare, and atropine sulfate from Calbiochem; and 3-quinuclidinyl benzilate (QNB) was obtained from Roche. Autoradiography. 3-Quinuclidinyl benzilate (QNB) was tritiated by New England Nuclear (NEN) and purified in our laboratories as described by Yamamura and Snyder (1974). The final material was 95-97% pure by thin-layer chromatography and had a specific activity of 1.8 Ci/ mmole. 13HlAlprenolol (32.6 Cilmmole) was obtained from NEN. Cultures were incubated in complete medium with varying concentrations of the radioactive ligands (up to 2 x 10e8M [3H]QNB for 1 h at 37°C or up to 4 x 10-8M [3H]alprenolol for 20 min at 2o”C!), washed five times with ice-cold medium for a total of 10 min, -fixed with 2.5% glutaraldehyde in 66 mM Na-K phosphate buffer, pH 7.2-7.4, for 60 min, rinsed with buffer plus 3% sucrose and then with distilled water, and finally airdried (all at 4°C). To ensure a uniform emulsion coating over dried cells, a monolayer stripping film (Land et al., 1977) was used based on the “flat substrate” method calibrated for electron microscope autoradiography (Sal-

256

DEVELOPMENTAL BIOLOGY

peter and Bachmann, 1973). A monolayer of Ilford L4 emulsion, as judged by purple interference color, was formed on a collodion-coated slide. In the darkroom, under Wratten OA safelight filter (yellowgreen), the collodion plus emulsion was stripped onto a distilled water surface, using one or two drops of 10% hydrofluoric acid as a stripping aid. The film was then lifted from below onto a square of filter paper (Whatman No. 50) (from which a small hole had been cut) and was placed emulsion side down over the cells. Circles scratched on the bottom of the petri dishes marked formerly beating single-heart cells. The filter paper was allowed to dry down on the cells for about 2-3 min, and the tissue culture dish was then dipped into a water dish and inverted. This caused the filter paper to imbibe water and fall off, leaving the emulsion layer adhering to the cells. Occasionally, the emulsion layer did not lie smoothly on the cells. This could be determined visually while holding the dish up to the safelight before the emulsion was fully dry. At this stage, any defective emulsion layer could be washed off and replaced. Using this procedure, the emulsion is directly in contact with the cells, and the collodion backing is on the other side of the emulsion. This allows absolute calibration of emulsion sensitivity, since no correction for self absorption in the collodion-backing layer is necessary. However, the close proximity of emulsion to cells causes a risk of chemography (i.e., chemical interaction between tissue and emulsion). To avoid this, a thin carbon layer is evaporated over the cells before emulsion coating. Furthermore, before the emulsion is developed, the collodion film has to be removed. This is done by a 5-min wash in 100% ethanol, followed by a few ethanol rinses and then air drying. The emulsion is then developed with D-19 (Kodak) at room temperature for 4 min, rinsed in distilled water, then fixed with nonhardening fixer (Gevaert) for 2 min, and finally rinsed in water and

VOLUME 57, 1977

air dried. A cover slip is mounted on the autoradiogram with a drop of glycerol, and grains are counted using a calibrated ocular at 630 x light optics. Since the emulsion layer has the same thickness and packing density as previously calibrated for EM autoradiography (deep purple interference color) (Salpeter and Szabo, 19731, using this developer, one can assume that the emulsion sensitivity to tritium radiation will be the same as that found in the previously mentioned study, i.e., approximately one-fourth or one grain per four radioactive decays for D19 development and Ilford L, monolayer of emulsion. [Such layers were, in fact, calibrated for sensitivity to lz51 by Land et al. (1977) and were indeed found to be identical to calibrations made on the EM autoradiographic level.] The EM autoradiographic calibration is applicable to light autoradiographic conditions, only provided that the labeled source is no thicker than -2000 A, at which time self absorption will begin to be a problem. For the present study, we assumed that the receptor antagonists produce primarily a surface label and could thus fulfill the above requirement. Dishes labeled with 13Hlalprenolol were exposed at 4°C for 11-16 days, and those labeled with [3H]QNB were exposed under the same conditions for 26-30 days. Autoradiographic-developed grain counts were used to calculate receptor density using the following formula (Salpeter and McHenry, 1973):

R = (gdlt) x A/(&C),

VI

where R is the number of receptors per unit area of tissue, g is the number of developed grains for that same unit area of the tissue, d is the number of decays to give one developed grain (from sensitivity value), t is the exposure time, A is Avogadro’s number, S, the specific activity of the labeled antagonist, and C the number of decays per curie (C and t; A and So must be expressed in the same units).

LANE ET AL.

Cholinergic

Electrophysiology. Electrophysiological measurements were made at room temperature, 20-22”C, in Tyrode’s solution of the following composition (in mmole/liter): NaCl, 138; KCI, 2.7; MgC12, 1.8; CaCl,, 1.8; NaH,P04, 0.42; glucose, 5.5; Hepes (N-2hydroxyethylpiperazine-N’-2-ethane-sulfonic acid), 18. The pH was adjusted to 7.4. To minimize the effects of evaporation, the solution was replaced every 15-20 min. The electrodes used had dc impedances of 50-80 mR. Only even-tapered electrodes with tips beyond the resolution of the light microscope (co.4 pm in diameter) were used. The electrodes were connected via a capacity-neutralized preamplifier (Model 701 from WP Instruments, Inc.) equipped with a bridge circuit. Electron microscopy. Cells were fixed in 2.5% glutaraldehyde plus 0.2% 0~0, in 66 mJ4 Na-K phosphate buffer (pH = 7.4) for 20 min at 2”C, followed by 2.5% glutaraldehyde in the same buffer for an additional half-hour. The cells then washed several times in phosphate buffer plus sucrose, postfixed in 1% OsO,, stained with 1% uranyl acetate (aqueous) for 1 h, and dehydrated in graded alcohol. A thin layer of Epon 812 was then poured over the cells and allowed to polymerize overnight at 60°C. The cells to be sectioned were identified under the light microscope and circled either at this time or before fixation. The desired area, including the epon and tissue culture dish, was sawed out, and the Epon layer was separated from the plastic dish. The cells (which remained in the Epon) were then re-embedded in flat rubber molds for sectioning at right angles to the dish or were glued onto Epon blocks for sectioning parallel to the plane of the dish. Pale gold sections were cut, stained with uranyl acetate and lead citrate, and examined with a Philips electron microscope. RESULTS

Morphology Beating cardiocytes were seen as single isolated cells, as double cells, or as multi-

and Adrenergic

Receptors

257

ple clusters, lying either flat, side-by-side, or overlapping. The distribution of cell types varied with culture age. Cultures l-3 days of age contained 70-80% cardiocytes, 40-50% of which were beating during any given observation period. Beat rates of different cells varied from 18-175 beats/min. The rate of any given cell remained stable to within -+4 beats/min over a 4-h observation period. Single cells or doublets were distributed throughout the dishes. Large flat cells, -200 km in diameter, composed the major portions of the noncardiocyte population. Cultures 4-8 days of age contained approximately 60% cardiocytes, again with about 40-50% beating at any one time. Single cardiocytes were observed less frequently during this period, as most were joined in synchronously beating clusters of two or more cells. Between Days 8 and 14 in culture, large sheets or clusters of beating and nonbeating cardiocytes were observed. It was difficult on the light microscope level to determine the ratio of cardiocytes to noncardiocyte cells in overgrown clusters. Electron microscopy suggests that the cardiocytes constituted the core of beating clusters, with noncardiocyte cells forming the outer layers. Cardiocytes divided most frequently at 2-4 days in culture. Cells stopped beating during division and resumed beating 1.5-2 h after division was completed. About one or two cells could be seen dividing over a lh observation period out of a population of -200 cells in a random field. In cultures from g-day in utero mice, we also found surviving isolated neurons which have been described in detail elsewhere (Lane et al., 1976). Single beating cardiocytes obtained from all ages studied have a similar morphology in culture. Figures 1, A and B, indicate the two types of single beating cells commonly observed: one flat, the other spindle-shaped. We are as yet not sure whether they represent two distinct cell types or whether they represent differ-

258

DEVELOPMENTAL

BTOLOGY

ent phases of development of the same cell type. In pharmacological response and range of beat rate, both types are indistinguishable.

FIG. IA and 1B. (A and B) Beating single cardiocytes, culture ‘s independent of mouse age. 420 x .

VOLUME

57, 1977

Figures 2 and 3 show representative electron micrographs of single and clustered cardiocytes. The degree of specialization, both intracellular and cell-to-cell,

2 days in culture.

Same morphology

seen in yol

FIG. 2. Electron micrograph of beating cardiocyte from g-day in utero mouse, grown 3 days in culture. Note rough endoplasmic reticulum and single strand of myofilaments with z bands (arrow). 20,200 x

LANE

ETAL.

Cholinergic

end Adrenergic

Receptors

259

Although paradoxical responses have been reported, the heart rate in viuo is decreased primarily be acetylcholine and increased by epinephrine and norepinephrine (Carrier and Bishop, 1972; Higgins et aZ., 1973; Koelle, 1975). We wanted to determine whether single isolated cardiocytes in culture could respond to autonomic agents and whether both adrenergic and cholinergic receptors could coexist on the same cell. We found that isolated cells could respond in a dose-dependent fashion to the three autonomic agents tested (DLepinephrine, nL-norepinephrine, and carbamylcholine),4 as illustrated in Fig. 4.

We found that, in the single cells in uitro, a maximum decrease in beat rate is obtained with 10e4 M carbamylcholine, and a half-maximal reduction is obtained at 2 x 10e6 M (Fig. 4). In contrast, large cell clusters responded at much lower concentrations. Maximum reduction was seen at 10esM with a half-maximal reduction at 3-4 x lo-‘* M. Some of the clusters stopped beating at lops M. Since cholinergic agonists can affect a target cell via either a nicotinic or a muscarinic ACh receptor and, in the normal heart, these act via the latter receptor (see review by Koelle, 1975), we determined the nature of the in vitro cardiocyte receptor system using specific antagonists of both types of receptors (Trautwein, 1963; Lee et al., 1972; Yamamura and Snyder, 1974; Innes and Nickerson, 1975a). Full inhibition of the effects of (1O-5M) carbamylcholine on single cells is seen with the muscarinic antagonists, atropine (lop6 M), QNB (2 x 10mgM), or scopolamine (10m8 M; 50% inhibition at lop9 M). In contrast, the nicotinic antagonists, d-tubocurare (1O-g-1O-5 M) or a-bungarotoxin (lo-’ M) were without effect. None of the antagonists tested by themselves in the absence of agonists had any effect on the beat rate. Thus, these results indicate that mouse cardiocytes contain a functional muscarinic cholinergic receptor in culture. The same cardiocytes also responded to nL-epinephrine (DL-Epi) and m-norepinephrine (DL-NEpi) with a dose-dependent beat rate increase. A plateau for both agents was reached at 10e6 M; 50% of the maximum increases are obtained at 1 x 1O-g to 1 x lo-* M for DL-Epi and 2-3 x lo-’ M for DL-NEpi (Fig. 4). DL-Epi and DL-NEpi can affect a target cell via either

4 Carbamylcholine is a cholinergic agonist with about 1 x IO-’ to 3 x 10m5M apparent dissociation constants in nicotinic (Higman et al., 1963; Nastuk et al ., 1966) and muscarinic (Furchgott and Bursztyn, 1967; Bolton, 1973) systems. We used carbamylcholine instead of acetylcholine (ACh) to study cholinergic interactions because of its virtual resistance

to hydrolysis by serum and endogenous esterases. We did not use acetylcholine in conjunction with esterase inhibitors (e. g., physostigmine) due to the atropine-like action of such e&erase inhibitors in muscarinic cardiac systems (Ertel et al., 1971; Sastre and Lane, unpublished).

seemed to depend more on the age of culture than on the ages of the mice from which the hearts were taken. In all beating heart cells, myofilaments with z bands were seen. These findings are consistent with others reported in the literature (Cedergren and Harary, 1964; Zachei and Caravita, 1972; Purdy et al., 1972; Fishman, 1972). The greatest degree of differentiation reminiscent of adult heart in uivo was seen in the overlapping cardiocyte clusters prominent in older cultures. Specializations included subsarcolemmal cisternae (McNutt and Fawcett, 1969) and gap junctions (nexuses) constituting a part of the developing intercalated disks (Dewey and Barr, 1964; Revel and Karnovsky, 1967; McNutt and Fawcett, 1969; Gilula, 1975; see Fig. ?B). The development of gap junctions in cultured cells taken from g-day mouse embryos is at variance with the situation in vivo where, according to Hirakow and Gotoh (1975), these junctions do not develop until postnatal stages. Functional Agents

Responses

to

Autonomic

260

DEVELOPMENTAL BIOLOGY VOLUME57, 1977

FIG. 3A. Electron micrograph of synchronously day in utero mouse. Note relatively undifferentiated 18,000 x.

an (Y- or P-adrenergic receptor (Innes and Nickerson, 1975b). We examined this question on the isolated mouse cardiocytes with CY-and /3-adrenergic agonists and antagonists. Figure 5A illustrates the inhibition of the DL-NEpi-induced increase in beat rate by the a-adrenergic antagonist phentolamine. We note that a full inhibition is not seen with this antagonist, even

beating cardiocyte “doublet,” intercalated disk (arrows)

3 days in culture, from 9and sparse myofilaments.

at 1O-5M. However, when lo+ M, propran0101, a p-adrenergic antagonist, was added in addition to phentolamine, the beat rate returned to baseline. In general, the response to DL-NEpi was reduced by 60% with lop6 M propranolol and by 40% with lo+ M phentolamine. The response to DLEpi, on the other hand, appears to be mediated somewhat more by the cY-adrenergic

LANE

ET AL.

Cholinergic

and Adrenergic

Receptors

261

FIG. 3B. Electron micrograph of synchronously beating cardiocytes from a ll-day in culture heart cell cluster derived from a O-day in utero mouse. Subsarcolemmal cisterane (asterisks), intercalated disks, and nexuses (arrow) are prominent, and myofilaments are more extensive than those in younger cultures. 32,200 x.

receptor, since its response was reduced 40% by lo+ M propranolol and 60% by lop6 M phentolamine. For both DL-NEpi and DL-Epi, one could abolish the entire response only by addition of both phentolamine and propranolol. The antagonists

by themselves had no effect on beat rate. These results are consistent with at least two possibilities: (i) that each cardiocytes possesses both (Y- and p-adrenergic receptors, or (ii) that they possess an “intermediate” or “mixed” receptor. To help

262

DEVELOPMENTAL BIOLOGY

FIG. 4. Response of single isolated cardiocytes to autonomic agents. Cells from (A) g-day in utero: (B) 17-day in utero; or (C) l-day postnatal hearts were tested after 2 days in culture. A base beat rate was determined in the absence of any agent. The agents were then added in increasing concentrations until a plateau of the response was obtained. The difference in beat rate at the plateau from the base rate is considered 100% response. The values at the lower concentrations are computed with respect to the 100% response. Values shown are the means k SE, pooling 7-10 cells tested for each point.

VOLUME 57, 1977

bamylcholine, the clusters of cardiocytes tested were again more sensitive to DLEpi and DL-NEpi than were the isolated single cells. However, monitoring beat rates of cardiocyte clusters is difficult because of their tendency to fibrillate at concentrations of DL-Epi and DL-NEpi 2 lop8 M. Occasionally, some quiescent cardiocytes resumed beating in response to DLNEpi or DL-Epi. Responsiveness to autonomic agents is not always a stable property in vitro. Although no cells that were sensitive to carbamylcholine were ever seen to be insensitive to the adrenergic agonists, the reverse was sometimes seen. Occasional cells were also insensitive to both agents. On the other hand, considerable stability in the responsiveness of individual cells is illustrated by several identified beating cardiocytes (or their progeny) that responded in the same dose dependent fashion over a three day period. Electrophysiology

distinguish between these possibilities, the It was not possible to obtain stable recordings from isolated single cardiocytes. effects of the predominantly a-agonist Lphenylephrine and of the predominantly Intracellular recordings were, however, P-agonist nL-isoproterenol were investimade from beating heart clusters (Fig. 6, gated. Figure 5B demonstrates that the /3- A and B), in which we saw the presence of spontaneous “atrial” and “Purkinje”-like agonist m-isoproterenol-induced increase in beat rate is fully antagonized by the p- action potentials (Hoffman and Craneantagonist propranolol and unaffected by field, 1960). We could record from these the a-antagonist phentolamine (10e6 M). cells for several hours and found that they contracted in a 1:l relation with the action Conversely, Fig. 5C shows that the increase in beat rate is induced by the cr- potentials. We never observed uncoupling agonist L-phenylephrine and is only of excitation and contraction as reported by Sperelakis (1972). Three types of cells slightly affected by the P-antagonist prowere obtained as far as their response to pranolol (lop6 M), whereas the major porIn the first type, tion of the response is abolished by the cy- electrical stimulation. neither the frequency nor the amplitude of antagonist phentolamine (lop6 M). The the action potentials were affected by cursimplest explanation for these observarent of either polarity. In the second type, tions is that the same cardiocyte in culture possesses pharmacologically specific (Y- the action potentials were unaffected by current (Fig. 6C), but exand P-adrenergic receptors, and that, as depolarizing hibited a marked decrease in frequency found in other tissues (Innes and Nickerupon application of hyperpolarizing curson, 1975b), DL-Epi and DL-NEpi can actirent (Fig. 6D). In the third type, the action vate both. potentials were quite sensitive to currents As in the case with the response to car-

8 7 6 5 -Log[Phentolamine] M

9

8

7

6

-Log[lsoproterenol M

6 ]

- Log[Phenylephrine] M

FIG. 5. (A) Inhibition of DL-NEpi-induced beat rate increase by the o-adrenergic antagonist phentolamine and the P-adrenergic antagonist propranolol. The base beat rate was elevated in the presence of 1O-5 M DL-NEpi. Phentolamine was added in increasing concentrations, reducing the beat rate until a plateau of response was reached. At the plateau point, propranolol (- $ -1 at 10e6 M was added, reducing the beat rate to baseline. (B) Inhibition of beat rate increase induced by the p-adrenergic agonist isoproterenol with A base beat rate was established, then the p-adrenergic agonist propranolol but not phentolamine. isoproterenol was added in increasing concentrations until a plateau was reached. Addition of the ccadrenergic antagonist phentolamine (A) at 10e6 M produced no effect, whereas addition of the p-adrenergic antoganist propranolol (-0-j at 10m6M reduced the beat rate to base line. (Cl Inhibition of beat rate increase induced by the predominantly ru-adrenergic agonist L-phenylephrine by propranolol and phentolamine. A base beat rate was established; then the predominantly a-adrenergic agonist L-phenylephrine was added in increasing concentrations until a plateau was reached (lo-’ Ml. At this point, the p-adrenergic antagonist propranolol (- 5 -) was added at 10e6 M causing a small beat rate reduction. The cu-adrenergic antagonist phentolamine (A) at 10m6M reduced the remaining response to baseline. Values shown for 5A, B, and C are the means + SE, pooling 6-10 cells tested for each point.

FIG. 6. Intracellular recordings and also monitors applied current. cases are 20 mV (or 5 nA of current C and D, and 5 set for E and F. “ventricular”-like action potential; frequency, whereas (D) illustrates the action potential frequency; (E) to de- and hyperpolarizing current.

from cardiocytes in clusters. The upper trace represents zero potential The lower trace measures membrane potential. Vertical divisions in all for the upper trace). Horizontal divisions are 0.2 set for A and B; 2 set for Figure 6A illustrates an “atrial” action potential; (B) a “Purkinje” or (C) same cell as (D) depolarizing current (up to 2 nA) fails to affect the that passing hyperpolarizing current (about 1 nAf causes a reduction in and (F) two examples of cells which responded with a change in frequency 263

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DEVELOPMENTAL BIOLOGY

of either polarity; the frequency was increased and the amplitude reduced upon depolarization, and, conversely, the frequency was reduced and the amplitude increased upon hyperpolarization (see Fig. 6E and 6F). Types 1 and 3 are similar in response characteristics to cardiocytes described by Sperelakis (1972).

Autoradiography Autoradiography was employed both qualitatively and semiquantitatively. Qualitative studies were used to localize muscarinic ACh and p-adrenergic receptors. The extent of nonspecific binding was first assessed by the labeling of cells other than heart cells and by the labeling of heart cells after preincubation to saturation with a high concentration of a nonradioactive agonist or antagonist of the specific receptor to be labeled. The labeling of the muscarinic ACh receptor with its specific inhibitor, r3HlQNB (Yamamura and Snyder, 19741, was competitively blocked by nonradioactive QNB, atropine, or scopolamine, but not by a-bungarotoxin. The labeling of the /I-adrenergic receptor with its antagonist [3Hlalprenolol (Alexander et al., 1975) was competitively blocked by the nonradioactive antagonist, propranolol. L3H]QNB gave very low nonspecific binding, even at concentrations of 2 x lop8 M, i.e., 10x that found to block maximally the effects of carbamylcholine. Background grain densities on noncardiocytes remained at