cardiac muscarinic cholinergic receptors · rinic cholinergic drugs that inhibited specific binding...

THE JOURNAL OF BIOWGICA~ CHEM,STRY Vol. 253, No 9, issue of May 10, pp. 3251-3258, 1978

Prmted in U.S.A.

Cardiac Muscarinic Cholinergic Receptors BIOCHEMICAL IDENTIFICATION AND CHARACTERIZATION*

(Received for publication, May 31, 1977, and in revised form, December 20, 1977)

JEREMY Z. FIELDS,+ WILLIAM R. ROESKE, EUGENE MORKIN, AND HENRY I. YAMAMURA§

From the Departments of Pharmacology and Internal Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724

The parasympathetic nervous system modulates heart rate and myocardial contractility. We have labeled the parasympathetic muscarinic cholinergic receptors in rabbit, rat, and guinea pig hearts using the specific high affinity ligand, [“Hlquinuclidinyl benzilate (L3HlQNB). Specific [“HIQNB binding in rabbit heart homogenates was defined as the difference in binding between the absence and presence of atropine (1 PM). At all L3HIQNB concentrations studied, specific binding increased linearly with tissue concentration in the range of 0.025 to 0.5 mg of protein/ml. Analyses of saturation isotherms at progressively decreasing tissue concentrations gave an apparent dissociation constant, K,,app, of 27.1 pm which was similar to the K,,app for rat brain homogenates (20.0 PM). The rate constants at 37°C for formation and dissociation of the QNB. receptor complex in rabbit heart were 1.03 x 10y M-’ min-’ and 2.45 x lo-’ min.‘, respectively. The mean value for the dissociation constant from the pairs of rate constants (K,, = k- Jk,, = 26.8 PM) was similar to the value determined from saturation isotherms. Stereospecificity of the receptor was demonstrated in experiments in which [“HIQNB binding was inhibited by dexetimide in the nanomolar range (K, = 0.69 nM) and by levetimide, its stereoisomer in the micromolar range (Ki = 1.45 FM). Values of K, (nanomolar) for muscarinic cholinergic drugs that inhibited specific binding were: atropine 1.05, oxotremorine 144, acetylcholine 490, carbamylcholine 528. The nicotinic cholinergic agents cY-bungarotoxin and nicotine were ineffective in displacing r3H]QNB binding at concentrations of 5 and 100 PM, respectively. The antiarrhythmic agents quinidine, lidocaine, and procainamide were also inhibitory with K, values (micromolar) of

* Portions of this research was SuDDorted bv an institutional mant and a grant-in-aid from the American Heart Association, Ar%ona Affiliate, and by United States Public Health Service Grants MH- 27257 and HL-i1486 and Program Project Grant HL-20984. This work has been presented in part at Federation of American Societies of Experimental Biology and American Society for Clinical Investi- gation and abstracts have been published (1, 2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734Wsolely to indicate this fact.

$ Supported by United States Public Health Service Postdoctoral Fellowship MH-05248 from the National Institute of Mental Health.

§ Recipient of a United States Public Health Service Research Scientist Development Award, Type-II (MH-00095) from the Na- tional Institute of Mental Health.

2, 18, and 48, respectively. The tricyclic antidepressant, imipramine, also was an inhibitor with a value for K, of 0.17 FM. Ouabain, propranolol, phentolamine, dopamine, and norepinephrine were ineffective in inhibiting QNB binding at 100 PM concentrations. In the rabbit heart, receptor densities (picomoles per g of protein) were: whole heart 57.2, left atrium 302, right atrium 200, ventricular septum 58, right ventricle 53, and left ventricle 37. We conclude that: 1) a population of specific, saturable, high affinity, muscarinic cholinergic receptors in the mammalian heart has been identified and characterized; 2) these binding sites appear to be similar to muscarinic cholinergic receptors in the brain; 3) the regional distribution of these receptors is consistent with classical physiological observations and with the relative distribution of acetylcholine and choline acetyltransferase within the heart; 4) cardiotonic agents, tricyclic antidepressants, and antipsychotic neuroleptics can interact with these myocardial cholinergic receptors.

The parasympathetic nervous system exerts an important regulatory control over heart rate and myocardial contractility (3). The negative chronotropic and inotropic effects of vagal stimulation and acetylcholine infusion on atria1 tissue have been established in the intact animal, isolated heart, and muscle bath preparations (3-10). Similar effects on ventricular performance have also been demonstrated (3, 4, 7). These effects of vagal stimulation and acetylcholine are blocked by atropine. The receptor for the post-ganglionic vagal innervation has been classified as muscarinic cholinergic (3).

Biochemical studies of these cardiac muscarinic cholinergic receptors would permit correlation of physiological studies with receptor affinities and densities and would aid the

correlation of drug mechanisms with interactions at the receptor level. Previous attempts to characterize biochemically the muscarinic cholinergic receptors have included stimulation of guanylate cyclase by muscarinic agonists (11, 12) and measurements of acetylcholine and its biosynthetic enzyme, choline acetyltransferase (13, 14). These latter measurements were complicated by the presence or’ cardiac parasympathetic gan- glia containing cholinergic nicotinic nerve endings necessitat- ing comparisons with denervated hearts (3, 4, 15, 16).

A direct ligand-receptor binding assay for the cardiac muscarinic cholinergic receptor would avoid the limitations of the

previous methods. Recently an assay using radiolabeled

3251

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3252 Cardiac Muscarinic Cholinergic Receptors

13H]quinuclidinyl benzilate ([“HIQNB) to demonstrate the muscarinic cholinergic receptor in the rat brain and in the longitudinal muscle of the guinea pig ileum has been devel- oped (17-19).

In this communication, we report the use of this ligand binding assay to demonstrate and characterize a single population of myocardial muscarinic cholinergic receptors in the guinea pig, rabbit, and rat myocardium. We used the assay to compare the myocardial receptors with the central nervous system receptors, to establish regional densities of receptors in mammalian heart, and to examine drug interactions at the receptor level.

MATERIALS AND METHODS

Tissue Preparation -Male New Zealand white rabbits (1.5 to 2.5 kg), male Sprague-Dawley rats (150 to 300 g), and male Hartley guinea pigs (300 to 800 g) were killed and the pertinent organs were quickly excised. Hearts were perfused through the aorta with 0.9% NaCl (4°C) until the coronary arteries were cleared of blood. Addi- tional blood clots, connective tissue, large vessels, and fat were trimmed and removed. Dissection of the rabbit heart for regional distribution was performed at 4°C. The free wall of the right ventricle was incised and removed leaving the floor of the right atrium and the intraventricular septum intact. The right atrium, including its floor, and the intra-atria1 septum then were removed followed by the free wall of the left atrium and its floor. Finally, the intraventricular septum was dissected from the free wall of the left ventricle.

Hearts were minced with scissors, mixed with 9 volumes of 0.32 M sucrose and homogenized at setting 5 on a Polytron (Brinkmann Instruments) with three 15-s bursts separated by 30-s pauses. Ho- mogenates of rat brain and of the longitudinal muscle of the guinea pig’ileum were prepared as described previously (17, 18). The heart homogenates were filtered through four layers of cheesecloth and utilized in the r3H]QNB1 binding assay. Protein content was determined by the method of Lowry et al. (20) using bovine serum albumin as standard.

Drugs and Chemicals -Tritium-labeled QNB (13 Ci/mmol) was obtained from New England Nuclear (NEN) and its purity checked by thin layer chromatography (17, 18). The authenticity of the material was confirmed by chromatography with a sample of nonra- dioactive QNB (Roche Co.) as standard. Dexetimide and levetimide, which are the stereoisomers of benzetimide, were gifts from Elliott Richelson, Mayo Clinic, Rochester, Minn. The QNB analogp-OH- QNB was a gift from Steven Flanagan, Friedrich Miescher-Institute, Basel, Switzerland.

Binding Assay-The incubation medium used for binding was 0.05 M (Na/K)-phosphate buffer, pH 7.4 (17). Specific r3H1QNB binding was experimentally determined from the difference between counts bound in the absence and presence of 1 pM atropine sulfate. In assays where either oxotremorine (100 pM) or unlabeled QNB (0.01 /LM) was used as an inhibitor to define specific binding, the results were identical. Aliquots of homogenates (0.025 to 0.5 mg of protein), 13H1QNB, and other drugs were incubated at 37°C with agitation. Incubations were carried out routinely for 60 min, unless otherwise indicated. Incubation medium was routinely added to a total volume of either 2 or 10 ml. All experimental points were determined in duplicate. Each incubation was terminated within 10 s by filtering the suspension through a GFlB glass fiber filter (Whatman) positioned over a vacuum. The filter was rinsed four times with 5 ml of ice-cold buffer. r3HlQNB retained on the filter was extracted for 16 h with 8 ml of scintillation fluid. The fluid was prepared by mixing 2 liters of toluene (Baker), 1 liter of Triton X- 100 (NEN), and 16 g of Omnifluor (NEN). Radioactivity was counted in a Searle Mark II liquid scintillation counter (45% efficiency).

Typically, 100 to 500 cpm of total filter binding per assay tube was obtained, about 90% of these counts were specific L3H1QNB binding. Small amounts of binding to the filter accounted for much of the nonspecific binding. Conditions were used to limit total binding to less than 5% of the radioactivity in the medium so that the concentration of free liquid did not change appreciably during the binding assay.

’ The abbreviation used is: QNB, quinuclidinyl benzilate.

Analysis of Data-For the analysis of binding data, the following simple model was assumed:

k H+ReRH (1)

where H is [3H]QNB, R is a high affinity binding site of the muscarinic cholinergic receptor, and RH is the bimolecular complex formed. Using the equilibrium (e) concentrations for each species and the relationship for the dissociation constant:

(2)

replacing (R), in Equation 2 by [CR), - (RH),,], where CR),. is the molarity of the total number of binding sites, and making the usual rearrangement (21) yields the rectangular hyperbola:

(31

(H), was estimated by (H),, the initial free molar concentration 13HlQNB, since incubation conditions were adjusted such that the concentration of bound ligand remained below 5% of added ligand. Equation 3 was transformed to a linear form according to the method of Scatchard (221 yielding:

1 + (R),

When the binding data are plotted in this manner, the negative of the slope of the regression line gives the dissociation constant, K,>. From the y intercept, CR),, the total receptor density in the cardiac tissue, R,,, (picomoles per g of tissue), can be calculated.

The rate constant for formation of (RH), k,,, was determined from the reversible second order equation:

0, WIT - VW. (R)T 1

(II)r. [@We - NOI 1 where (RH) is the molar concentration of receptor complex at time t. (R), in this case is the initial free molar receptor concentration derived from the y intercept of Scatchard plots and the incubation volume. The rate constant of dissociation, km,, was calculated from:

k-,=(i).ln[E]

where (RH), is the equilibrium level of complex formed just before addition of displacer (1 PM atropine). The half-time for dissociation was calculated from a rearrangement of Equation 6:

t I,% = WWk-, (7)

The K, can be calculated independently of equilibrium methods from the ratio of the rate constants:

KU = k-,lk+, (8) The ability of a drug to inhibit specific 13H1QNB binding was

estimated by IC,,; i.e. the concentration of the compound which inhibits binding by 50% under the particular experimental conditions. In many recent binding studies, a value for the inhibition constant, K,, has been calculated from the IC,, and from Cheng et al. (23):

where K, is the dissociation constant for [3H]QNB binding. In practice, this equation should be valid when the receptor concentration, CR),, is below 0.1 K,. When this is not the case, the K,] will be an apparent K, (K,app) and fractional occupancy of receptor sites, f, in relation to added ligand, (H),, will depend on the total receptor concentration, CR),, (Equation 44 of Ref. 24):

(H),.- f +f 0, (K,J (1 - fl [ 1 &I

(10)


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Cardiac Muscarinic Cholinergic Receptors 3253

In the present work therefore, we have used Equation 9 and the K, value determined from kinetic experiments (see “Results”) only to normalize a set of IC,,, values for a given drug that have been determined at varying (H), concentrations. Decreasing the receptor concentration to below 0.1 K, was not possible because insuffIcient counts would be bound on each filter to measure accurately.

The Hill coefficient, for inhibition of [SH]QNB binding by unlabeled QNB and by other drugs was determined from:

I = n.lw cm. (11)

where (R),. and (RH) are the equilibrium concentrations of receptor complex in the absence and presence of inhibitor, respectively.

Regional differences in cardiac receptor densities were determined from the y intercepts of Scatchard plots for each region and then compared using Student’s t test (25).

RESULTS

To insure proper kinetic conditions and to optimize and maximize specific 13H]QNB binding to the cardiac receptors, we examined the effect of varying the protein and electrolyte composition of the medium. Specific 13H]QNB binding was linear with tissue concentration (Fig. 1) up to 0.5 mg of protein/ml when the free ligand concentration was varied from 5 to 500 PM. 13H]QNB binding was not altered detectably when other buffers, such as Krebs-Ringer phosphate or (Na/ K)-Tris-Cl, were substituted for the (Na/K)-phosphate buffer. Specific I 3H]QNB binding remained constant over a pH range from 6.8 to 8.0. 13H]QNB binding changed by less than 10% when the concentration of K+ and Na+ was varied from 0 to 100 mM and the concentration of Ca’+ and Mg2+ was varied from 0 to 10 mM.

Biochemical Characteristics of j3H]QNB receptor Com- plex -Two important aspects of the identification and characterization of the receptor are: 1) the demonstration of a saturable population of receptor sites, and 2) the determination of a high affinity dissociation constant, K,,, consistent with values found in physiological experiments (18). The K,, was evaluated by two different methods.

In the first method, the saturability of specific 13H]QNB binding in cardiac homogenates was measured as a function of the added 13HlQNB concentration (Fig. 2). The saturation isotherm resembled a rectangular hyperbola, suggesting that

PROTEIN (mg/ml)

FIG. 1. Effect of tissue concentration on specific r3HlQNB binding to rabbit heart homogenates. Specific 13HlQNB binding was determined as explained under “Materials and Methods.” Total r3HIQNB concentration was 40 PM. Total volume = 2 ml, temperature = 3i”‘C, pH = 7.4. Incubation time was 60 min. All points were determined in triplicate. Incubations were started by the addition of tissue.

[SH]QNB (pM)

2. A typical experiment of specific and nonspecific 13HlQNB . . . . - binding to rabbit heart homogenates. Protein concentration was 0.50 mg/ml, CR& = 5.9 PM. Specific L3HlQNB binding (0) was experimentally determined as the difference between total binding and nonspecific binding (0) in parallel assays in the absence and presence of 1 pM atropine sulfate. Assay conditions were similar to Fig. 1 except 10 ml total volumes were used. The inset shows a Scatchard plot derived from the specific 13HIQNB binding data of Fig. 2. Correlation coefficient (r) = 0.979; dissociation constant K,,app = 41.8 PM; R,,, = 54.9 pmol/g of protein where the ordinate is bound ligand (picomoles per g of protein) and the abscissa is bound over free ligand (liters per g of protein).

cardiac homogenates contained a single population of saturable high affinity muscarinic cholinergic receptors. No evidence was seen for a second population of saturable binding sites of lower affinity when the 13H]QNB concentration was raised to 1 nM. However, the nonspecific component of binding contin- ued to increase linearly with increasing concentrations of 13H]QNB. These data are compatible with the model described by Equation 1 and were replotted as a single straight line according to the method of Scatchard (Equation 4; Fig. 2) (22). Correlation coefficients for this straight line in over 30 experiments ranged between 0.85 and 0.99, again indicating a single population of sites.

Saturation isotherms were repeated at progressively lower concentrations of tissue per assay tube and the K,,app values were plotted as a linear function of receptor concentration (see Fig. 3 and Equation 10) (24). The molar receptor concentration, (Rjr, was determined by dividing the value from the y intercept of the Scatchard plot (femtomoles bound) by the incubation volume. The dependence of K,,app on receptor concentration in rat brain homogenates also is shown in Fig. 3. The K,, values at “infinitely low” receptor concentration (y intercept) were 27.1 PM for the rabbit heart and 20.0 PM for the rat brain. The extrapolated K,, value of 27 PM has been obtained for [“HIQNB binding in bovine retina.2

Determination of the time necessary to reach equilibrium even at the lowest 13H]QNB concentrations (10 PM) used in Scatchard plots indicated that the binding reaction had equil- ibrated by 60 min. In addition, the saturation isotherms were repeated at longer incubation times (90 min, 120 min) and the slopes and y intercepts of the Scatchard plots were not different from values obtained for 60-min incubations.

In the second method, K,, was calculated from the rate constants of association and dissociation (Equations 5 to 8). In

2 R. E. Hruska, R. White, J. Asari, and H. I. Yamamura, manuscript submitted.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Fig. 4, the association of l”H]QNB with the rabbit heart receptor at 37°C reached equilibrium by about 40 min. When data from experiments such as those in Fig. 4 were entered into Equation 5, the average h,, was 1.03 x lo” M-’ min’ (S.E. = 0.18, n = 5). Similarly, a value for h-, of 2.45 x 10e2 min’ (S.E. = 0.19, n = 5) was obtained by using Equation 6. The K,, value (26.8 ? 3.3 PM; n = 5) calculated in this manner for the five pairs of rate constants agreed very closely with values obtained from saturation isotherms extrapolated to infinitely low receptor concentrations (27.1 PM) (Fig. 3).

In the case of binding of some polypeptide hormones, such as growth hormone (26) and insulin (27), cooperative binding effects have been observed. If this were true for l”HlQNB binding, it would not be possible to calculate a value for K,, from a single regression line of Scatchard plots or a Km, from

L 1 I I 1 1 20 40 60

(RI, (PM)

FIG. 3. Effect of receptor concentration (RX. on the value of the apparent dissociation constant, K,,app. Determinations of K,,app similar to that shown in Fig. 2 were made at progressively lower receptor concentrations: The lower curve (a), is for K,,app uer.sz~s receptor concentration using homogenates of rat brain; the upper cnrve (0) is for the rabbit heart.

TIME (mid

FIG. 4. Rate of association and dissociation of IYHIQNB binding as a function of time. Total binding, 0, was determined in the absence of atropine. Nonspecific binding, n , was determined in the presence of 1 ELM atropine. At the arrow, atropine (1 PM) was added to a parallel set of tubes and both total binding and dissociation of the PHIQNB receptor complex, 0, were monitored for an additional 90 min. The assay contained 0.05 mg of protein/ml and the concentration of I”HlQNB was 80 PM.

Equation 3. In the present case, however, over the concentration range of l”H]QNB studied, no indication was seen for multiple classes of binding sites or for cooperativity. As noted, in over 30 Scatchard plots at varying receptor concentrations. the correlation coefficient ranged between 0.85 and 0.99. Furthermore, an experiment was performed in which a single batch of 1”HlQNB receptor complex was distributed to parallel flasks and dissociation was initiated by either dilution, addition of unlabeled QNB, addition of atropine, or by addition of ACh. No differences were found in km, or in t,,,. This suggests that the determination of h-, is not a function of the concentration of either unlabeled QNB or of other agonists or antagonists and further lessens the likelihood of cooperative binding effects. It is interesting to note that growth hormone and insulin are physiological agonists, whereas QNB is an antagonist that produces only a blockade of the receptor.

For a unimolecular dissociation reaction, Equation 7 per- mits calculation of the half-time for dissociation which was 29.2 min (S.E. = 2.2; n = 5) at 37°C (Fig. 5). The analogous t,,, value for dissociation of the rat brain receptor complex was about 60 min (17). Bound lSH]QNB did not measurably disso- ciate for up to 60 min at PC, the temperature at which the filters were routinely rinsed. This extremely slow dissociation constitutes a major advantage of using 13HlQNB in the muscarinic cholinergic receptor binding assay.

The Ki value for unlabeled QNB was approximated by the inhibition of 13H]QNB binding using unlabeled QNB. This procedure gives satisfactory estimates of the K, provided that the initial free [“HlQNB ligand concentration and the receptor concentration are less than 0.1 of the true K,, (24). In an experiment (shown in Fig. 7) in which an initial I”H]QNB concentration of 7 PM (about 0.25 K,,) and (RX- = 1.7 PM (CO.1

K,,) were used, the K,app was about 36 PM; this K, value approaches the K,, values determined by saturation isotherms (27.1 PM) and kinetic parameters (26.8 PM).

Inhibition of [“H/QNB by Receptor Agonists and Antago- nists -Two other characteristics expected of a physiological receptor are stereoselectivity and pharmacological specificity. The pattern of drug inhibition of specific 13H1QNB binding was used to identify the receptor as muscarinic cholinergic and as stereospecific. Figs. 6 to 9 depict drug inhibition experiments in which the IC,,, was obtained at the concentration of the drug that inhibited 50% of the specific I”HlQNB

binding. Using Equation 9, the IC,,, values were normalized (Table I).

The stereospecificity of QNB receptor binding was tested by

TIME (mid

FIG. 5. Rate of dissociation of specific PHIQNB binding. Data of Fig. 4 were replotted semilogarithmically UCIXLS time. r = 0.99; tIl, = 30 min.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8 lated using Equation 11 (Fig. 8). The Hill coefficients for

3 antagonists were about one: QNB, 0.92 * 0.05; p-OH-QNB,

m50 1.01 + 0.10; atropine, 0.85 & 0.06. The Hill coefficients for

s 0 ATRDPINE l DEXETIMIDE

agonists were lower: oxotremorine, 0.61 t 0.04; ACh, 0.64 -C 0 l LEVETIMIDE 0.04. This distinction between agonists and antagonists has

‘I’

L 25 also been seen for [“HIQNB binding to other mammalian

if

tissues (28). . The nicotinic cholinergic drugs, a-bungarotoxin and nico-

G

!i

tine, did not inhibit I”H]QNB binding at concentrations of 5

f;,,,’ (:f’ 10-S

and 100 PM, respectively. Most classical noncholinergic drugs

16” had little or no effect on I”H]QNB binding (Table I). Surprisingly, agents such as benzylquinonium, decametho-

= 100 . f O- z 75

333

atropine, had a K, value of 1.05 nM. The greater potency of antagonists as compared with agonists in displacing I”H]QNB is in agreement with previous observations in the brain (16:.

Hill coefficients for the inhibition of I”H]QNB binding by muscarinic cholinergic agonists and antagonists were calcu-

FIG. 6. Inhibition of specific r3HlQNB binding by the stereoiso- nium, and curare which were thought to be highly specific for

mers of benzetimide. 13H]QNB concentration was 80 PM. Protein the nicotinic cholinergic receptor, showed considerable po-

concentration was 0.05 mgiml. tency in the micromolar range in acting on the muscarinic cholinergic receptors of the rabbit heart (Table I): the K,

TABLE I

Relative potencies of drugs in reducing [3H]QNB binding in rabbit

heart homogenates

values were 0.10, 2.15, and 4.31 PM, respectively. These results suggest that these agents should not be regarded as

[“H]QNB was added to a final concentration of either 20, 40, or 80 PM. Tissue = 0.05 mg of protein/ml. All components of the medium were mixed and the incubation started by addition of an aliquot of the cardiac homogenate. Each K, value represents the average from separate IC,, determinations at each of the three 13HlQNB concentrations (using a different rabbit heart homogenate each time). The

o OXOTREMORINE . ACETYLCHOLINE . (I-SUNGAROTOXIN

IC,, values were normalized using Equation 9, except for the Kiapp . NlCOTlNE

for 3-quinuclidinyl benzilate which was determined at a L3HlQNB concentration of 7 PM and not normalized.

Drug KNAPP Drug KGPP - PM FM

Muscarinic 3. Choline uptake in- 3-Quinuclidinyl ben- 0.000036 hibitor

zilate Hemicholinium-3 3.43 p-OH-QNB 0.00070 Atropine 0.00105 4. Neuroleptics Oxotremorine 0.144 Chlorpromazine 0.17 Acetylcholine (phy- 0.490 Droperidol 1.57

FIG. 7. Inhibition of specific c3HJQNB binding from rabbit heart

aostigmine 1 FM) Spiroperidol 3.22 homogenates by cholinergic agents. L3H]QNB = 80 PM. Tissue = 0.05

Carbamylcholine 0.528 Haloperidol 27.6 mg of protein/ml. Incubations were started by addition of tissue.

Dexetimide 0.00069 Levetimide 1.45 5. Cardioactiv&

Atropine 0.00105 --

Nicotinid’ Imipramine 0.173 Benzylquinonium 0.1 Quinidine 2.04 Decamethonium 2.15 Xylocaine 16.2 ATROPINE

d-Tubocurarine 4.31 Procainamide 47.6 OXDTREMDRINE

” Nicotinic agents that were ineffective were: a-bungamtoxin at 5.0 ELM, hexamethonium at 10.0 FLM, and nicotine at 100.0 &LM.

* Cardioactive agents that were ineffective were: propranolol and ouabain at 100.0 PLM and dopamine, norepinephrine, phenylephrine, taurine, hista- mine, and leuo-alprenolol at 10 FM. Other agents that were ineffective at 10 FM were y-aminobutyric acid, glycine, picrotoxin, bicuculline, physostigmine, phenelzine, choline, dean& and 2,4-dinitrophenol.

g - comparing the inhibitory potencies of stereoisomers of the muscarinic antagonist, benzetimide (Fig. 6). Dexetimide (K, = 0.69 nM), was over 2000 times more potent than its stereoiso- I

mer, levetimide (K, = 1.45 FM). 16” IO- d IO-J

Acetylcholine (ACh), the physiological neurotransmitter [DRUG] (Ml

substance, was effective in inhibiting specific (“HIQNB bind- FIG. 8. Hill coefficients for inhibition of specific 13HlQNB binding ing with a K, value of 490 nM. Physostigmine (1 FM) was also to rabbit heart homogenates by muscarinic cholinergic agonists and

uresent whenever ACh was added (Fin. 7) and bv itself had no antagonists. Data such as those in Figs. 6, 7, and 9 were plotted

effect on binding. Other muscarinic agonists such as carba- according to Equation 11. (RH),,,,,, is numerically equivalent to (Rjr.

mylcholine and oxotremorine, gave K, values of 528 and 144 Hill coeff%ients are taken as the negative of the slope of the inhibition line. 0, unlabeled QNB; 0, p-OH-QNB; A, atropine; A,

nM, respectively, while the specific muscarinic antagonist, . . .

oxotremorine; 0, acetylcholme.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


completely specific for nicotinic cholinergic receptors. TABLE II Inhibition of 13H]QNB Binding by Neuroleptics and Car-

dioactive Agents -The 13H]QNB binding assay was utilized to determine drug interactions at the receptor level. We examined several commonly used cardiotonic agents as well as certain drugs which have been found to bind to muscarinic cholinergic receptors in the brain.

Comparison ofdensity ofmuscarinic receptors in hearts ofdifferent

species

Ouabain, a cardiac glycoside, did not inhibit QNB binding at concentrations as high as 100 PM. On the other hand, three commonly used antiarrhythmic agents, quinidine, lidocaine, and procainamide (Fig. 9) were inhibitory with K, values of 2.0, 18, and 48 PM, respectively.

[3H]QNB.receptor densities, R,,,, were obtained from the y intercepts of Scatchard plots (see Fig. 2 and “Materials and Meth- ods”). Values are mean 2 S.E.

Species Receptor density (N) pmollgprotein

Rabbit 51.2 k 4.3 14 Rat 144 2 13 9 Guinea pig 178 k 13 8

The tricyclic antidepressant, imipramine, which can be severely cardiotoxic (291, inhibited QNB binding with a Ki value of 0.17 PM.

TABLE III

In agreement with previous reports on 13H]QNB binding to mammalian brain (19, 30, 31), we found that many of the antipsychotic, neuroleptic drugs (i.e. phenothiazines and butyrophenones) are effective inhibitors of L3H]QNB binding. Chlorpromazine, droperidol, spiroperidol, and haloperidol had K, values of 0.17, 1.57, 3.2, and 28 PM, respectively. Haloperi- do1 had the weakest anticholinergic activity of the antipsychotic butyrophenones examined which was consistent with previous observations for central muscarinic receptors (31).

Species and Organ Differences in Muscarinic Cholinergic Receptors -We utilized the 13H]QNB binding assay to examine and compare the biochemical characteristics of muscarinic cholinergic receptors from cardiac homogenates of three commonly employed species of laboratory animals. K,)app and U%ax values were obtained from the slope and y intercept of

Regional distribution ofmuscarinic cholinergic receptors in rabbit

heart

YHIQNB receptor densities, R,,,, and percentage of total cardiac receptors. Values listed are from Scatchard plots determined on regions of each of five rabbit hearts and are means i- S.E.

Percentage of to- Region Receptor density tal cardiac reeep

tors

pmollgpmtein

Left ventricle 37.4 2 7.6 28.9 T 2.22 Right ventricle 52.7 2 10.2 15.2 k 1.01 Ventricular septum 58.1 A 6.5 21.7 i .74 Right atrium 200.0 k 13.5 17.8 i 1.32 Left atrium 302.4 k 30.9 16.4 k 2.86

Scatchard plots (see “Materials and Methods”). As shown in Table II, the rank order for cardiac muscarinic receptor densities was guinea pig 2 rat > rabbit. However, there were no detectable differences for K,,app in these species as determined either by saturation isotherms or by the kinetic method. For the rabbit heart, the receptor density value of 57.2 pmol/g of protein was equivalent to 4.47 pmol/g of tissue.

In addition, we confirmed the values reported previously for the density of muscarinic receptors localized in the longitudinal muscle of the guinea pig ileum (2390 pmol/g of protein) (18) and in the rat brain (910 pmol/g of protein) (19) using 13H]QNB of about lo-fold higher specific activity than was available previously.

Regional Distribution of Muscarinic Cholinergic Recep- tors -Regional tissue distribution of ligand binding should

parallel the effectiveness of neurotransmitters and of related agonists and antagonists in pharmacological and physiological experiments. Saturation isotherms and Scatchard analysis was carried out for each region. Values of receptor density for L3H]QNB binding in homogenates from five regions of the rabbit heart are shown in Table III. Atria1 tissue contained 6 to 9 times more cholinergic muscarinic receptors per g of protein than did ventricular tissue. Interestingly, the greatest concentration of receptors was in the left atrium even though the right atrium was dissected so as to contain both the sinoatrial node and atrioventricular node. Although septal and ventricular homogenates contained a lower concentration of receptors per g of protein, because of their weight, these regions contained significant percentages of the total myocardial receptors (Table III). These data are consistent with the relative influence of vagal stimulation and acetylcholine infu- sions on atria1 and ventricular performance (4, 7).

FIG. 9. Inhibition of specific L3H]QNB binding to rabbit heart homogenates by cardiotonic agents. L3H1QNB concentration was 80 PM. Tissue concentratiax was 0.05 mg of protein/ml.

The values from saturation isotherms for K,,app for all regions were within the range of values for whole heart homogenates: 24 to 75 PM and were consistent with the regression line in Fig. 3. The range of K,,app values for each region were 24 to 56 PM, left ventricle; 32 to 57 PM, right ventricle; 41 to 72 PM, ventricular septum; 23 to 51 PM, right atrium; and 28 to 78 PM, left atrium. Thus, the regional variation is due to a change in receptor density rather than a change in affinity.

DISCUSSION

The present study describes the specific, saturable, high affinity muscarinic cholinergic receptors in the mammalian heart. The binding characteristics of these receptors are similar to those of the muscarinic receptors in the central nervous system (17-19). The regional distribution of the cardiac receptors is consistent with classical physiological observations (3- 10). Cardiotonic agents, antipsychotic neuroleptics, and tri-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


cyclic antidepressants are potent, competitive inhibitors of specific 13H]QNB binding.

In agreement with earlier studies in rat brain and guinea pig ileum, the cholinergic muscarinic receptors in the heart consist of a single population of saturable binding sites (17, 18). However, the extrapolated K,, determined from saturation isotherms (27.1 PM) for cardiac homogenates is more than an order of magnitude lower than previously reported for saturation isotherms for rat brain homogenates (17). This difference stems, in part, from the dependence of K,)app on the concentrations of receptor added in the assay (24). Thus, the use of lower specific activity [3H]QNB in earlier studies (17) required the use of greater tissue concentration. With the higher specific activity 13H]QNB now available, it was possible to repeat the determinations of the K,, at receptor concentrations as low as 2 PM. Furthermore, these values were extrapolated to infinitely small receptor concentrations (y intercept of Fig. 3) to determine the true K,,. The K,, for both rat brain and rabbit heart are then in excellent agreement. Values for K,, determined by the kinetic method (26.8 PM), which is inde- pendent of tissue concentration, were in agreement with the values of K,, determined from saturation isotherms.

As expected (Equation 10; Ref. 24), we found K,, to be dependent upon (R),.. However, the slopes (2 to 3) of theK,,app uersus (RX. plot (Fig. 3) are not similar to that predicted (0.5) by simple consideration of the sequestering effect of specific binding on the free ligand concentration (Equation 10). The higher slopes suggest some additional interference with the binding process which we are not able to explain at the present time. We note that the higher slope was found even for rat brain homogenates (Fig. 3) which had been washed twice. We further note that the K,, value determined from the y intercept of the K,app versus (RX. plot agrees very closely with the K,, determined from the ratio of the rate constants despite the unexpected higher slope. Slopes greater than 0.5 have also been found for 13H]QNB binding in other tissues and for several other receptor binding assays studied in our laboratory. These include [3H]spiroperidol binding to a brain dopaminergic receptor and cu-lZI-bungarotoxin binding to a brain nicotinic cholinergic receptor.3

Several aspects concerning the drug inhibition of QNB binding by cholinergic agents also require further comment. It is noteworthy that the potent nicotinic cholinergic antagonist, c*-bungarotoxin, fails to inhibit 13H1QNB binding even at 5 PM. This concentration is about 10,000 times the half- maximal binding concentration of cr-‘2”I-bungarotoxin to nicotinic cholinergic receptors (32). Nicotine (100 PM) is also ineffective in inhibiting [3H]QNB to the cardiac muscarinic receptors. In agreement with previous observations, the muscarinic cholinergic antagonists, atropine and QNB, were more potent in inhibiting 13H]QNB binding than the muscarinic agonists, acetylcholine, oxotremorine, and carbamylcholine (17, 18, 33). The greater affinity of antagonists as compared with agonists for receptor sites has been noted in studies of the muscarinic cholinergic receptor in other tissues. A possible explanation for this phenomenon is that QNB binds to an antagonist conformation of the receptor (34, 35). Alterna- tively, agonist binding may exhibit negative cooperativity (24.

The stereospecificity of the QNB receptor site was demonstrated using the stereoisomers of benzetimide, dexetimide, and levetimide. Pharmacologically, the antagonist, dexetim-

3 Unpublished observations.

ide, is reported to be about 6000 times more effective than its stereoisomer, levetimide (36). In our binding studies, dexetimide was about 2000 times more potent than levetimide in inhibiting QNB binding.

The classical nicotinic cholinergic agents, curare, decamethonium, and benzylquinonium, demonstrated surprising potency in inhibiting QNB binding. Curare at pharmacological concentrations has been reported to possess a vagolytic effect (37). Thus, these agents at high concentrations appear to interact with both muscarinic and nicotinic receptors.

Hemicholinium-3, a choline uptake inhibitor, also has been shown to effect the muscarinic receptors in smooth muscle (38, 39). The potency for the presynaptic inhibition of high affinity choline transport, however, is about 100 times greater (38).

We note that, as with a number of other receptor binding assays, the Hill coefficients for inhibition by antagonists were close to 1.00, while Hill coefficients for agonists were signifi- cantly lower, being about 0.5.

Further, it is interesting to note that p-OH-QNB is about 10 times less potent than QNB. In a recent report (40), a binding assay for the muscarinic cholinergic receptor was carried out using ‘““I-labeledp-OH-QNB. Although higher specific activi- ties can be obtained using iodinated compounds, in this case, that advantage is partially offset by the lower affinity of the p-OH-QNB for the binding site which may result in greater nonspecific binding.

The regional distribution of muscarinic cholinergic receptors was consistent with previous physiological experiments. The negative inotropic and chronotropic effects of acetylcholine in the atria are well established both for the intact heart and in isolated tissue (3-10). The demonstration of a low but significant concentration of 13H]QNB binding sites in the ventricles also is consistent with the small negative inotropic effect observed in the ventricles upon vagal stimulation or acetylcholine infusion into the coronary arteries (4, 7). Fur- ther support for parasympathetic innervation of the ventricle is provided by recent reports demonstrating a high concentration of acetylcholinesterase at the postganglionic parasympathetic nerve ending, associated with the ventricular conduction system (41, 42). Moreover, reports of the atrioventricular distributions of acetylcholine, choline acetyltransferase, and acetylcholinesterase (13, 43-45) are similar to the regional distribution of muscarinic cholinergic receptors reported here.

The inhibition of [3H]QNB binding obtained with clinically useful cardiotonic and psychotropic agents suggests that the cardiac muscarinic receptors may be involved in the molecular mechanism of action or with the side effects of these drugs. The tricyclic antidepressants, such as imipramine, are thought to exert their pharmacological effects at synapses in the brain (43), presumably prolonging the effectiveness of norepinephrine released at the adrenergic synaptic cleft (46). In agreement with earlier studies (30), we have shown that imipramine blocks the muscarinic receptor at concentrations at or below the range of clinically attained plasma levels (29, 47). This action may therefore play a role in the clinical cardiotoxicity of tricyclic antidepressants (29, 48, 49).

The rank order of the extrapyramidal side effects of antipsychotic agents is the inverse of the rank order of their anticholinergic potency (31). That is, thioridiazine, the most potent anticholinergic neuroleptic in brain, appears to have minimal extrapyramidal side effects. Haloperidol, the weakest anticholinergic, possesses marked extrapyramidal side effects. We find that a number of these neuroleptics also interact with myocardial muscarinic receptors with the same rank order as


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


found previously for the central muscarinic receptors (31). The cardiac glycoside, ouabain, fails to inhibit QNB bind-

ing. The well known vagal effect of this cardiac glycoside apparently is not a direct effect on the receptor. In contrast, the three widely used antiarrhythmic agents, quinidine, lido- Caine, and procainamide, exhibit interactions with the cardiac muscarinic receptor. Quinidine has been reported to possess a

vagolytic effect (50). It is important to note that the K, values of these antiarrhythmic agents are of the same order as clinically achieved plasma levels. It is possible that at least part of the therapeutic actions of these antiarrhythmic agents on heart rate and intracardiac conduction could be mediated initially through muscarinic cholinergic receptors, particu- larly those associated with the conducting tissues of the heart (41, 42).

Acknowledgments-We would like to express our apprecia- tion to Tom McManus, Janet Cohn, and Maria Valenzuela for their technical assistance and to Miss Ann Vallefuoco for her secretarial assistance in typing this manuscript.

1.

2.

3.

4.

5. 6.

7. 8.

9.

10.

11.

12.

13. 14.

15.

16. 17.

18.

19.

REFERENCES

Fields, J. Z., Roeske, W. R., Morkin, E., and Yamamura, H. I. (1977) Fed. Proc. 36, 979

Roeske, W. R., Fields, J. Z., Morkin, E., and Yamamura, H. I. (1977) CZin. Res. 25, 457 Abstr.

Higgins, C. B., Vatner, S. F., and Braunwald, E. (1973) Phar- macol. Reu. 25, 119-155

Priola, D. V., Spurgeon, H. A., and Geis, W. P. (1977) Circ. Res. 40, 50-56

Priola, D. V., and Fulton, R. L. (1969) Circ. Res. 25, 265-275 Buccino, R. A., Sonnenblick, E. H., Cooper, T., and Braunwald,

E. (1966) Circ. Res. 19, 1097-1108 Levy, M. N., and Zieske, H. (1969)Am. J. Physiol. 216, 890-897 DeGeest, H., Levy, M. N., Zieske, H., and Lipman, R. I. (1965)

Circ. Res. 17, 222-235 Furchgott, R. F., Sleator, W., and DeGubareff, T. (1960) J.

Pharmacol. Exp. Thu. 129, 405-416 Friedman, W. F., Buccino, R. A., Sonnenblick, E. H., and

Braunwald, E. (1967) Circ. Res. 21, 573-582 Lee, T. P., Kuo, J. F., and Greengard, P. (1972) Proc. Natl.

Acad. Sci. U. S. A. 69, 3287-3291 Gardner, R. M., and Allen, D. 0. (1976) J. Pharmacol. Exp.

Thu. 197, 412-419 Brown, 0. M. (1976) Am. J. Physiol. 231, 781-785 Roskoski, R., Jr., Schmid, P. G., Mayer, H. E., and Abboud, F.

M. (1975) Circ. Res. 36, 547-552 Napolitano, L. M., Willman, V. L., Hanlon, C. R., and Cooper,

T. (1965) Am. J. Physiol. 208, 455-458 Jacobowitz, D. (1967) J. Pharmacol. Ezp. Ther. 158, 227-240 Yamamura, H. I., and Snyder, S. H. (1974) Proc. N&Z. Acad.

Sci. U. S. A. 71, 1725-1729 Yamamura, H. I., and Snyder, S. H. (1974) Mol. Pharmacol. 10,

861-867 Snyder, S. H., Chang, K. J., Kuhar, M. J., and Yamamura, H.

I. (1974) Fed. Proc. 34, 1915-1921

20.

21.

22. 23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

36.

37.

38.

39.

40.

41.

Flanagan, S. D., and Storni, A. (1976) Abstracts of the Sixth Annual Meeting of the Society for Neuroscience, Toronto, Canada, November 7-11,1976, Vol. I, Part 2, p. 782 (Abstr.)

Kent, K. M., Epstein, S. E., Cooper, T., and Jacobowitz, D. M. (19741 Circulation 50. 948-955

42. Kent, K. M., and Epstein, S. E. (1976) Cardiology 61, 61-74 43. Kilbinger, H. (1973) J. Neurochem. 21, 421-429 44. Kilbinger, H., and Loffelholz, K. (1976) J. Neural Transm. 38,

45.

46. 47.

48.

49.

50.

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275

Goldstein, A., Aronow, L., and Kalman, S. M. (1969) Principples ofDrug Action. D. 70. Harrier & Row, New York

S&chard, G. (19491 Ann. N: Y. Acad. Sci. 51, 660-672 Cheng. Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22,

3099-3108 Cuatrecasas, P., and Hollenberg, M. (1976) Adu. Protein Chem.

30, 251-451 Goldstein, A. (1969) Biostatistics; An Zntroductory Text, p. 59,

MacMillan, N.Y. Lesniak, M. A., and Roth, J. (1976) J. Biol. Chem. 251, 3720-

3729 Gavin. J. R.. III. Roth. J.. Neville. D. M.. Jr.. DeMevts. P.. and

Buell, D. N. (i974)Z%&. Natl. .&ad. Sci. U’. S. A. “71, 84-88 Wastek, G. J., Johnson, P. C., Stern, L., and Yamamura, H. I.

(1978) Mol. Pharmacol., in press Spiker, D. G., Weiss, A. N., Chang, S. S., Reinstch, J. F., and

Biggs, J. T. (1976) Clin. Pharmacol. Thu. 18, 539-546 Snyder, S. H., and Yamamura, H. I. (1977) Arch. Gen. Psychia-

trv 34. 236-239 Snyder, S. H., Banerjee, S. P., Yamamura, H. I., and Green-

berg, D. (1974) Science 184, 1243-1253 Speth: R. C., Chen, F. M., Kobayashi, R., Lindstrom, J., and

Yamamura, H. I. (1977) Brain Res. 131, 350-355 Birdsall, N. J. M., and Hulme, E. C. (1976) J. Neurochem. 27,7-

16 Snyder, S. H. (1977) N. Engl. J. Med. 296, 266-271 Snyder, S. H., Pasternak, G. W., and Pert, C. B. (1976) in

Handbook of Psychopharmacology (Iversen, L. L., Iversen, S. D., and Snyder, S. H., eds) Vol. 5, pp. 329-360, Plenum Press, New York, N.Y.

Soudijn, W., Van Wijngaarden, I., and Arieens, E. J. (1973) Eur. J. Pharmacol. 24, 43-48

Hughes, R., and Chappel, D. J. (1976) Br. J. Anaesth. 48, 847- 852

Yamamura, H. I., and Snyder, S. H. (1973) J. Neurochem. 21, 1355-1374

Bertolini, A., Greggia, A., and Ferrari, W. (1967) Life Sci. 6, 537-543

9-14 Dietrich. H. A.. Kaffel. H., Kilbinger. H.. and Loffelholz, K.

(1976) J. Pharmacol. .&p.‘Ther. 1199, 236-245 Glowinski, J., and Axelrod, J. (1964) Nature 204, 1318-1319 Bigger, J. T., Glardina, E. G. V., Perel, J. M., Kantoo, S. J.,

and Glassman, A. H. (1977) N. Engl. J. Med. 296, 206-208 Elonen. E.. Mattila. M. J.. and Saarnivaara. L. (1974) Eur. J.

Pharmacbl. 28, 178-188 Dumovic, P., Burrows, G. D., Vohra, J., Davies, B., and

Scoggins, B. A. (1976) Arch. Toxicol. 35, 255-262 Moe, G. K., and Abildskov, J. A. (1975) in The Pharmacological

Basis of Therapeutics (Goodman, L. S., and Gilman, A., edsl 5th Ed, p. 688, MacMillan, New York


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J Z Fields, W R Roeske, E Morkin and H I Yamamuracharacterization.

Cardiac muscarinic cholinergic receptors. Biochemical identification and

1978, 253:3251-3258.J. Biol. Chem.

http://www.jbc.org/content/253/9/3251.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/253/9/3251.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/253/9/3251.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;253/9/3251&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/253/9/3251.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=253/9/3251&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/253/9/3251.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/253/9/3251.citation.full.html#ref-list-1

http://www.jbc.org/

cardiac muscarinic cholinergic receptors · rinic cholinergic drugs that inhibited specific binding...

Documents