Characterization of muscarinic cholinergic receptors in the lower esophageal sphincter of the cat: Binding of [3H]quinuclidinyl benzilate

GASTROENTEROLOGY

77X25-1234.1979

Characterization of Muscarinic Cholinergic Receptors in the Lower Esophageal Sphincter of the Cat: Binding of [“HlQuinuclidinyl Benzilate T. J. RIMELE,

W. A. ROGERS,

and T. S. GAGINELLA

Division of Pharmacology, College of Pharmacy, and Division of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

We have studied the binding of the potent muscarinic antagonist rH]quinucJidinyJ benzilate to characterize muscarinic receptors in the lower esophageal sphincter of the cat. Specific binding of rH]quinucJidinyJ benzilate reached equilibrium in 25-30 min at 37°C was linear with tissue protein concentration, and was saturable. Estimates of the apparent equilibrium dissociation constant (KD) of 1.1 f 0.1 nM and maximum receptor density (B,,S of 147 + 5.5 fmoJ/mg protein were determined from Scatchard plots of the binding data. The Hill coeficient for binding was 1.06 f 0.07, indicating the absence of cooperative interactions. From kinetic analysis of the data association (k,) and dissociation (kz) rate constants of 6.3 x IO’ M-’ min-’ and 5.0 x 10m3 min-‘, respectively, were calculated. Inhibition of binding was stereoselective, with the (-)-isomer of quinuclidinyl benzilate being 37-fold more potent in inhibiting specific binding than the (+)-isomer. Various muscarinic agents inhibited binding in accordance with their estimated affinities for the muscarinic receptor; nicotinic and noncholinergic drugs had virtually no affinity for rH]quinucJidinyJ benzilate binding sites. In functional studies, atropine and (A)-quinuclidinyl benzilate antagonized bethanechol-induced contraction of the lower esophageal sphincter in accordance with their abilities to inhibit specific binding of rH]quinucJidinyJ benzilate. The regional distribution of specific binding along the length of the esophagus paralleled the distribution of smooth muscle in the tunica muscularis. Binding to duodenal, jejunal, iJeaJ, and nonsphincter

esophageal muscle was similar to that in the sphincter, but was fourfold greater to fundic muscle.

The smooth muscle of the lower esophagus, and in particular the lower esophageal sphincter (LES), is responsive to a variety of neurohumoral agents, hormones, and drugs. In their review, Goyal and Rattan’ have emphasized the complexity of the functional control of the LES. Although physiologic and pharmacologic studies have added to our understanding of esophageal function, quantitative details on relative populations of receptors, and most importantly their functional dominance, remains obscure. Biochemical techniques for the characterization of a variety of hormone and drug receptors have developed rapidly over the past few years. Numerous studies have documented the feasibility of characterizing receptors by direct binding of high specific activity radiolabeled ligands.“-’ One such agent is [3H]quinuclidinyl benzilate ([3H]QNB), a potent reversible muscarinic cholinergic antagonist that has been used to demonstrate muscarinic receptors in a wide range of tissues.7-‘3 The present study was designed primarily to characterize the binding of [“H]QNB to muscarinic receptors in the LES of the cat in terms of specificity, affinity, density, and stereoselectivity, and to determine if differences in binding exist in different regions of the gut. Materials Physiologic

Received November 21.1976. Accepted July 16,1979. Address requests for reprints to: T. S. Gaginella, Ph.D., Ohio State University, College of Pharmacy, 500 West 12th Avenue, Columbus, Ohio 43210. T. J. Rimele is an AFPE Albert H. Diebold Memorial Fellow. Dr. Gaginella is the recipient of Research Career Development Award 1 K04 AM 69471-01 from the National Institutes of Health. This work was supported in part by NIH Grant 1 R01 AM 21932. We wish to thank Mrs. Helen Kelly and Ms. Kathy Brooks for expert secretarial assistance. 0 1979 by the American Gastroenterological Association 9616-5065/79/121225-lO.$Q2.60

and Methods Definition

of the LES

Experiments were done essentially as described by Christensen et a1.14,15Adult cats of both sexes (1.5-4.5 kg) received sodium pentobarbital (40 mg/kg) by the intra-

peritoneal route for anesthesia. The esophagus was measured in situ, removed, opened longitudinally, and pinned to its in situ length in a Petri dish containing wax immersed in Krebs buffer (pH 7.4 at 37’C) aerated with 95% O&Y% CO,. The buffer had the following composition (millimolar): NaCl, 115.5; KCl, 4.6: NaH,PO,, 1.2; NaHCO,, 21.9;

CaCl,,

2.5; MgSO,,

1.2; and glucose,

11.5. The mucosa

1226

GASTROENTEROLOGY Vol. 77,No.6

RIMELE ET AL.

was removed, and the muscle cut into transverse strips (2.0 X 0.3 cm) with a razor blade. Tissue samples were taken such that the middle strips represented the area of the mucosal esophagogastric junction. Strips were placed into 40-ml jacketed tissue baths containing 35 ml of Krebs solution, aerated with 95% O,-5% CO, and held at 36.537.5’C. The strips were held by an electrode-clip like that described by Anuras et al.” Responses of the circular muscle strips to electrical field stimulation (10set trains of 50-60 V at 10 Hz and 0.5 msec duration), and the stimulus signal were recorded simultaneously on a Grass model 79 recording polygraph. Three regions in the area of the LES could be distinguished by their responses to electrical field stimulation. Strips from the stomach contracted; this response was both tetrodotoxin and atropine sensitive. Strips from the esophageal body contracted briefly at the end of the stimulus period; this response was also tetrodotoxin and atropine sensitive. Strips from the esophagogastric junction relaxed during stimulation, and this response was sensitive to tetrodotoxin but not atropine. In all animals tested, sphincteric responses (relaxation) occurred only in the most distal part of the esophagus. Therefore, unless otherwise stated, it was this region, the distal 1.5 cm of the esophagus (measuring from the mucosal esophagogastric junction) that was used for the binding assays. Pharmacologic Studies Receptors in the LES

on Muscarinic

Transversely cut muscle strips from the distal 1.5 cm of the esophagus were mounted in 10 ml jacketed tissue baths containing Krebs solution aerated with 95% O,5% CO, and held at 36.5-37.5’C. Each strip was anchored by one end to a glass tissue holder, and the other end was attached to a force-displacement transducer (Grass, model FT-03C) for isometric recording of tension with a Grass model 7 recording polygraph. The strips were allowed to relax to a constant base-line tension of 2 g at which time they were challenged with a single concentration (1 x 10e4 M) of bethanechol, washed thoroughly, and again allowed to achieve constant baseline tension. Two cumulative concentration-response curves to bethanechol were obtained, the first in the absence of antagonist (atropine or (*)-QNB), and the second after 45 min of exposure to either atropine or (*)-QNB. Contraction values were plotted as percentages of the maximal response to bethanechol. Means were calculated as the geometric mean (antilog of the arithmetic mean of the logarithms of individual ED50 or K, values). Changes in tissue sensitivity were monitored using the method of Furchgott.“.” The pA, values (-log KB) for atropine were estimated by the method of Arunlakshana and Schild,” using three concentrations of atropine. The apparent dissociation constant (KB) for (+)-QNB was calculated (using a single concentration of (-+)-QNB) as described by Furchgott.1’.‘8

Preparation chest

of Tissue

for Binding

Studies

Under pentobarbital (40 mg/kg) anesthesia, the was opened, and the esophagus was cut 5-6 cm

above the phrenoesophageat ligament. This section, including a rim of adjacent stomach, was removed, cleaned of extraneous tissue, and placed in ice-cold 50 mM tris (hydroxymethyl)aminomethane (Tris) buffer (pH 7.4 at 37OC). The section was opened longitudinally, stretched to its in situ length and the distal 1.5 cm transferred to a Petri dish containing ice-cold Tris buffer and pinned in place, mucosal surface up, on dental wax. The mucosa and submucosal layers were removed with forceps and scissors. The remaining tissue was blotted, weighed, placed into a IO-ml beaker with 5 ml of ice-cold Tris buffer and minced to a fine suspension with scissors. This suspension was transferred to a 25-ml test tube and homogenized on ice for 1.5 min in 15 ml of ice-cold Tris buffer using a Brinkmann Polytron (model PT-10) at setting 7. Protein was determined by the method of Lowry et al.,zo using bovine serum albumin as a standard.

rH]QuinucJidinyJ

Benzilate

Binding

In general, tissue homogenate (300-506 ~1, containing 300-906 pg protein) was added to 2 ml polystyrene cups (Kew Scientific, Columbus, Ohio) and incubated at 37°C for 30 min in a Dubnoff incubator with constant shaking (100 cycles per min), with -1.0 nM [3H]QNB (29.4 Ci/mmol) and specific drugs (where indicated) in a total volume of 1.5 ml of 50 mM Tris buffer (pH 7.4 at 37°C). After incubation, samples were filtered under vacuum through Tris-prewetted Whatman GF/B glass fiber filters. The filters were washed five times with 2 ml of ice-cold Tris buffer (total filtration time 30-40 set), placed into scintillation vials and allowed to air dry for 30 min. To each vial, 0.5 ml of tissue solubilizer (0.6 N quaternary ammonium hydroxide solution) was added, and the vials were capped tightly and shaken (50 cycles per min) in a Dubnoff incubator at 50°C for 30 min. The samples were then allowed to cool to room temperature, at which time 50 ~1 of glacial acetic acid and 10 ml of Thrift-Solve (Kew Scientific) were added. The vials were then mechanically shaken for at least 1 hr on an Eberbach shaker (240 cycles per min) and maintained at lO“C for 6 hr before being counted in a Beckman LS-345 liquid scintillation counter. Appropriate corrections were made for quench which was monitored by an automatic external standard. Counting efficiencies for tritium were routinely between 29 and 31%. Radioactivity bound to tissue and filters in the presence of 5 x lo-'M atropine was defined as nonspecific binding. Radioactivity nonspecifically bound was subtracted from total radioactivity bound to tissue and filters to obtain “specific” binding. Typically, total binding was about 3000 dpm, 7565% of which was specific. Binding to the filters (less than 1% of total radioactivity added) accounted for most of the nonspecific binding. Conditions were used to limit total binding to less than 5% of the radioactivity added to each incubation so that the concentration of free ligand did not change appreciably during the binding assay. There was no detectable binding to the plastic incubation cups. Specific binding increased linearly with increasing tissue concentration over the range of 0.2-2.0 mg of protein. Binding assays were, therefore, always conducted with less than 900 pg protein.

MUSCARINIC

December 1979

Sources

t)L-[benzilic+“H]quinuclidinyl benzilate, mol wt obtained from Amersham), was used for experiments shown in Figure 3. In all other experiments, nt.-[benzilic-4,4’-“Hlquinuclidinyl benzilate (mol wt 337.4) with a higher specific activity (29.4 Ci/mmol, New England Nuclear), was used. The radiochemical purity of [3H]QNB was routinely checked by thin layer chromatography using silica gel-GF plates (New England Nuclear), and a solvent system of chloroform:acetone:diethylamine (60: 30: 10). The percent radiochemical purity was found to be never less than 95%. Other drugs were obtained and used in the forms indicated: atropine sulfate, bethanechol chloride, pilocarpine HCl, hexamethonium bromide, decamethonium bromide, dichloroisoproterenol HCl, phenylephrine HCl, histamine dihydrochloride, 5hydroxytryptamine creatinine sulfate, arecoline HCl, carbachol, isoproterenol HCl, physostigmine, and propantheline bromide (Sigma Chemical Co.); l,l-dimethylphenylpiperazinium iodide (DMPP) (Aldrich Chemical Co.). The following drugs were kindly provided by the companies noted. Propranolol HCl, (Ayerst Laboratories); phentolamine HCl (Ciba); cimetidine and isopropamide iodide (Smith Kline & French Laboratories); haloperidol and McN-A-343-11 [4-(m-chlorophenylcarbamoyloxy) - z - butynyltrimethylammonium chloride](McNeil Lab; methscopolamine bromide (Upjohn Co.); metoclopramide andglycopyrrolate (A. H. Robins Co.); dicyclomine HCl (Merrell National); anisotropine methylbromide,(EndoLaboratories);tridihexethylchloride(Lederle Laboratories); and clidinium bromide, racemic, (+)-and (-)QNB HCl (Hoffman-La Roche, Inc.). Tetrodotoxin was purchasedfromCalbiochem. 337.4, (16.4 Ci/mmol,

Inhibition

Studies

on Muscarinic

16’ Figure 1 Typical

3 x10-’

0

0

lo+

3 X10-+

0

1o-5

by Atropine

of Binding

At 37”C, specific [“H]QNB binding occurred rapidly. Binding reached a half-maximal value in about 9 min and attained a plateau by 25-30 min. In contrast, nonspecific binding was time-independent (Figure 4). Kinetic data from Figure 4 were used to calculate, according to the method of Williams and Lefkowitz,’ a second order forward rate constant, k, of 6.3 X lo7 M-’ min-’ (Figure 5A). Dissociation of specifically bound [“HIQNB was very slow, following first order kinetics with a rate constant, k,, of 5.0 x lo-” min-’ (Figure 5B). Nonspecific binding did not dissociate over the time period tested. The ratio k,/k,, an estimate of the apparent dissociation constant K,, was 0.08 nM.

The LES muscle strips accommodated slowly to the applied 2-g tension, relaxing to a constant base-line in lo-20 min. Stretch-induced contractions were never seen. Bethanechol always produced rhythmic contractions superimposed on a tonic con-

??

of Binding

Kinetics

in the LES

0

1227

Atropine inhibited the binding of [“HIQNB in a concentration dependent manner (Figure 3), reaching maximum inhibition at about 5 x lo-” M. Specific binding was generally between 75 and 85% of total binding.

Kesults Beceptors

IN LES

traction; at higher concentrations, the response was mainly tonic (Figure 1). The mean ED50 for bethanechol (calculated from individual ED5Os obtained from 20 LES strips) and 95% confidence limits were 1.3 X 10e5 and (7.0 X lo-“-Z.3 X lo-‘) M, respectively. Atropine and (+)-QNB caused parallel shifts to the right in concentration response curves of bethanechol (Figure 2). Changes in tissue sensitivity ranged from 0.1 to 0.6 log units, and concentration ratios were corrected for these changes.” A mean (+ SEM) pA, (n = 3) value of 8.91 + 0.06 (corresponding to a K, of 1.2 x lo-’ M) with a mean (*SEM) slope of 0.99 + 0.05 was determined for atropine. The mean K, and 95% confidence limits calculated for (k)-QNB (n = 3) were 1.9 X lo-’ and (5.2 X lo-‘“-6.1 X lo-‘) M, respectively.

of Drugs

Pharmacologic

RECEPTORS

0

3x1d5

a

0

1o-4 3x1o-4

0

0

go- 3 3~16~5~

0

.

lo-hASH

.

WASH

contractile response (polygraph tracing) of a circular muscle strip from the LES to cumulative additions of bethanechol. Basal tension (star) was 2 g. S, and S, refer to different amplifier sensitivities. Note the lack of spontaneous activity before addition of bethanechol and the rhythmic activity superimposed on tonic contractions induced by cumulative additions of hethanechol.

RIMELE

1226

GASTROENTEROLOGY

ET AL.

[3H]QNB binding. In contrast, nicotinic and noncholinergic drugs failed to alter specific binding even at high concentrations. The muscarinic agonists, arecoline, pilocarpine, bethanechol, and carbachol, had a lesser affinity for the binding site than did the antagonists. Specific binding was stereoselective, with (-)QNB being W-fold more potent than (+)-QNB at inhibiting specific binding of [3H]QNB (Figure 7).

Binding as a Function of rH]QNB Concentration The binding curve had two components, one that was saturable and one nonsaturable (Figure 6A and B). A Scatchard plot of specifically bound [“H]QNB revealed the presence of one specific binding site over the ligand concentration range tested (Figure 6C). The mean f SEM for the apparent K, from three experiments was 1.1 f 0.1 nM. The calculated maximal number of binding sites (B,,,) from three experiments was 147 f 5.5 fmol/mg protein. A plot of the binding data according to the Hill equation revealed a straight line with a Hill coefficient nH = 1.04, indicating the absence of cooperative interactions (Figure 6D). The Hill coefficient from the three experiments was 1.06 f 0.07.

Binding to Various Regions of the Gut The distribution of smooth muscle along the esophagus differed among the cats used, but specific binding of [“H]QNB paralleled the relative amount of smooth muscle at any given level of the esophagus (Figure 6). The degree of specific binding in separated circular and longitudinal muscle layers of the LES was similar (Table 2). Fundic muscle taken from just below the gastroesophageal junction had about four times the specific [“HIQNB binding capacity of the esophageal smooth muscle, but binding along the small bowel was similar to that in the esophagus (Table 2).

Effect of Drugs on rH]QNB Binding A variety of drugs inhibited specific [3H]QNB binding to varying degrees (Table 1). The muscarinic antagonists, QNB, propantheline, methscopolamine, and atropine were the most potent inhibitors of

SETHANECHOL

100

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t g

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LT k

60-

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50-

g

40-

2

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8

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CONCENTRATION (M)

-I’1 Figure

t)

QNB

(3~0-~M)

i

-

1;0-0

IO-'

10." BETHANECHOL

OS

Vo!. 77, No. 6

O4

CONCENTRATION (M)

0'

D2

2. Data from representative experiments illustrating response of the LES to bethanechol in the absence and presence of antagonists. A, Antagonism by atropine: (-*) Control, (-0-) 10e9 M, (-A-) 10-s M, (-A-) 10m7 M. The inset is a Schild plot” of these data. CR represents the concentration ratio and the pA, (6.96) is the -log of the intercept of the line (drawn by linear regression, r = 0.97) with the abscissa. B. Antagonism by (k)-QNB. A Ks of 6.5 x 10mXo M for (k)-QNB was determined by the method of Furchgott.”

December

1979

MUSCARINIC

RECEPTORS

IN LES

1229

ATROPINE

NONSPECIFI C

J

Figure

ATROPINE

( M)

Inhibition of [“HIQNB binding by atropine. Three hundred microliters of LES homogenate (500-720 pg protein) were incubated in a total volume of 1.8 ml of 50 mM Tris buffer with 1.3 nM [3H]QNB (16.4 Ci/mmol) and various concentrations of atropine for 30 min at 37°C. Points represent total [3H]QNB binding minus filter binding. Each point represents the mean + SEM from three experiments, each performed in triplicate. Control indicates incubations without atropine.

4. Time-course for association of specific (0-O) and nonspecific (O-0) binding of [“HIQNB to the LES of the cat. Aliquots of tissue homogenate (-650 pg protein) were incubated with -1.5 nM [“HIQNB in 50 mM Tris buffer. Specific and nonspecific binding were determined at various times at 37°C as described in Materials and Methods. Association was begun by addition of tissue and terminated by filtration. The data shown are from three experiments, each done in duplicate. Figure

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TIME Cm)

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40

60

80 TIME

IO0 120 (m!n)

‘40

160

5. Kinetic plots of specific [SH]QNB binding. A. Data from Figure 4 (first seven points) were used to determine X (amount of specific [3H]QNB bound at time t) and X,, (amount of specific [3H]QNB bound at equilibrium). The line, determined by linear regression (r = 0.98), has a slope of koh. The forward rate constant k,, was calculated from k, = (kob - k,)/ [QNB] where k, is the rate constant for dissociation (Figure 58) and lQNB1 is the concentration of [SH]&JB il.5 nM). 8. Time-course for dissociation of specific [3H]QNB binding. Aliquots of tissue homogenate (-200 pg protein) were incubated with 1.5 nh4 [SH]QNB in 50 mM Tris buffer for 30 min after which a large excess of atropine (1 x lo-” M) was added to prevent rebinding of the free radioligand. The time of atropine addition is defined as t = 0. At the indicated times, specific

[“HIQNB binding was determined and compared with binding immediately before the addition of atropine. The data are from four experiments, each done in duplicate. The line was determined by linear regression (r = 0.98). X,, represents the amount of specific binding immediately before the addition of the atropine, and X the amount of specific binding at each time after its addition.

Discussion Binding of [“Hlquinuclidinyl benzilate to the smooth muscle from the LES of the cat possesses the characteristics expected of binding to muscarinic cholinergic receptors. The binding satisfies criteria previously established for characterization of muscarinic receptors from pharmacologic studies on intact tissues,‘.7.“.17~1Y.21~23 and other ligand binding studies.“-‘3 These criteria include saturability, reversibility, tissue specificity, stereoselectivity, and

high affinity of the ligand. Binding should also relate to biologic activity. In agreement with studies in other tissues,7-‘3 muscarinic receptors in cat LES appear to be present as a single population of saturatable sites. The apparent K, from Scatchard plots is in good agreement with values for guinea pig lung,‘” longitudinal muscle of the guinea pig ileum,7 and bovine retina.25 In addition, the maximum density of muscarinic receptors in the LES (147 fmol/mg protein) is virtually the same as that found in the guinea pig lung” (153

1230

Figure

RIMELE

GASTROENTEROLOGY

ET AL.

6. [3H]QNB binding as a function of increasing concentrations of [3H]QNB. Homogenates of LES muscle (800 gg protein) were incubated, as described in Materials and Methods, with various concentrations of [3H]QNB. Nonspecific binding was measured in the presThe ence of 5 x lOme M atropine. data are from a representative experiment performed in duplicate. A. Total and nonspecific binding. B. Specific binding, determined by subtracting nonspecific from total binding at each concentration. C. Scatchard plot of specifically bound [3H]QNB. D. Hill plot of binding data, with a Hill coefficient of nH = 1.04. The mean + SEM for the apparent Ko from a total of three experiments was 1.1 + 0.1 nM; maximal number of binding sites (B,,,) and the Hill coefficient were 147 f 5.8 fmol/mg protein and nn = 1.06 f 0.07, respectively.

2

0

4

6

8

IO

12

14

[L3H1 QNB]hM)

[[3HtONB](nM) I 2

z

2 oI

I

1

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fmol/mg

<

4

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isl6-

D nH=104

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40-

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m 5

20

~

2

-02-

2 -

Vol. 77, No. 6

0

20 L3H]

-04

406080CO120140160 3NB

fmol/mg protein) and bovine retina” (140 fmol/mg protein). It has been previously emphasized,3*28.27 and we have confirmed, that the interaction of ligand with receptor occurs rapidly. The half-life for association of [“H]QNB was estimated to be 9.5 min; however, dissociation was very slow, with a half-life of 145 min. Slow dissociation of [3H]QNB has been observed in other binding’~“~“~‘”and functional studcharacteries7,2s and is related to the hydrophobic istics of QNB.” Our kinetically derived K, was lower than the K, estimated from equilibrium saturation data. The uncharacteristically slow dissociation of the ligand from receptor sites may partially account for this discrepancy. Kinetic K,‘s are less dependent upon receptor (tissue) and ligand concentrations than K,‘s determined from saturation isotherms.“~” If we had chosen different protein and ligand concentrations for these studies, agreement between the two values might have been better. However, the K, we determined stoichiometrically is in good agreement with the K, for biologic activity of QNB in the LES. Although specific [3H]QNB binding was saturable and inhibited by atropine, this is not sufficient evidence to ensure that binding is to “true” muscarinic receptors. One would expect estimates of binding (IC50) and pharmacologic (ED50 and KB) affinities to

BOUND (fmolhg

proten)

-J -9 LCG[[3H1

-8 QNB]

-7 (M 1

be in agreement if they are calculated using the same tissue (provided receptor reserve is small). Indeed, the relative binding affinities for the muscarinic drugs we used closely parallel binding affinities for muscarinic receptors in a wide variety of tissues.7~10.12.25 For example, QNB is more potent than atropine in inhibiting specific [3H]QNB binding in cat LES, rat brain,’ and guinea pig ileum.7 In contrast, nicotinic and noncholinergic drugs had little or no affinity for the [3H]QNB binding sites in these tissues. Our pA, value for atropine (8.91) is in excellent agreement with values obtained by others from gut tissue.l’ In addition, the pharmacologic (functional) estimate of atropine’s affinity (K, = 1.2 X 10m9 M) agreed with the affinity (IC50 = 3 x lo-" M) estimated from the binding study. Furthermore, the K, for (+)-QNB (1.9 x 10e9M) is in good agreement with its binding IC50 (1.0 X lo-'M), as is the ED50 (1.3x 10e5M) of bethanechol with its IC50 (3 x lo-‘M). Another criterion for receptor identification is stereoselectivity. Ideally, if one obtains the same isomerit activity ratios for a given pair of stereoisomers in intact tissues, and in binding experiments, proof of the receptor can be accepted more readily.“” The stereoselective binding we observed agrees with the relative pharmacologic activity of the enantiomers of QNB in other tissues.‘0.31.32 When, as in QNB, the asymmetric center of anticholinergic esters is situ-

December

Table

1979

1.

Relative

MUSCARINIC

Potencies

of Drugs that Inhibited

Specific TH] QNB Binding to Homogenates of Cat LES” Drug (-)-QNB (+)-QNB Propantheline Methscopolamine Atropine Clidinium Isopropamide Glycopyrrolate Tridihexethyl (+)-QNB Dicylclomine Anisotropine Arecoline Pilocarpine Beth,anechol Carbachol McN-A-343-11

IC50(M)b 6.0 1.0 1.5 1.7 3.0 5.0 1.2 1.3 1.5 2.5 7.0 1.7 3.5 7.0 3.0 3.5 2.0

X x x x x x x x x X x x x x x x x

lo-“’ 1o-y 10-y 10-s 10-g 1o-9 lo-* 10-8 lo-” lo-' lo-* 10-7 10-e 10-S 10-S 10-S

10-4

No inhibition at 10 CM Dichloroisoproterenol, histamine, 5_hydroxytryptamine, propran0101, hexamethonium, phentolamine, haloperidol, isoproterenol. metoclopramidc Less than 15% inhibition at 10 pM Phenylephrine, decamcthonium. cimetidine, DMPP, physostigmine ’ Tissue homogenates (250-400 pg protein) were incubated with 0.5-1.0 nM [“H]QNB for 30 min at 37’C. b Molar concentration of drug which inhibited specific [3H] QNB binding by 50%. IC50 values were determined from a log-probit plot, using four to five different concentrations of the inhibitor. Values are the means of at least two experiments (each in duplicate or triplicate).

ated in the alcoholic moiety great differences in potency between enantiomers are not seen.31 Thus, the relatively small difference in potency we observed (Figure 7) could be expected. Specific binding paralleled the relative amount of smooth muscle at any given level of the esophagus.

RECEPTCIRS

IN LES

1231

This finding corresponds with the regional variations in response of cat esophageal muscle to stimulation by acetylcholine, carbachol, and methacholine.“” It therefore appears that [3H]QNB binding will not distinguish the LES from adjacent regions of the esophagus. However, esophageal and fundic tissue can be easily distinguished by the greater [3H]QNB binding capacity in the fundic region. The binding characteristics in the small bowel appear to be very similar to those in areas of the esophagus where smooth muscle is present. by binding A question that cannot be answered studies is the physiologic significance of the muscarinic receptors in the LES. However, bethanechol, a selective neuroeffector muscarinic agonist, increases LES pressure in normal subjects and patients with chronic gastroesophageal reflux.34-37 Bethanechol also produces contraction of the LES in anticholinergic cat ‘a and opossum.39 Conversely, agents either have no effect on basal LES pressure or reduce it only slightly.“-“” In addition to neuroeffector receptors, a second type of muscarinic site in the LES has been postulated. Stimulation of this receptor by McN-A-343-11 causes relaxation via an intramural inhibitory neuron.e’ McN-A-343-11 exerts a preferential action on ganglionic muscarinic receptors,’ although it has some activity in smooth muscle preparations devoid of ganglion cells.45 On the other hand, muscarinic antagonists such as atropine and propantheline (and presumably QNB) have similar effects on both receptor types.’ Since our preparation contained intramural nerves, we cannot eliminate the possibility that some binding may have occurred to neural elements of the sphincter. However, from our saturation data, only one binding site (over the ligand concentration range tested) seems probable. it one Figure

7. Inhibition of specific [3H]QNB binding by the (-)- and (+)- stereoisomers of quinuclidinyl benzilate. Homogenates of LES muscle (500 pg protein) were incubated with 1.3 nM [“HI- QNB and various concentrations of (-)- and (+)-QNB for 30 min at 37°C. EAch point represents the mean f SEM of four experiments, each done in duplicate, or triplicate, except for three points (lo-'",lo-'2, 10UfiM) which represent the mean of two experiments, each in duplicate. The isomeric activity ratio (IAR) is defined as the ratio of the concentrations of (-)and (+)-QNB that inhibited specific binding by 50%. The mean IAR and 95% confidence limits (in parentheses) were calculated from the IAR’s from the four separate experiments.

1232

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GASTROENTEROLOGY

ET AL.

Vol. 77, No. 6

16cm

14cm

12cm

10cm

8cm

6cm

4cm

2cm

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GEJ

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25

L

11

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. ib

13Hl QNB specifically bound (f mol /mg protein)

Figure 8. Regional distribution of specific [3H]-QNB binding to the outer muscular coats of the cat esophagus. The esophagus was marked in situ every 2 cm moving proximal from the gastroesophageal junction. The mucosa and submucosal layers were removed. Homogenates of the tunica muscularis were incubated with 1.5 nM 13H]-QNB for 30 min at 37°C. Bars depict data from four separate experiments, each in triplicate. The star indicates the mean binding at a given region of the esophagus. GEJ refers to the gastroesophageal junction. Photomicrographs (H & E X 42) are representative and were taken from one of the four cats used in this series of experiments. C and L refer to circular muscle and longitudinal layers of the tunica muscularis, respectively.

December

1979

Table 2.

MUSCARINIC

Specific rH] QNB Binding to Various of the Cat Alimentary Tract” Specific (fmoI/mg

Region LESb Circular muscle of LESb.d Longitudinal muscle of LESb Stomach (fundus) Duodenum’ Jejunum Ileum

65.0 63.5 60.6 240.2 81.5 82.8 81.1

Regions

binding protein)

-+ 6.4 (12)’ f 9.1 (4) f 12.2 (4) + 32.1 (4) +- 13.5 (4) f 8.7 (3) f 10.5 (3)

” Tissue homogenates (250-500 pg protein) were incubated with 1.5 nM [‘H] QNB for 30 min at 37°C. Specific binding was determined as described in Materials and Methods. Values were determined at a single ligand concentration rather than B,,,. They are proportional to B,,, if one assumes the same affinity for QNB in the various regions. b Refers to tissue taken from the distal 1.5 cm of the esophagus, measuring from the esophagogastric mucosal junction. ’ Mean f SEM from the number of experiments shown in parentheses. d Contains tissue that relaxed upon electrical field stimulation before the binding assay. c Samples from the small bowel were taken from the mid-point of each segment.

assumes that [“HIQNB has the same affinity for muscle and neural receptor types, then one would observe only one binding site. In conclusion, our experiments demonstrate that ligand binding studies are an effective means of directly measuring parameters of receptor occupancy in esophageal smooth muscle. Furthermore, comparison of these parameters with those measured in diseased or functionally abnormal tissue may provide insight into the pathophysiology of esophageal motor disorders.

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