Abstract
Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction.
Located at the junction of the tubular esophagus and the saccular stomach, the gastroesophageal junction (GEJ) is the area of transition from positive pressure in the abdominal cavity to the respiratory oscillations of negative and positive pressure in the thoracic cavity. It has the dual function of ensuring passage of a swallowed bolus and preventing gastroesophageal reflux. The existence of an anatomical sphincter at the GEJ has been disputed for more than half a century.
The existence of a sphincter at the GEJ was proposed in an observational and anatomical study of cadavers (Lerche, 1950). With the use of manometry in 1956, this same area was described as a high-pressure zone (HPZ) rather than an anatomical sphincter (Code et al., 1956). Since then, much has been written about the HPZ in the lower esophagus. It is mainly composed of pressures from the extrinsic crural diaphragm and the intrinsic muscles of the stomach and the lower esophagus (McCray et al., 2000).
In 1979, the arrangement of the smooth muscles around the GEJ was first described as consisting of “clasp” fibers at the lesser curvature and “sling” fibers at the greater curvature of the stomach, suggesting that these muscle fibers might produce the HPZ at the GEJ (Liebermann-Meffert et al., 1979). This theory laid the foundation for further studies on the physiology, pathology, and pharmacology of the GEJ. Over the span of the next three decades, the formation and regulation of the HPZ were closely studied with the help of high-resolution endoscopic ultrasound, esophageal manometry, autopsies, and animal experiments (Burleigh, 1979; McCray et al., 2000). Differences have been reported between human clasp and sling stomach muscle fibers in the sensitivity and maximal responses to acetylcholine, dopamine, phenylephrine, and isoproterenol (Tian et al., 2004). Differences in response to electric field stimulation (EFS) have also been reported between both the clasp and sling fibers shown to relax to EFS, whereas the areas caudal to this (stomach) contracted under EFS (Burleigh, 1979).
Muscarinic receptors belong to the G protein-coupled receptor family. Five subtypes designated M1 to M5 exist. No completely specific agonists or antagonists are known for any of the subtypes; however, some very specific toxins have been identified. The potency of subtype-selective muscarinic receptor antagonists suggests that in most smooth muscles, contraction is primarily mediated by the M3 receptor subtype (Caulfield, 1993; Caulfield and Birdsall, 1998). M2-mediated contractile responses have been shown in smooth muscle cells isolated from the cat esophagus (Biancani et al., 1997). However, M3 receptors predominantly mediate contraction of smooth muscle cells isolated from the cat lower esophageal sphincter (LES) circular smooth muscle. In an experimental model of esophagitis created by perfusing the esophagus with HCl, the affinity of antimuscarinic drugs is altered and is intermediate between their reported M2 and M3 affinities (Biancani et al., 1994).
Under certain experimental conditions, several studies have shown that the M2 receptor subtype contributes to the contractile response. These include alkylation of M3 receptors with increased intracellular levels of cAMP in the rat bladder (Hegde et al., 1997; Braverman and Ruggieri, 1999), guinea pig ileum (Ehlert and Thomas, 1995), and trachea (Thomas and Ehlert, 1996) or after alkylation without increasing intracellular cAMP levels in other tissues such as the guinea pig gallbladder (Braverman et al., 2000) and colon (Sawyer and Ehlert, 1998). In some experimentally induced pathologies, an increased contractile role for the M2 receptor subtype is evident. These include a cat model of experimentally induced esophagitis (Sohn et al., 1997), the denervated rat bladder (Braverman et al., 1998), and a model of acute cholecystitis in the guinea pig gallbladder (Braverman et al., 2000).
The aim of the present study was to determine which muscarinic receptors mediate contraction of human clasp, sling, LEC, MEC, and MEL muscle fibers and to quantify the density of total and M2 and M3 muscarinic receptor subtypes in these tissues. This information may suggest useful targets for the development of drugs to treat disorders of the GEJ.
Materials and Methods
Materials. All drugs and chemicals were obtained from Sigma-Aldrich (St. Louis, MO), except darifenacin (which was a generous gift from Pfizer Central Research, Sandwich, Kent, UK), digitonin (Wako Pure Chemicals, Osaka, Japan), and pansorbin (Calbiochem, San Diego, CA).
Human stomachs with the attached esophagus were obtained, with consent, from brain-dead organ transplant donors through either the National Disease Research Interchange (Philadelphia, PA) or the International Institute for the Advancement of Medicine (Jessup, PA). Peritoneal fat was removed, and dissection began using microscissors to remove the most superficial longitudinal fibers in a circular pattern around the esophagus. The deeper circular fibers were removed next, moving from the greater curvature toward the lesser curvature. The exact location of the sling and clasp fibers was identified at the greater and lesser curvature of GEJ, respectively, once the superficial longitudinal fibers were removed. Sling muscle fibers were removed from a relatively straight section of the greater curvature. Clasp fibers were obtained 2 to 3 cm distal to GEJ along the lesser curvature. The LEC fibers were obtained from the thickened area of the esophagus approximately 1 to 2 cm proximal to the stomach. The MEC and MEL fibers were obtained from the esophagus 10 cm proximal to the stomach. The muscles were further divided into individual strips, each measuring 1 to 2 mm in width and 8 to 10 mm in length. Care was taken to ensure the orientation of the muscle fibers parallel to the muscle strips. The muscle strips were then suspended with 0.5 g of tension in tissue baths containing 10 ml of modified Tyrode's solution (125 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.8 mM CaCl2, 0.5 mM MgCl2, 23.8 mM NaHCO3, and 5.6 mM glucose) and equilibrated with 95/5% O2/CO2 at 37°C.
Bethanechol Response Curves. After equilibration to the bath solution for 30 min, the strips were incubated for 30 min in the presence or absence of one of three concentrations of the competitive M2-selective antagonist methoctramine (1E-7, 1E-6, or 1E-5 M) or the competitive M3-selective antagonist darifenacin (3E-8, 1E-7, or 3E-7 M). Dose-response curves were derived from the peak tension developed after the cumulative addition of nonsubtype-selective muscarinic receptor agonist bethanechol. Bethanechol concentrations at half-log intervals from 1E-8 up to 1E-2 M if required to reach maximal contraction were used with approximately 3 min between addition of successive concentrations. Either vehicle or one concentration of methoctramine or darifenacin was used for each muscle strip. Dose ratios were determined based on the average of the responses of vehicle (H2O)-treated strips. EC50 values were determined for each strip using a sigmoidal curve fit of the data (Origin; OriginLab Corp., Northampton, MA), and Schild plots were constructed.
Immunoprecipitation. Immunoprecipitation of muscarinic receptors from the individual dissections was performed using subtype-selective antibodies. The specificity of these antibodies and methods has been described in detail previously (Braverman et al., 2007). This immunoprecipitation assay makes use of tandem specificity: the specificity of [3H]QNB binding to only muscarinic receptors and the specificity of the individual antibody binding to only the given subtype. If the antibody binds to other proteins that do not bind [3H]QNB, then those proteins would not be detected in the assay. Likewise, if [3H]QNB binds to other proteins that do not bind to the antibody, then those proteins would not be detected by the assay. In brief, the tissues were homogenized at 100 mg/ml in ice-cold Tris-EDTA (TE) buffer, with 10 μg/ml of the following protease inhibitors: soybean and lima bean trypsin inhibitors, aprotinin, leupeptin, pepstatin, and α2-macroglobulin. Twenty microliters of the nonsubtype-selective muscarinic receptor antagonist [3H]QNB (49 Ci/mM, approximately 4000 cpm/μl) per milliliter of assay homogenate was added and incubated at room temperature for 30 min, with inversion every 5 min. Samples were pelleted via centrifugation at 20,000g for 10 min at 4°C, and the pellet was solubilized in TE buffer containing 1% digitonin and 0.2% cholic acid (1% TEDC), with the above-mentioned protease inhibitors at 100 mg of wet weight per ml. Samples were incubated for 50 min at 4°C, with inversion every 5 min, and then centrifuged at 30,000g for 45 min at 4°C. The supernatant containing the solubilized receptors was incubated overnight after addition of the M2 antibody, the M3 antibody, or vehicle at 4°C.
To determine total receptor density, samples were desalted over Sephadex G-50 minicolumns with 0.1% TEDC. M2 and M3 receptors were precipitated by adding 200 μl of pansorbin and incubated at 4°C for 50 min, with inversion every 5 min. The precipitated receptors were pelleted via centrifugation at 15,000g for 1 min at 4°C, and the pellet was surface washed with 500 μl of 0.1% TEDC. Fifty microliters of 72.5 mM deoxycholate/750 mM NaOH was added and incubated for 30 min at room temperature. The pellet was resuspended in 1 ml of TE buffer and neutralized with 50 μl of 1 M HCl. Radioactive counts were determined by liquid scintillation spectrometry. Protein content was determined by a Coomassie Blue dye binding protein assay using bovine serum albumin as a standard. Receptor density (mean ± S.E.M.) is reported as femtomoles of receptor per milligram of solubilized protein.
Statistics. All statistical differences were determined by a non-parametric statistic (Wilcoxon rank sum/Mann-Whitney U test) because of nonhomogenous variances.
Results
Immunoprecipitation. Five different dissections of human gastroesophageal smooth muscle were studied. These sections were clasp, sling, LEC, MEL, and MEC. For each dissection, we determined total, M2, and M3 muscarinic receptor densities using immunoprecipitation (as described under Materials and Methods), and we did this as a prelude to subsequent studies of bethanechol-induced contraction, which are also described below. The results of the receptor density determinations are shown in Table 1. The rank order of total receptor density in the five different smooth muscle dissections was sling > LEC > clasp > MEL ≈ MEC fibers. The M2 receptor subtype density followed a similar pattern as total receptor density with sling > clasp > LEC > MEC ≈ MEL fibers. However, the M3 receptor subtype density was 60 to 83 fmol/mg protein for the sling, LEC, MEC, and LEC fibers but approximately 10-fold less (8 ± 2 fmol/mg protein) for the clasp fibers.
TABLE 1.
Total, M2, and M3 muscarinic receptor density (femtomoles per milligram of solubilized protein) for different dissections of human GEJ muscles Total muscarinic receptor density was determined by total [3H]QNB binding, whereas M2 an M3 receptor density was determined using subtype-selective immunoprecipitation (as described under Materials and Methods). Results are reported as mean ± S.E.M. for at least duplicate determinations from two individual organs for clasp and sling fibers, whereas n = 3 donors for LEC, MEL, and MEC fibers. Statistical differences were determined using nonparametric statistics with a Mann-Whitney U test.
| Muscle | Total | M2 | M3 | M2/M3 Ratio |
|---|---|---|---|---|
| Clasp | 228 ± 20a | 116 ± 16b,c,d | 8 ± 2a,b,c,d | 14.5 |
| Sling | 353 ± 7b,c,d | 171 ± 6b,c,d | 60 ± 14 | 2.85 |
| LEC | 244 ± 12c,d | 73 ± 7 | 83 ± 13 | 0.88 |
| MEC | 190 ± 7 | 59 ± 9 | 69 ± 9 | 0.86 |
| MEL | 209 ± 10 | 54 ± 4 | 78 ± 3 | 0.69 |
(P < 0.05 if not bold, and P < 0.01 if bold)
, Significantly different from sling fibers
, Significantly different from LEC fibers
, Significantly different from MEC fibers
, Significantly different from MEL fibers
Concentration-Effect Relationships. Representative tracings of bethanechol concentration-response experiments for each muscle fiber are shown in Fig. 1. Spontaneous activity was sometimes observed; however, this did not interfere with the determination of the effect of bethanechol, and the antagonists had no effect on baseline spontaneous activity. Each muscle section was studied for isometric tension development in response to bethanechol and each demonstrated a dose-related response to this agonist. For example, Fig. 2 shows the graded concentration-effect relationship for bethanechol in clasp fibers. Also shown in this figure are the curves for graded doses of this agonist with three different fixed concentrations of darifenacin, a relatively selective M3-competitive antagonist. Shown in Fig. 3 are the curves for graded doses of this agonist with no antagonist and with two different fixed concentrations of methoctramine, a relatively selective M2-competitive antagonist. The fitted curves show an obvious dose-dependence; furthermore, they also show rightward shifts resulting from each antagonist dose. These log plots show approximate parallelism (indicative of competitive inhibition). However, the relatively low potency calculated by Schild analysis for darifenacin in clasp fibers (pA2 = 7.8 ± 0.2) compared with the reported darifenacin affinity at M3 receptors (pKb = 8.65) and M2 receptors (pKb = 7.20) suggests that M2 receptors mediate contraction. In contrast, the low potency calculated for methoctramine in clasp fibers (pA2 = 6.3 ± 0.2) compared with its reported affinity at M2 receptors (pKb = 8.05) and M3 receptors (pKb = 6.60) suggests that M3 receptors mediate contraction (Caulfield, 1993; Caulfield and Birdsall, 1998). Darifenacin potency (pA2) calculated by Schild analysis is 8.0 ± 0.1, 8.2 ± 0.2, 8.2 ± 0.1, and 8.4 ± 0.2, and methoctramine potency (pA2) is 6.8 ± 0.2, 6.2 ± 0.2, 5.7 ± 0.2, and 5.6 ± 0.3 in sling, LEC, MEC, and MEL fibers, respectively.
Fig. 1.
Original tracings of bethanechol concentration-response experiments from the various smooth muscle components of the human GEJ.
Fig. 2.
Concentration-response curves for bethanechol-induced contraction of human clasp fibers in the presence of various concentrations of darifenacin (DAR). Inhibition of bethanechol induced human clasp fiber contractions with increasing concentrations of the M3-selective antagonist darifenacin causes parallel rightward shifts in the concentration-response curve. Results are shown as percentage of the maximal response shown in Table 2. Control, n = 14 strips from four donors; 30 nM DAR, n = 6 strips from two donors; 100 nM DAR, n = 5 strips from two donors; and 300 nM DAR, n = 7 strips from two donors.
Fig. 3.
Concentration-response curves for bethanechol-induced contraction of human clasp fibers in the presence of various concentrations of methoctramine (METH). Inhibition of bethanechol induced human clasp fiber contractions with increasing concentrations of the M2-selective antagonist methoctramine causes parallel rightward shifts in the concentration-response curve. Results are shown as percentage of the maximal response shown in Table 2. Control, n = 14 strips from four donors; 1 μM METH, n = 3 strips from one donor; and 10 μM METH, n = 3 strips from one donor.
These potencies in clasp and sling fibers suggest that the bethanechol effect is mediated by both M2 and M3 receptors; hence, using Schild plot analysis that is based on the assumption that one receptor is mediating the effect is inappropriate. For that reason, and to add clarity to the relative contribution of each receptor subtype, we transformed each bethanechol concentration to receptor occupations of both M2 and M3 receptors. That transformation is based on mass-action binding which, at equilibrium, gives receptor occupation = [A][R]/([A] + KA), where [A] denotes the agonist concentration, [R] is the receptor concentration, and KA is the agonist dissociation constant (reciprocal of affinity). For this purpose, we used published values of KA for bethanechol as follows: KA for M2 = 1.7 E-4 M derived using cloned human M2 receptors expressed in Chinese hamster ovary cells (McKinney et al., 1991) and KA for M3 = 1.1 E-4 M derived from human astrocytoma cells, which predominantly express M3 receptors (Evans et al., 1985). The concentration-effect curve in clasp fibers is shown Fig. 4 in which the abscissa scales show the simultaneous values of M2 and M3 occupancy that follow from the bethanechol concentrations that were used. It is noted that the M2, M3 occupation pair that gives 50% of the maximum tension is the pair (8.8, 0.9). However, from this graph it is not apparent that occupancy of both M2 and M3 receptors occurs simultaneously, resulting in contraction. This critical point is more clearly evident in an alternative view of this dual receptor occupation-effect (Fig. 5), which is a three dimensional plot with the effect shown as the height above the M2-M3 occupation plane.
Fig. 4.
Bethanechol-induced clasp fiber contraction as a function of M2 and M3 receptor occupancy. The human clasp fiber bethanechol concentration-response curve was converted into occupation response curves for the M2 and the M3 receptor subtypes. The y-axis is the percentage of the maximal bethanechol effect, and the lower x-axis shows the density of M2 receptor occupied by bethanechol, whereas the upper x-axis shows the density of M3 receptors occupied. Receptor occupation = [A]/([A] + KA), where [R] denotes the receptor concentration (R was obtained from the immunoprecipitation results shown in Table 1), KA is the agonist dissociation constant (reciprocal of affinity), and [A] is the agonist concentration. For this purpose, we used published values of KA (Evans et al., 1985; McKinney et al., 1991) for bethanechol as follows: KA for M2 = 170 μM and KA for M3 = 110 μM.
Fig. 5.
Three-dimensional graph of bethanechol-induced clasp fiber contraction as a function of M2 and M3 receptor occupancy.
Antagonist Effects. The presence of a fixed concentration of a competitive antagonist reduces the agonist occupancy to a lower quantity given by the equilibrium equation of Gaddum (1937): receptor occupation = [A][R]/[A] + KA(1 + [B]/KB), where [B] is the antagonist concentration and KB is its dissociation constant. Of course, this holds at each receptor with each receptor's applicable values of KA and KB. Thus, the presence of the antagonist yields bethanechol occupancy at M2 and M3, each calculated from the above-mentioned equation, thereby giving a view of occupation of this receptor pair and its corresponding effect. This relation is shown in the three-dimensional plot (Fig. 6). This graph, for clasp fibers, was generated using published affinity values (Caulfield, 1993; Caulfield and Birdsall, 1998), from three different doses of darifenacin (pKB M3 = 8.65, pKB M2 = 7.2, thus relatively selective for M3) and two different doses of methoctramine (pKB M3 = 6.6, pKB M2 = 8.1, thus relatively selective for M2). The use of the two antagonists in several different fixed concentrations yielded an array of M2, M3 occupancy values and their associated effects.
Fig. 6.
Surface plot of clasp fiber contraction as a function of M2 and M3 receptor occupancy. Subtype-selective antagonists alter the number of M2 and M3 receptors occupied by bethanechol that yield a given effect level. Using the formula for occupancy of an agonist in the presence of an antagonist [receptor occupancy = AR/(A + Ka(1 + B/Kb))] and published antagonist affinity values (Caulfield, 1993; Caulfield and Birdsall, 1998), the M2 and M3 occupancy-effect curves in the presence of three concentrations of darifenacin and two concentrations of methoctramine were derived. A surface plot showing the effect of combinations of M2 and M3 occupancy in human clasp fibers is overlaid. The surface plot was constructed by transformation of the individual data points into a matrix using a random gridding method with Kringing correlation (Origin, OriginLab Corp.).
A more global view of these results is provided in the form of a response surface, also shown in Fig. 6, indicating that both M2 and M3 receptors have a significant role in mediating contraction in clasp fibers. This is based on the occupancy-effect relationship in the presence of the antagonists. In the presence of darifenacin, where very few M3 receptors are occupied by bethanechol, the occupancy-effect relationship is more dependent on M2 occupancy than on M3 occupancy. This can be seen on the surface plot in Fig. 6 where the occupancy effect curve in the presence of darifenacin is almost parallel with the axis of M2 occupancy and shows very little deflection along the M3 occupancy axis. In contrast, in the presence of methoctramine, where very few M2 receptors are occupied by bethanechol, the occupancy-effect relationship is more dependent on M3 occupancy than on M2 occupancy.
Other Gastrointestinal Muscle Fibers. The analysis of occupancy-effect relations described above for the clasp fibers was also conducted on the human sling, LEC, MEC, and MEL smooth muscle fibers. For each muscle group, a surface plot, similar to that of the clasp fibers, was generated. The surface plot for sling fibers (Supplemental Fig. 1), which have more M2 receptors than M3 receptors (Table 1), is similar to the surface plot for clasp fibers, which also have more M2 than M3 receptors. The surface plot for LEC fibers, which have more M3 receptors than M2 receptors, has a different shape (Fig. 7). The surface plots for MEC and MEL fibers, which also have more M3 receptors than M2 receptors, are similar to that for LEC fibers (Supplemental Figs. 2 and 3). In these muscle groups, the occupation-effect relationships demonstrate that contraction is more dependent on M3 occupation than M2 receptor occupation. This is demonstrated by the occupation-effect relationship of the LEC fibers shown in Fig. 7. When the M2-selective antagonist methoctramine is present, the occupation-effect relationship shows that contraction is dependent on occupation of M3 receptors. In addition, in the presence of darifenacin, contraction increases with increasing M2 occupancy, but only up to a point; maximal tension is only obtained when the bethanechol concentration is high enough to compete for occupation of the M3 receptors. This is demonstrated in Fig. 7 at the point labeled “a” by the deflection to the right, which means increasing M3 occupancy, of the occupation-effect curve in the presence of darifenacin.
Fig. 7.
Surface plot of LEC fiber contraction as a function of M2 and M3 receptor occupancy. The surface plot was constructed as described for Fig. 6. The point labeled as a denotes the rightward deflection toward increasing M3 occupancy for contraction of LEC fibers in the presence of the M3-selective antagonist darifenacin.
Discussion
The arrangement of the clasp/sling muscle fiber complex was first described in 1979 (Liebermann-Meffert et al., 1979) and was hypothesized to be a physiologic circular smooth muscle sphincter in the distal esophagus (Code et al., 1956). However, until recently, no intrinsic muscarinic receptor-mediated pressure in the proximal stomach has been demonstrated from the gastric sling/clasp fiber muscle complex. Along with this pressure generated, we observed a second muscarinic receptor-mediated pressure profile in the distal esophagus associated with the LEC. These distinct pressure profiles were discovered using simultaneous ultrasound and manometry (Brasseur et al., 2007). Thus, the importance of muscarinic tone within both the distal clasp/sling muscle fiber complex and the more proximal LEC is established.
Using the same techniques in gastroesophageal reflux disease patients, we found that the proximal pressure profile due to the LEC was present. However, the gastric sling/clasp fiber pressure profile was absent in all gastroesophageal reflux disease patients (Miller et al., 2009). Thus, our previous study demonstrated the importance of the intrinsic muscarinic gastric sling/clasp muscle fiber pressure profile to the antireflux barrier. Given the importance of these two distinct muscle complexes to the antireflux barrier, it is important to understand how these muscle complexes function. This includes any anatomic or physiologic differences between the muscle groups that generate the pressure to prevent reflux and the rest of the smooth muscles within the esophagus that do not contribute to the antireflux barrier. It was with these goals in mind that we undertook the current study.
The results presented above show that the density of muscarinic receptor subtypes is different in the different smooth muscle preparations of the human GEJ and esophagus. Both the clasp and the sling fibers, which work together to contract the GEJ to prevent reflux have a greater density of M2 than of M3 receptors similar to most other smooth muscles studied. In LEC, MEC, and MEL fibers, however, M3 receptor density is greater than M2 receptor density.
The bethanechol-induced maximal contraction is greater in the sling fibers than all other fibers studied (Table 2). This result is in general agreement with a previous study showing that human sling fibers contract significantly greater to acetylcholine than human clasp fibers (Tian et al., 2004). As shown here for the first time, sling fibers may contract greater than the other fibers because they have a greater total density of muscarinic receptors than all other muscles studied.
TABLE 2.
Maximal tension and bethanechol potency determined for the different dissections of human GEJ muscles Results are reported as mean ± S.E.M. Statistical differences were determined using nonparametric statistics with a Mann-Whitney U test.
| Muscle | BETH Max | BETH pEC50 |
|---|---|---|
| G | ||
| Clasp | 1.20 ± 0.17a,c (n = 14) | 5.08 ± 0.09c (n = 14) |
| Sling | 2.18 ± 0.24b,c,d (n = 37) | 4.98 ± 0.10b,c (n = 37) |
| LEC | 0.92 ± 0.09 (n = 29) | 5.19 ± 0.11c (n = 29) |
| MEC | 0.79 ± 0.07d (n = 24) | 4.34 ± 0.08d (n = 24) |
| MEL | 1.37 ± 0.21 (n = 10) | 4.80 ± 0.09 (n = 10) |
P < 0.05 if not bold, and P < 0.01 if bold
, Significantly different from sling fibers
, Significantly different from LEC fibers
, Significantly different from MEC fibers
, Significantly different from MEL fibers
Classical pharmacologic analysis of concentration-effect relationships was formulated before the concept of multiple receptor subtypes existed and is based upon the assumption that one receptor mediates one effect. Because of this assumption, there is no theoretical framework that allows Schild analysis to yield meaningful conclusions in the context of multiple receptors mediating a response. Schild analysis yielded conflicting conclusions with respect to which receptor subtype mediates contraction of clasp and sling fibers. The M3-selective antagonist darifenacin yielded an affinity intermediate between that reported for M2 and M3 receptors, suggesting that both receptors may mediate the contractile response. However, the M2-selective antagonist methoctramine yielded a low affinity, suggesting that M3 receptors mediate contraction. Thus, no definitive conclusions regarding the contribution of each subtype to the contractile response could be drawn using Schild analysis.
If contraction was mediated by purely M3 receptors, we would expect that darifenacin would have high potency and methoctramine would have low potency. Conversely, if the M2 receptor solely mediated contraction, we would expect that darifenacin would have relatively low potency, whereas methoctramine would be highly potent. The apparently contradictory results we obtained can be explained if both M2 and M3 receptor subtypes mediate contraction. The M3-selective antagonist darifenacin has only minimal effects until its concentration is high enough to block M2 receptors in addition to M3 receptors. The M2 subtype mediates contraction when low concentrations of darifenacin block M3 receptors, thus there is very little to no shift in the CRC in the presence of low concentrations of darifenacin. Because the rightward shifts in the CRCs in the presence of high concentrations of darifenacin are due mostly to occupation of M2 receptors, the result is parallel CRCs. The opposite is also true for methoctramine, in which M3 receptors mediate contraction until the methoctramine concentration is high enough to block both M2 and M3 receptors.
Using muscarinic receptor knockout mice, we have previously shown that contraction of the stomach body is mediated by both M2 and M3 receptor activation in an additive manner (Braverman et al., 2008). The M3 receptor alone can mediate a maximal cholinergic contraction; however, M2 receptors alone can only mediate a contraction of approximately 45% of M3 receptors. The type of analysis used in that report is dependent on determining the contribution of the individual receptor subtypes, as can be determined in receptor knockout mice. However, because of the lack of completely specific antagonists, the contribution of the individual receptor subtypes cannot be determined in human tissue. Thus, we constructed three-dimensional occupation-effect graphs to allow visualization of how agonist occupancy of M2 and M3 receptors relates to contraction. Based on this analysis, both M2 and M3 receptor subtypes are involved in mediating contraction of all muscle fibers studied here. However, in clasp and sling fibers, where the M2 receptor subtype is more abundant than the M3 receptor subtype, M2 receptors have a greater contribution to the contractile response than in the LEC, MEC, and MEL. In the LEC, MEC, and MEL fibers in which the M3 receptor subtype is more abundant than the M2 receptor subtype, M3 receptors have a greater contribution to the contractile response.
Analysis of the surface plot for the clasp fibers demonstrates that M2 and M3 occupancy is 8.8 and 0.9 fmol/mg solubilized receptor, respectively, at 50% of the maximal contraction. M2 density in clasp fibers is 116 fmol/mg solubilized receptor; thus, 7.6% (8.8/116) of all M2 receptors are occupied by agonist at the 50% effect level. M3 density in clasp fibers is 8 fmol/mg solubilized receptor; so, 11.3% (0.9/8) of all M3 receptors are occupied by agonist at the 50% effect level, a percentage similar to M2 receptors. Based on these occupancies, it seems there is a relatively large surplus of both M2 and M3 receptors in human clasp fibers. In the presence of high concentrations of darifenacin (1E-7 M), the percentage of M2 receptor subtypes occupied by bethanechol is increased compared with the percentage of M3 receptors occupied. Here, M2 and M3 occupancy at 50% of maximal contraction is 33.5% (38.9/116) and 4.25% (0.32/8), respectively, suggesting that there is a relatively small surplus of M2 receptors. Conversely, in the presence of methoctramine (1E-6 M), M2 and M3 occupancy at 50% of maximal contraction is 0.26% (0.3/116) and 7.5% (0.6/8), respectively, suggesting that there is a relatively large surplus of M3 receptors.
The contribution of each receptor subtype is probably dependent not only on the density of the individual subtypes but also on which intracellular signal transduction mechanisms are activated by each subtype. For example, in the feline esophagus, where the M2 receptor subtype predominantly mediates contraction, the M2 receptor activates phosphoinositide-PLC, phosphatidylcholine-PLC, phospholipase D, and cytosolic phospholipase A2 to produce contraction dependent on a protein kinase C. However, in the cat LES, similar to the human LEC reported here, the M3 receptor subtype primarily mediates contraction. In the cat LES, this contraction is mediated by activation of PLC, generation of inositol trisphosphate, release of intracellular calcium, activation of calmodulin, and finally activation of myosin light chain kinase. (Harnett et al., 1999). As demonstrated here, the contribution of M2 and M3 receptors is not the same in the different human smooth muscle preparations. In the cat esophagus, the M2 receptor subtype predominantly mediates contraction, but as demonstrated here, the M3 receptor predominantly mediates contraction in the human longitudinal and circular esophageal muscle layers. Thus, contribution of the individual subtypes to contraction of the same smooth muscles can be different between species.
In summary, the receptor density of each smooth muscle group within the esophagus differs according to the muscle location and function. Using a new method of analysis, it was found that both the M2 and the M3 receptors contribute to the tonic contraction of all of the smooth muscles of the esophagus. However, the M2 muscarinic receptor plays a greater role in mediating contraction of sling and clasp fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction.
Supplementary Material
Acknowledgments
We acknowledge the expert technical assistance of Elan S. Miller and Gabrielle N. Soussan in carrying out the contractility studies.
This work was supported by the National Institutes of Health [Grant R01-DK059500].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.108.148106.
ABBREVIATIONS: GEJ, gastroesophageal junction; HPZ, high-pressure zone; EFS, electric field stimulation; LES, lower esophageal sphincter; LEC, lower esophageal circular; MEC, midesophageal circular; MEL, midesophageal longitudinal; QNB, quinuclidinyl benzilate; TE, Tris-EDTA; TEDC, Tris-EDTA buffer containing 1% digitonin and 0.2% cholic acid; PLC, phospholipase C; DAR, darifenacin; METH, methoctramine; BETH, bethanechol; CRC, concentration response curve.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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