Introduction

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Five subtypes of the muscarinic receptor (M1-M5) encoded by five distinct genes have been identified by molecular cloning methods (Bonner et al., 1987, 1988; Peralta et al., 1987). In the gastrointestinal tract, many smooth muscles express both the M2 and M3 receptor subtypes. In most instances the M3 receptor mediates contraction, whereas the contribution of the M2 receptor remains uncertain (Eglen et al., 1996; Ehlert et al., 1997). Esophageal peristalsis is controlled by the combined effects of inhibitory and excitatory nerves. In vivo studies have shown that the excitatory contribution involves muscarinic receptors because peristalsis in the smooth muscle portion of the esophagus is inhibited by atropine in several species, including the human (Dodds et al., 1981; Paterson et al., 1991). Others have demonstrated that contractions produced by electrical field stimulation of intrinsic nerves in muscle strips from the human esophagus are also substantially antagonized by atropine (Tottrup et al., 1990; Preiksaitis et al., 1994). However, little is known about the expression or function of specific muscarinic receptor subtypes in the human esophagus (Eglen et al., 1996; Ehlert et al., 1997).

The contribution of muscarinic receptor subtypes in controlling peristalsis in animals also is not completely resolved. Previous receptor ligand binding studies demonstrated specific binding of radiolabeled antagonist to muscarinic receptors in cat esophageal muscle (Rimele et al., 1979; Keshavarzian et al., 1992). Keshavarzian et al. (1992) demonstrated that receptors in the cat esophagus did not show characteristics of the M1 subtype, but neither of these studies provides specific information regarding the presence of the other receptor subtypes. Previous in vivo studies in the cat and the opossum demonstrated that the cholinergic contribution to peristalsis involved mainly the M3 receptor (Gilbert and Dodds, 1986; Blank et al., 1989). We recently examined the effects of several muscarinic receptor antagonists on carbachol-stimulated contraction of longitudinal and circular muscle (CM) strips from the cat esophagus and also found a pattern of selectivity consistent with a major functional role for the M3 receptor (Preiksaitis and Laurier, 1998). On the other hand, in dispersed myocytes isolated from the circular muscle layer of the cat esophagus, it was shown that acetylcholine-induced cell shortening involved the M2 receptor subtype (Sohn et al., 1993).

In the present study, we set out to characterize muscarinic receptors in the CM and longitudinal muscle (LM) layers of the smooth muscle portion of the human esophagus. Based on the results of receptor ligand binding and in vitro muscle contraction studies, we have concluded that the human esophagus is similar to other gastrointestinal smooth muscles (Eglen et al., 1996; Ehlert et al., 1997). The M2 muscarinic receptor is most abundantly expressed, but the antagonism of muscle contraction shows a pattern consistent with the M3 receptor subtype. However, further analysis using RT-PCR, immunoloblotting, and immunocytochemistry (ICC) reveals that human esophageal smooth muscle expresses all five muscarinic receptor subtypes, indicating that the pharmacology of this tissue may be more complex than previously recognized.

	Experimental Procedures

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Patients and Tissue Retrieval. Tissue collection was carried out in accordance with the policies of the University of Western Ontario Review Board for Health Sciences Research Involving Human Subjects and conformed to the Helsinki Declaration. Patient consent was obtained for removal of tissues. Tissues were obtained from patients undergoing total removal of the esophagus because of cancer as previously described (Preiksaitis et al., 1994; Sims et al., 1997). The tissue specimen was immediately chilled on ice in the operating room and transported to the laboratory where it was placed in ice-cold Krebs' solution. The length of the esophagus was measured and tissue samples were obtained from the distal third of the esophagus at least 2 cm proximal to the gastroesophageal junction and at least 2 cm away from macroscopically evident disease. Specimens with large tumors involving the area of interest or with Barrett's epithelium overlying the muscle layer were not used in this study. Muscle specimens were free of invasion by cancer cells. A square (1 × 1 cm) of the muscularis propria was dissected by separating the LM and CM layers and cutting them into several strips, ensuring that the long axis of each strip followed the direction of the muscle fibers. Some strips were prepared for isometric tension recording and others were used for preparation of dispersed muscle cells and ICC, as detailed below. At the time of esophageal resection a small portion of each muscle layer was frozen on dry ice and stored at 70°C and used subsequently for preparation of homogenates for ligand binding, immunoblotting, and RNA isolation as described below. In some cases, we were able to obtain a complete cross-section of the esophageal body, which was immediately frozen in OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN) on dry ice and stored at 70°C to be used for receptor autoradiography. Krebs' solution had the following composition: 143 mM sodium, 5.0 mM potassium, 2.5 mM calcium, 1.2 mM magnesium, 128 mM chloride, 2.2 mM phosphate, 25 mM bicarbonate, 1.2 mM sulfate, and 10 mM glucose, and was equilibrated with 5% CO₂ and 95% O₂.

Receptor Binding and Autoradiography. Frozen tissue samples were thawed at 4°C and homogenized in 10 volumes of binding buffer containing 0.32 M sucrose with 10-s bursts (Brinkman Polytron) followed by 1-min cooling on ice repeated three times. The homogenate was filtered through buffer-soaked cheesecloth and centrifuged at 1500g. The supernatant was retained and the pellet was rehomogenized, repeating the above-mentioned steps. The combined supernatants were centrifuged at 40,000g for 20 min. The pellet was resuspended in 10 volumes of binding buffer, yielding the final membrane homogenate used in the binding assay. The binding assay was initiated by the addition of 200 µl of homogenate, adjusted to contain 1 to 2 mg of protein to a final volume of binding buffer containing 25 to 500 pM [³H]QNB. Nonspecific binding was determined for each [³H]QNB concentration by including 10 µM atropine. Antagonist inhibition curves were obtained with 100 pM [³H]QNB and 12 to 16 different concentrations of unlabeled muscarinic antagonists. Binding assays were done in triplicate or quadruplicate at 37°C for 2 h. The binding buffer had the following composition: 120 mM NaCl, 5 mM KCl, 4 mM MgCl₂, 1 mM CaCl₂, 1 mM EDTA, 50 mM Tris, pH 7.4. The binding reaction was terminated by the addition of 10 ml of ice-cold Tris buffer (50 mM Tris, 1 mM EDTA, pH 7.4) followed by rapid vacuum filtration (Skatron FilterMat 11734; Skatron Instruments Inc., Newmarket, UK) and washed with additional buffer. Bound radioactivity was determined by scintillation counting (Scintiverse cocktail, Beckman LS5000TA counter) at 47% efficiency.

RT-PCR. Total RNA was isolated separately from each muscle layer by acid guanidinium thiocyanate-phenol-chloroform extraction. RNA samples were run out on agarose gels to verify integrity. One microgram of total RNA from each sample was reverse transcribed for 90 min at 42°C using random hexamers and Superscript RT (Life Technologies, Gaithersburg, MD). The cDNA was diluted 2.5 times, and 5 µl was used in each 50-µl PCR reaction. PCR reactions were carried out for 35 cycles with 2.5 mM MgCl₂, 0.2 mM dNTPs, 2 µM of each primer pair, and 0.2 µl of Taq DNA polymerase (ID Labs Biotechnology, London, Ontario, Canada) in the reaction mixture. The timing of each cycle was 0.5 min at 94°C, 0.5 min at 58°C, and 1 min at 72°C, followed by a final 7-min extension at 72°C. The PCR oligonucleotide primers (Life Technologies) used to amplify cDNA and the expected product sizes are listed in Table 1. PCR primers for -actin were used to confirm fidelity of the PCR reaction and to detect genomic DNA contamination. The amplified products (10 µl) were analyzed by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The identity of PCR products was verified by sequencing (Robarts Research Institute Core Molecular Biology Facility, London, Ontario, Canada).

Immunoblotting. Tissue was homogenized (Brinkmann Polytron) in 10 mM HEPES buffer (pH 7.0) containing 1 mM dithiothreitol and Mini Complete protease inhibitor cocktail tablets (1 tablet in 25 ml of buffer; Boehringer Mannheim, Indianapolis, IN). Crude protein homogenates were denatured by boiling for 15 min, separated on 10% polyacrylamide gels (50 µg/lane) and electrophoretically transferred to Hybond-ECL nitrocellulose membranes (Amersham, Piscataway, NJ) in buffer containing 25 mM Tris, 250 mM glycine, and 20% methanol (pH 8.3). Membranes were blocked overnight at 4°C with 4% goat serum in 30% goat's milk in Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 (TBS-T). After three washes in TBS-T (10 min), membranes were incubated with primary anti-mAChR antibodies in 1% goat serum, 30% goat's milk in TBS-T (2 h at 22°C). Rabbit polyclonal anti-mAChR subtype-specific antibodies (Research and Diagnostic Antibodies, Berkeley, CA) were used to probe blots at the following dilutions: 1:2000 for anti-M1, 1:2000 for anti-M2, 1:3000 for anti-M3, 1:2500 for anti-M4, and 1:500 for anti-M5. These antibodies have been shown to be highly specific with no cross-reaction between other receptor subtypes or other unrelated peptides, at concentrations greater than used in the present study (Ndoye et al., 1998, Buchli et al., 1999). After five washes in TBS-T (10 min), the membranes were incubated (2 h at 22°C) with anti-rabbit horseradish peroxidase-conjugated IgG antibody (final dilution 1:3000). Antibody binding was detected by enhanced chemiluminescence, with blots exposed to radiographic film for 2 to 15 s (ECL; Amersham). For negative controls primary antibodies were omitted or preadsorbed with the appropriate peptide immunogen and processed as described above.

Preparation of Dispersed Muscle Cells and Immunocytochemistry. Muscle cells were dispersed by enzymatic digestion using a modification of our previous method (Sims et al., 1997). Muscle strips (~1 × 5 mm) were incubated in 2.5 ml of dissociation solution at 4°C for 4 to 12 h. One milliliter of dissociation solution contained 0.2 mg of collagenase (blend type F; Sigma, St. Louis, MO), 2.5 mg of papain (Sigma), 2 mg of bovine serum albumin, 0.4 mg of 1,4-dithio-L-threitol, 10 mM taurine, and 0.5 mM EDTA (adjusted to pH 7.0). Tissues were incubated at room temperature for 30 min and then at 31°C for 60 min. Tissues were transferred to PBS prewarmed to 31°C, washed twice, and dispersed by trituration with a fire-polished Pasteur pipette. One drop of cell suspension was placed on a Superfrost Plus microscope slide (Fisher Scientific, Nepean, Ontario, Canada). After allowing 30 min for cells to settle and adhere, the slide was rinsed in PBS and fixed in 50% acetone/methanol at room temperature for 3 min, and then stored at 70°C until used for ICC.

Recording of Mechanical Activity. Muscle tension recording was done as described previously (Preiksaitis et al., 1994, Sims et al., 1997). Muscle strips (~0.2 × 1 cm) were mounted vertically in 10-ml jacketed organ baths containing Krebs' solution held at 37°C and continuously bubbled with 5% CO₂ and 95% O₂. Using silk ties, one end of the strip was fixed to a tissue holder and the other end to a Grass FT03 isometric force transducer coupled to a Grass 79E chart recorder (Grass Instruments, Quincy, MA). After a 1-h equilibration period individual strip length was adjusted until the optimum response to stimulation with 1 µM carbachol was obtained. All other determinations were done with strips held at this length. Concentration-response curves were produced by exposing strips to 10⁸ to 10⁵ M carbachol in a cumulative manner, each incremental concentration being added when the response to the previous concentration reached a maximum. The cumulative concentration-response curve for carbachol was similar to that obtained with single concentrations of drug with complete washout of the effect between challenges. Muscle strips were exposed to each concentration of muscarinic receptor antagonist for a minimum of 30 min before being rechallenged with carbachol. Usually one concentration of antagonist was tested in each muscle strip to determine the effects on complete carbachol concentration-response relations. In some cases no more than two increasing concentrations of antagonists were tested in sequence, yielding identical results to those obtained with single antagonist concentrations. Because carbachol is also active at nicotinic and muscarinic receptors on parasympathetic ganglia, the nitric-oxide synthase inhibitor N^G-nitro-L-arginine (100 µM) and hexamethonium (10 µM) were present throughout all antagonist experiments to minimize these effects (Preiksaitis and Laurier, 1998). Neither 100 µM N^G-nitro-L-arginine nor 10 µM hexamethonium, alone or in combination, had a significant effect on the maximum tension response or the EC₅₀ for carbachol. Results were expressed as a percentage of the maximum carbachol tension response in each strip.

Data Analysis and Statistics. [³H]QNB saturation and competition experiments were analyzed using GraphPad Prism, version 2.0 (GraphPad Software Inc., San Diego, CA). Using this program, K_d and B_max for saturation experiments are determined by nonlinear curve-fitting by the least-squares method. Antagonist inhibition experiments were analyzed similarly, K_i being calculated by the method of Cheng and Prusoff using the formula K_i = EC₅₀/(1 + [ligand]/K_d). All data were tested to determine the best fit for one or two binding sites based on the F test. Carbachol concentration-responses and the effects of muscarinic antagonists were analyzed using GraphPad Prism, version 2.0. Schild plots were generated to determine pA₂ values with the lines fitted by least-squares linear regression. The slope of a straight line was considered to be not different from unity if the 95% confidence interval for the slope included 1. Results are reported as mean ± S.E. The number of esophageal specimens studied is indicated by n. Statistical comparisons were made with the Student's t test, with P < .05 considered significant.

Drugs and Materials. [³H]QNB (specific activity, 43 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Methoctramine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and pirenzepine were obtained from Research Biochemicals International (Natick, MA). All drugs were prepared as concentrated stock solutions and diluted into Krebs' buffer just before use such that drugs were added to the 10-ml tissue bath in a volume of 10 to 100 µl. All other reagents or drugs used were obtained from Life Technologies, Sigma, or as indicated.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Receptor Binding Studies. Figure 1 shows an autoradiogram demonstrating [³H]QNB binding in a whole-mounted cross section of the distal third of the human esophagus. [³H]QNB specific binding was seen in three muscle layers: the muscularis mucosae, and the LM and CM layers of muscularis propria, identified by comparison with the H&E-stained image of the section used to obtain the autoradiogram. An adjacent section incubated with [³H]QNB plus 10 µM atropine showed no specific binding. Further analysis of [³H]QNB binding was carried out using muscle homogenates. A representative saturation isotherm of [³H]QNB binding to LM and CM homogenates is shown in Fig. 2. Nonspecific binding (in the presence of 10 µM atropine) accounted for <10% of total binding. Specific binding was saturable and best fit by a single population of binding sites. The experiment was repeated for a total of seven esophageal specimens and results are summarized in Table 2. There was no statistically significant difference in either the affinity of [³H]QNB binding (K_d) or the number of binding sites in the LM and CM layer (Table 2).

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Autoradiogram of [3H]QNB binding to cross section of distal human esophagus. A, H&E-stained cryostat cross section of human esophagus showing muscularis mucosae (MM), and LM and CM layers of muscularis propria. B, autoradiogram of section in A showing selective binding of 0.5 nM [3H]QNB to MM, LM, and CM. C, no binding was evident in adjacent section incubated with 0.5 nM [3H]QNB plus 10 µM atropine, demonstrating specificity of [3H]QNB binding.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Saturation isotherm of [3H]QNB binding in smooth muscle homogenates from the longitudinal and circular muscle layers. Data shown are the means ± S.E. for triplicate determinations obtained from one sample. Nonspecific binding (in the presence of 10 µM atropine) accounted for >10% of total binding. Specific binding was saturable and best fit a single population of binding sites. The experiment was repeated for a total of five esophageal specimens and results are summarized in Table 2.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent inhibition of [3H]QNB binding by muscarinic receptor subtype-selective antagonists. Data shown are the means ± S.E. of four esophageal specimens assayed in triplicate. Note that the three antagonists tested produced similar inhibition in longitudinal muscle (filled symbols, solid line) and circular muscle (open symbols, dashed line). [3H]QNB (100%) bound refers to specific binding with 100 pM [3H]QNB. The results of analysis of these experiments are given in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Comparison of affinities (pKi) of muscarinic antagonists in human longitudinal and circular esophageal muscle estimated by inhibition of [3H]QNB binding in muscle homogenates The pKi values (log Ki) were determined by nonlinear regression of antagonist competition for the total specific binding of 100 pM [3H]QNB using a two-site model. Ki values were calculated as described under Experimental Procedures. Values presented here were determined from the data shown in Fig. 3 and are the means ± S.E.M. obtained for four esophageal specimens. The mean Hill coefficient (nH) for the one-site model in each case is significantly different from 1.

Identification of Muscarinic Receptor Subtypes by RT-PCR. RT-PCR analysis was done using primers based on the known human gene sequences encoding the five muscarinic receptor subtypes (Table 1). Transcripts for all five mAChRs were identified in both the LM and CM layers (Fig. 4). The identity of the amplified products was confirmed by direct DNA sequencing. The identification of a single RT-PCR product of predicted size for -actin messenger RNA rules out contamination by genomic DNA because the -actin primers were designed to span an intron. Although quantitative RT-PCR was not done, messenger RNA encoding the M2 receptor was readily detected and appeared to be most abundant, whereas M5 appeared to be the least strongly expressed.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Identification of mRNA encoding messenger RNA for five muscarinic receptor subtypes (M1-M5) in both LM and CM. PCR products of the expected sizes were obtained (Table 1). Identity of the products was confirmed by sequencing. -Actin primers spanned two introns, so finding of a single band of 660 base pairs verifies that genomic DNA was not present. Control, no cDNA present; ladder, molecular mass markers.

Immunoblotting and Immunocytochemistry of Muscarinic Receptors. Protein expression of mAChR subtypes was determined by immunoblot analysis using specific polyclonal antibodies directed at each of the five mAChR subtypes and whole homogenates of the LM and CM layers (Fig. 5). We identified immunoreactive protein bands corresponding to each receptor subtype, and estimated their molecular masses to be M1 = 55 kDa, M2 = 52 kDa, M3 = 67 kDa, M4 = 54 kDa, and M5 = 80 kDa. For each receptor antibody, a dominant single band was observed that was suppressed by preincubation of the primary antibodies with the respective peptide immunogen. Some nonspecific labeling of other proteins occurred with the M1, M3, and M4 receptor antibodies. These other protein bands did not correspond to other muscarinic receptor subtypes based on their molecular masses. No protein bands were observed if primary antibody was omitted and immunoblots were incubated only with secondary anti-rabbit horseradish peroxidase-conjugated IgG antibody (Fig. 5).

View larger version (73K): [in this window] [in a new window]   Fig. 5.   Identification of muscarinic receptor subtypes in LM and CM by immunoblotting. Subtype-specific antibodies bound to protein bands in the LM and CM. Where multiple bands appear, only the dominant band (arrows) was suppressed by adsorption of the antibody with the appropriate immunogen peptide. Specific bands did not appear in experiments in which the primary antibody was omitted (control).

View larger version (71K): [in this window] [in a new window]   Fig. 6.   Immunocytochemistry of mAChR subtypes in dispersed human esophageal smooth muscle cells. Subtype-specific antibodies bound to cells acutely isolated from the longitudinal muscle layer demonstrating the presence of M1, M2, M3, M4, and M5 receptors (shown in green). When secondary fluorescein isothiocyanate-labeled antibody was used without the primary antibody no significant binding was observed (control). In this example of cells isolated from circular muscle, nuclei are stained red with TO-PRO-3. Similar results were obtained for cells isolated from circular muscle.

Muscle Contraction Studies. Previous studies have shown that although M2 is the most abundant muscarinic receptor in gastrointestinal smooth muscle, it is M3 that plays the dominant role in mediating the contraction response (Eglen et al., 1996; Ehlert et al., 1997). In view of our results showing that human esophageal smooth muscle contains multiple muscarinic receptor subtypes, we next examined carbachol contractions in muscle strips to identify which receptor(s) might contribute to the functional response. Carbachol produced a concentration-dependent increase in tension in LM and CM muscle strips (Fig. 7). The mean log EC₅₀ was significantly different for LM (5.88 ± 0.07, n = 10) and CM strips (6.67 ± 0.08, n = 10, P < .001), the basis of which was not further studied in the present series of experiments. The maximum response to carbachol in both muscle layers was completely blocked by 10 µM atropine (data not shown). The effects of 4-DAMP, pirenzepine, and methoctramine were similar in both muscle layers. The pattern of antagonist selectivity suggested that the functional contractile response in both muscle layers was mediated by the M3 receptor. This was confirmed by Schild analysis (Fig. 8). 4-DAMP, pirenzepine, and methoctramine produced parallel rightward displacements of the carbachol concentration-response curves (Fig. 7), and Schild analysis yielded regression lines with slopes not significantly different from unity (Fig. 8; Table 4). The resulting pA₂ values were similar in the LM and CM, and demonstrated a high affinity of the receptor mediating carbachol contractions for 4-DAMP, a low affinity for methoctramine, and an intermediate affinity for pirenzepine (Table 4). In some preparations methoctramine has been found to require prolonged tissue contact to exert its full antagonist effect (Barocelli et al., 1993). No difference in the suppression of contraction to 1 µM carbachol by methoctramine was found after 2 h of contact time compared with 30 min (data not shown, n = 2).

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Effects of 4-DAMP, pirenzepine, and methoctramine on carbachol contractions in longitudinal and circular muscle strips. Carbachol produced a concentration-dependent increase in muscle tension that was inhibited by these antagonists, producing parallel rightward shifts in the carbachol concentration-response curves. Symbols represent the following antagonist concentrations: , none; ×, 109 M; , 108 M; , 107 M; , 106 M; , 105 M; and , 104 M. Values shown represent the means ± S.E. for four specimens.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Schild plots for data shown in Fig. 6: antagonism by 4-DAMP (,), pirenzepine (,), and methoctramine (,) of carbachol-mediated contraction in longitudinal muscle (solid symbols) and circular muscle (open symbols) strips. Lines shown are best fit by least-squares linear regression. The resulting slopes and x-intercepts (pA2 values) are summarized in Table 4.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We examined the expression and function of muscarinic receptor subtypes in human esophageal smooth muscle. [³H]QNB specifically labeled muscarinic receptors in the LM and CM, with both muscle layers showing comparable agonist affinity and receptor density. Antagonist competition revealed two populations of binding sites. Site 1 accounted for 60 to 70% of the total specific [³H]QNB binding and displayed an antagonist affinity profile consistent with the M2 receptor. RT-PCR, immunoblotting, and ICC revealed the additional expression of M1, M3, M4, and M5 receptor subtypes, which may account for the smaller component of the total [³H]QNB binding. Antagonist effects on contraction of muscle strips showed a pattern consistent with the M3 receptor for both LM and CM.

The present findings do not support the view that gastrointestinal smooth muscles express only M2 and M3 receptors (Eglen et al., 1996; Ehlert et al., 1997), a conclusion based mainly on data derived from ligand binding experiments. As others have pointed out (Ehlert et al., 1997), it is difficult to adequately resolve multiple receptor subtypes using this approach, especially if receptor densities are low. The major binding site we identified showed an antagonist competition pattern consistent with the M2 receptor (Eglen et al., 1996; Ehlert et al., 1997). M2 and M4 receptors have similar affinities for 4-DAMP and methoctramine, but are distinguished by the fact that pirenzepine has a lower affinity for M2, similar to what we find for site 1 (Eglen et al., 1996). The affinity of 4-DAMP and methoctramine for site 2 are consistent with what has been reported for the M3 receptor (Eglen et al., 1996). However, the affinity of pirenzepine for this site is higher than anticipated for the M3 subtype and also higher than predicted for the M1 receptor for which this antagonist is known to have highest selectivity (Ehlert et al., 1997). It seems likely that the accuracy of the affinity data regarding the lesser population of binding sites is limited by the ligand-binding approach because of the presence of multiple receptors. Two previous studies in cat esophagus report similar [³H]QNB binding characteristics to what we report here, but neither provided information regarding the contribution of specific receptor subtypes (Rimele et al., 1979; Keshavarzian et al., 1992).

Using a combination of molecular and immunological approaches, we demonstrate the presence of all five muscarinic receptors in human esophageal smooth muscle. Immunocytochemistry revealed the expected localization of the receptors in the plasma membrane of acutely isolated esophageal smooth muscle cells. This is an important finding because it confirms that the muscarinic receptors are intrinsic to the smooth muscle, although we cannot rule out a contribution from other cellular elements in the intact tissue such as enteric nerves (Lambrecht et al., 1999). This could account for the apparent paradox of abundant M2 receptor expression in the intact tissue, whereas the contraction response is consistent with a major role for the M3 receptor. Several groups have identified muscarinic receptor subtypes M1-M4 in gastrointestinal smooth muscle (Dorje et al., 1991; Wall et al., 1991; Heinig et al., 1997), however the present study is the first to show the presence of M5.

Our molecular mass estimates for receptors M1-M4 are in agreement with reports from other tissues (McLeskey and Wojcik, 1990; Ndoye et al., 1998), and receptor immunolabeling was suppressed by preincubation with peptide immunogen, confirming antibody specificity (Ndoye et al., 1998; Buchli et al., 1999). The anti-M5 antibody labeled a peptide band with an estimated molecular mass of ~80 kDa. This is higher than would be predicted from the known structure of the M5 receptor (66 kDa), but less than that reported by others (95 kDa, Ndoye et al., 1998; Buchli et al., 1999). Muscarinic receptors have been reported to form dimers and multimers through disulfide linkages (Zeng and Wess, 1999), but this is unlikely to account for the higher molecular mass because experiments were done under reducing conditions. The greater than anticipated molecular mass of M5 might result from post-translational modification, genetic polymorphism, or the existence of splice variants, all of which have been reported for G-protein-coupled receptors (Bockaert and Pin, 1999). Although its function is unclear, the M5 receptor has recently been found in a variety of non-neuronal tissues (Gil et al., 1997; Ndoye et al., 1998; Bany et al., 1999; Buchli et al., 1999; Elhusseiny et al., 1999; Tayebati et al., 1999). Interest in the role of this receptor has been stimulated by the observation that its anatomical distribution in the brain differs from other muscarinic receptor subtypes (Reever et al., 1997), and that it also contains unique structural features that allow it to be activated constitutively in the absence of ligand (Burstein et al., 1998). Whether this feature may have a role in esophageal smooth muscle remains to be determined.

Schild analysis of muscarinic antagonists showed similar results for CM and LM, indicating that the same receptor subtype mediates contraction in both muscle layers. The pA₂ values calculated for 4-DAMP, methoctramine, and pirenzepine are similar to those reported for other gastrointestinal smooth muscles in which the contraction response has been shown to be M3 mediated (Eglen et al., 1996; Ehlert et al., 1997). Furthermore, these values are in good agreement with those found in human smooth muscle from colon (Kerr et al., 1995), airway (Watson et al., 1995), and urinary bladder (Harriss et al., 1995), where cholinergic contraction also has been attributed to activation of the M3 receptor. Although 4-DAMP displays only ~10-fold greater affinity for M3 over M2 receptors (Eglen et al., 1996; Ehlert et al., 1997), our conclusion that contraction in the human esophagus is mediated by the M3 receptor is supported by the observation that methoctramine is a poor antagonist of carbachol responses. In agreement with the present findings, methoctramine yields a low pA₂ value for M3-mediated responses in other human smooth muscle types (Harriss et al., 1995; Watson et al., 1995; Norel et al., 1997). In the present study, Schild-plot slopes for methoctramine were not different from unity and prolonged exposure to methoctramine did not result in any difference in antagonist effectiveness, indicating that inadequate tissue equilibration (Barocelli et al., 1993), or allosteric interaction of methoctramine with the receptor (Eglen et al., 1988) was not observed. The relatively low pA₂ for methoctramine and intermediate pA₂ for pirenzepine also make it unlikely that carbachol responses in this tissue are mediated by the M1 or M4 receptors (Eglen et al., 1996; Ehlert et al., 1997). We cannot exclude a contribution by the M5 receptor because it shares a similar antagonist profile with the M3 receptor. A recent report highlighted the similarity of the pharmacological profiles of the M3 and M5 receptors expressed in CHO-K1 cells (Watson et al., 1999).

We previously demonstrated that cholinergic excitation in muscle strips and single cells acutely isolated from CM and LM of the human esophagus involves the release of Ca²⁺ from intracellular stores (Sims et al., 1997). This effect could be mediated by the M1, M3, and/or M5 receptor because these mAChRs preferentially activate phospholipase C, leading to the release of Ca²⁺ from intracellular stores (Eglen et al., 1996; Ehlert et al., 1997). However, we also found a component of Ca²⁺ influx accompanying cholinergic stimulation in acutely isolated cells or muscle strips from the human esophagus (Sims et al., 1997). In airway and ileal smooth muscle, stimulation of the M2 receptor can activate a nonselective cation channel with the resulting depolarization activating voltage-dependent Ca²⁺ channels thus contributing to the contraction response (Wang et al., 1997; Zholos and Bolton, 1997). This effect depends on concurrent M3 receptor activation, complicating the analysis of muscarinic receptor antagonists. The possibility that a similar mechanism may contribute in esophageal smooth muscle requires further study, but seems unlikely given the poor potency of methoctramine in antagonizing carbachol contractions in the present study.

Several previous reports from animal studies also support a major role for the M3 receptor in esophageal contraction. In both the opossum and the cat, peristalsis was potently inhibited by 4-DAMP, whereas pirenzepine had no effect (Gilbert and Dodds, 1986; Blank et al., 1989). We previously found that cholinergic contraction of esophageal smooth muscle strips from the cat displayed a pattern of sensitivity to subtype-selective antagonists similar to that reported here for human esophageal smooth muscle and therefore also consistent with a M3 receptor-mediated response (Preiksaitis and Laurier, 1998). However, Sohn et al. (1993) found that in acutely dispersed CM cells of the cat esophagus, antagonist effects on acetylcholine-induced cell shortening were consistent with an M2-mediated response. Thus, the functional contribution of muscarinic receptor subtypes in smooth muscle may be influenced by assay conditions or physiological factors.

In summary, this study provides the first account of the expression and function of muscarinic receptor subtypes in human esophageal smooth muscle. Pharmacological analysis of the contractile response indicates a major role for the M3 receptor in mediating the response. However, this smooth muscle also expresses all of the remaining muscarinic receptor subtypes. The functional significance of the remaining subtypes is not yet known.