The experimental protocol has been approved by the Research Ethics Committee of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Written informed consent was obtained from the patients. Muscle strips were collected from 28 patients who underwent esophagectomy for mid-third esophageal carcinoma in the Department of Thoracic Surgery at this hospital from March 2012 to August 2012. There were 16 males and 12 females, with an average age of 58 years (range, 50 to 67 years). Patients with a history of GERD, achalasia, scleroderma, or other diseases associated with a disorder of the LES were excluded from the study. Each specimen was resected en bloc in the operating room, and the fresh specimen was placed immediately in ice-cold Krebs solution. The Krebs solution had the following composition (in mM): sodium, 143.0; potassium, 5.0; calcium, 2.5; magnesium, 1.2; chloride, 128.0; phosphate, 2.2; bicarbonate, 24.9; sulfate, 1.2; and glucose, 10.0. Specimens were not included in this study if any segment that was required for study contained a macroscopically visible tumor.

In the laboratory, the fresh specimens of the gastroesophageal junction were opened along the long axis of the esophagus and the greater curvature of the stomach. Specimens were pinned to a wax plate in the presence of Krebs solution at 37°C to maintain its approximate in situ dimensions, with a continuous supply of mixed gas of 95% O₂ and 5% CO₂. The mucosa and submucosa were then removed by dissection.

The sling and clasp fibers were identified as thickened bands of circular oriented smooth muscle in the gastric cardia, adjacent to the greater and lesser curvature of the stomach, respectively. The sling and clasp muscle strips were prepared using a previously described method (5,6). In addition, circular muscle fibers from the esophagus above the LES, and circular muscle fibers from the gastric fundus below the LES, were dissected for use as control specimens. These circular muscle strips were obtained 3 cm proximal and distal to the gastro-esophageal junction. All specimens were removed carefully to ensure that the myenteric plexus and the longitudinal muscle in the wall of the esophagus and stomach were excluded. The dissected muscle strips from the four parts were frozen in liquid nitrogen and stored at −80°C for subsequent RNA extraction.

Tissue was homogenized in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) at a ratio of 100 mg tissue to 1 ml TRIzol, and then centrifuged at 12,000 × g for 5 min. Total RNA was extracted by acid guanidinium thiocyanate-phenol-chloroform extraction. The quality of the RNA was verified by agarose gel electrophoresis using ethidium bromide staining. First-strand cDNA synthesis (reaction volume, 20 μl) using 2 mg RNA was performed by RT reaction in the presence of RevertAid Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). RT was conducted with 0.5 mg oligo(dT)₁₈, using diethypyrocarbonate (DEPC)-treated water to achieve a volume of 11 μl, and the reaction mixture was incubated at 70°C for 5 min, prior to being chilled on ice. Next, 4 μl 5X reaction buffer (Fermentas), 2 μl 10 mM 4 dNTP mix and 20 units RNasin (both from Fermentas) were added, using DEPC-treated water to provide a reaction volume of 19 μl, and the mixture was incubated at 37°C for 5 min. Finally, 200 units (1 μl) RevertAid M-MuLV reverse transcriptase was added, and the reaction mixture was incubated at 37°C for 50 min, before the reaction was stopped and held at 70°C for 15 min.

PCR amplification of the cDNA was performed using primers designed specifically to match the 5-HT receptor mRNA (primers listed in Table I). In each PCR reaction, 2 μl cDNA reaction mixture was used. PCR was performed in a 20-μl reaction volume. The amplification conditions were: 5 min initial denaturation at 95°C, then 36 cycles of 95°C for 35 sec, 60°C for 35 sec and 72°C for 45 sec followed by 72°C for 5 min. A negative control in which all the components of the reaction were added, except the cDNA template was tested in parallel with each sample to identify any risk of false positive results. The amplified products were electrophoresed on a 1.5% agarose gel and PCR products were determined using the Gel-Pro gel image analysis system (Media Cybernetics, Silver Spring, Maryland, USA). Densitometry for analysis of the PCR product bands was conducted by the imaging software. The relative expression level of each gene was normalized by the value of β-actin. The amplified products were analyzed by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining, with images captured by photography under a UV transilluminator. The integrated optical density (IOD) of the gel was calculated with Gel-Pro software (Media Cybernetics). The relative expression level of the mRNA of each 5-HT receptor type was normalized by the value of β-actin.

The qPCR experiments were conducted using an Applied Biosystem 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and data were analyzed with ABI 7500 (version 2.0.6) software. All oligonucleotide primers for qPCR were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3www.cgi) and synthesized by Invitrogen Life Technologies. The diluted cDNA (1 μl from each sample) was used as PCR template. The reaction composition contained: 1 μl diluted cDNA; 12.5 μl 2X TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China.), 10 μl ddH₂O, 0.5 μl forward primer and 0.5 μl reverse primer, and 0.5 μl Passive Reference Dye (TransGen Biotech) in a final volume of 25 μl. The cDNA was amplified by one cycle at 94°C for 30 sec, followed by 42 cycles of 95°C for 5 sec, 60°C for 34 sec and melt curve analysis. Reactions were performed in triplicate according to the manufacturers’ instructions (TransGen Biotech). Melting point and melting curve analyses were undertaken on each set of reactions to confirm that only a single product was produced. No primer-dimers were detected by melting point analysis and this was confirmed in preliminary runs with gel electrophoresis. The expression level of the target gene was calculated by the 2^−ΔΔCt method (7). The relative expression of target gene mRNA was indexed to the reference gene β-actin using the formula: 10,000 × 2^ΔCt, in which ΔCt = Ct_β-actin - Ct_{target gene}.

Total proteins were extracted from the muscle strips using a protein extraction kit (Solarbio, Beijing, China). Protein concentration was determined using a colorimetric bicinchoninic acid (BCA) protein assay reagent (Multisciences, Hangzhou, China). Following denaturation at 100°C for 10 min, aliquots of protein samples (30 μg) were separated by electrophoresis on SDS-polyacrylamide gel 10% separation gel and 4% pycnotic gel, separated at 150 V for 1 h, and transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked for 1 h with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) at room temperature, and incubated with an anti-human, polyclonal primary antibody (dilutions: rabbit anti-5HT_1AR, 1:300; rabbit anti-5HT_2AR, 1:400; rabbit anti-5HT_3AR, 1:500; rabbit anti-5HT₄R, 1:500; rabbit anti-5HT_5AR, 1:300; rabbit anti-5HT₆R, 1:300; rabbit anti-5HT₇R, 1:300; 1:10,000 for the rabbit anti-β-actin; Abcam Trading, Shanghai, China) at 4°C overnight. After washing three times with TBST at room temperature for 30 min in total, the membrane was incubated with a goat anti-rabbit IgG polyclonal secondary antibody (1:2,000, anti-rabbit IgG; Abcam Trading) for 1 h. After three washes with TBST, the membrane was analyzed using an infrared fluorescence imaging instrument (Odyssey Infrared Imaging System, American LI-COR, Lincoln, Nebraska, USA). The IOD value was calculated by the Gel-Pro software and the relative expression level of each protein was normalized by the value of β-actin.

Results are expressed as the mean ± standard deviation (SD). SAS software, version 9.2 (SAS Institute Inc., Cary, NC, USA) was used to conduct the statistical analysis. Differences in the mRNA and protein expression levels were analyzed with one-way analysis of variance, and the Student-Newman-Keuls multiple range (SNK-q) test was used to evaluate comparisons within groups. P<0.05 was considered to indicate a statistically significant difference.

Using RT-PCR, the mRNA expression levels of seven 5-HT receptors in the human LES were determined. Distinct bands of the expected sizes were detected for each of the seven 5-HT receptor mRNAs and their levels of expression appeared to differ. Similar results were obtained in all PCR assays performed on mRNA extracted from the 28 patients. The primer pairs designed to recognize the 5-HT_3AR and 5-HT₄R mRNA generated strong bands, indicating high expression levels of the 5-HT_3AR and 5-HT₄R mRNA. The 5-HT_1AR and 5-HT_2AR mRNA also generated comparatively strong bands. However, the primer pairs for the 5-HT₇R, 5-HT_5AR and 5-HT₆R mRNA produced relatively weak bands (Fig. 1).

To compare the expression levels of the seven different 5-HT receptor mRNAs, qPCR was performed. Significant differences were identified when the mRNA expression levels of the 5-HT receptors were compared in the same muscle strip (F, 78.281; P=0.000). The rank order of expression was as follows: 5-HT_3AR = 5-HT₄R > 5-HT_1AR = 5-HT_2AR > 5-HT_5AR = 5-HT₆R = 5-HT₇R. However, no significant difference was observed in the mRNA expression levels of the 5-HT receptors among the four types of muscle strip (F, 0.232; P=0.731; Fig. 2).

With the exception of 5-HT_5AR and 5-HT₆R proteins, the other five receptors were identified. Significant differences in the IOD values for the different 5-HT receptors in the same muscle strip were observed (F, 657.357; P=0.000). The rank order of the IOD values was as follows: 5-HT_3AR = 5-HT₄R > 5-HT_1AR = 5-HT_2AR > 5-HT₇R. No significant differences in IOD values were identified among the four type of muscle strip (F, 0.194; P=0.801; Fig. 3).