The Expression Of Dopamine Receptors In The Human Lower Esophageal Sphincter

Objective: The lower esophageal sphincter (LES) is a thickened region of the circular muscle layer located at the gastroesophageal junctionin human, extending over an axial distance of 2–3 cm. Liebermann-Meffer et al proposed that musculature of equivalent of the LES consists of semicircular or clasp fibers at the lesser curvature and sling fibers at the greater curvature. These two muscle fibers and the crural diaphragm form a high-pressure zone at the gastroesophageal junction and maintain sphincter closure by which the LES forms an uniqe one-way valve. On the one hand, the continuous contraction of the LES prevents reflux of the gastric contents into the esophagus, but on the other hand, the proper relaxation of the LES permits passage of food from the esophagus into the stomach or to vomit and hiccup. The functional regulation of contraction and relaxation of the LES is very complicated, it involves nervous system, humoral factor and spontaneous myogenic factors. Dopamine (DA) receptors as an important member of G protein-coupled receptor family are widely expressed in the central and periphery nervous system and play an important role. By now, five distinct DA receptor subtypes (D1R-D5R) have been found, these subtypes can regulate the contraction and relaxation of smooth muscle in different tissues by the regulation of the activity of adenylyl cyclase (AC). DA receptor subtypes are divide into D1-like receptor subtypes (D1R and D5R) and D2-like receptor subtypes (D2R, D3R, and D4R). D1-like receptor subtypes activate adenylyl cyclase, while D2-like receptor subtypes inhibit adenylyl cyclase. This study was conducted to explore the expression of mRNA and protein of the five DA receptor subtypes in sling fibers, clasp fibers, circular muscle strips of the esophagus and stomach with the use of reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The results of this study are proposed to establish the fundament for the next study of the importance role that DA receptors have played in the regulatory mechanism of the LES in human, so that we can demonstrate the regulatory mechanism of the LES much more properly, and provide theoretical bases for the clinical treatment of esophageal motility disorders.Methods:1 Thirty patients including twenty-three males and seven females undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in our hospital between December 2007 and October 2008 were selected in this study. Fresh specimens were obtained from the gastroesophageal junction in the operating room. After the mucosa and submucosa were removed by sharp dissection, the sling and clasp fibers of LES, and circular muscle strips of esophagus and gastric were obtained from various regions of the gastroesophageal junction and adjacent structures.2 One ml of Trizol Reagent per hundred mg of tissue was homogenated thoroughly with the tissue homogenizer under four℃,followed by the abstraction of Chloroform. The supernatant was precipitated with Isopropyl alcohol and washed with seventy-five percent Ethanol, and then the RNA was obtained from tissue. At the end of the procedure, the RNA was dissolved in Rnase-free DEPC water. After the identification of its purity and integrity with the use of ultraviolet spectrophotometer and 1% agarose gels, we carried out RT-PCR reactions using 5 DA receptors primers, in order to detect the expression of mRNA in the four muscle strips. The amplified products were analyzed by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The unit of integrated optical density (IOD) of the gel was calculated with the Gel-Pro software. The relative values of mRNA were expressed by the ratio of IOD value of DA receptor subtypes toβ-actin.3 One hundred ul TBS reagent per ten mg of tissue was homogenated thoroughly with the tissue homogenizer, and then the homogenate was centrifuged at one thousand×g for ten minutes. The supernatant was discarded and the membrane receptor protein was obtained with the use of Membrane Protein Extraction. The membrane receptor was quantitated and adjusted to the identical concentration, then the 5 DA receptor subtypes was separated by electrophoresis. At last the detection of the protein expression was operated using 5 DA receptor subtypes polyclonal antibody after the trarsmembrane. The IOD value was also calculated with Gel-Pro softwere.Results:1 According to ultraviolet spectrophotometer and 1% agarose gels, the value of A260/280 of total RNA was between 1.8 to 1.9, the width and brightness of 28S band were double than 18S band. The band of mRNA ofβ-actin was uniformity in 838bp. Transcripts for three DA receptor subtypes were identified in four muscle strips. They were D₁R, D₂R, D₅R, and the PCR product was consistent with expected size. Significant differences were demonstrated when comparing the ratios in the same muscle strips (F=5741.75, p=0.00). The rank order of expression extent was D₁R, D₅R, and D₂R. However, there was no significant difference in the four muscle strips in the same DA receptor subtype mRNA expression. There was no identification of mRNA of D₃R and D₄R in the four muscle strips.2 Three DA receptor subtypes were identified in their expression of protein, they were D₁R, D₂R, D₅R, with respective molecular, 51KD, 51KDand 53KD. There was a significant difference in IOD values for DA receptors in same muscle strip (F=8453.308, p=0.00). The rank order of the value was the same as the result of RT-PCR. There was no significant difference in IOD values of muscle strips in the same DA receptors. There were protein expression of D3R and D4R in the four muscle strips.Conclusions:1 The mRNA of three dopamine receptor subtypes are detected in the four muscle strips, they are D₁R, D₂R, D₅R. The rank order of their expression extent is D₁R, D₂R, and D₅R. There is no significant difference in the four muscle strips in the same dopamine receptor mRNA expression, the mRNA of D3R and D4R are negative in the four muscle strips.2 The protein of three dopamine receptor subtypes are detected in the four muscle strips, they are D₁R, D₂R, D₅R. The rank order of their expression extent is D₁R, D₂R, and D₅R. There is no significant difference in the four muscle strips in same dopamine receptor protein expression, and the protein of D3R and D4R are negative in the four muscle strips.