α₁-adrenoceptors Expression In Human Lower Esophageal Sphincter

Objective: Primary esophageal motor function disorder such asachalasia, nutcracker esophagus, high tensile LES is a kind of disease whichaffecting people’s living quality of life. These diseases are often accompaniedby some typical symptoms, pharyngeal difficulties etc.. In recent years, withthe popularity of the population ’s living standards improvement andesophageal manometry and24-hour PH testing and other inspectionequipment, the incidence of primary esophageal motor function disorders hasincreased. However, the etiology and pathogenesis of these diseases is not yetentirely clear. Nervous, hormonal regulation, psychological factors,inflammation, viral infections and autoimmune factors may be involved in thepathogenesis of primary esophageal motor function disorders.Lower esophageal sphincter(LES) is located at the esophagogastricjunction. It is shown that LES is composed of the sling fibers (SF) and claspfibers (CF) components, which is the important component forming LESpressure. This high pressure zone has important physiological significance. Asthe valve it played a key role in physiological anti-reflux. The disorder of LEScan lead to a variety of esophageal motor function disorder. Mechanisms ofregulation here is important for gastroesophageal reflux diseases andesophageal motility disorders.Α1adrenergic receptor (α1-AR) and its subtypes are G protein-coupledreceptors are widely expressed in human and mammals. Existing researchsuggests that α1-AR for the regulation of smooth muscle contraction isimportant, but previous studies are on α1-AR expression mainly in thevascular, genitourinary, liver and kidneys and other organs.Little informationis available on the distribution and function of the human LES α1-AR subtypereceptors and their expression in gasrointesinal tract. The expression of α1-AR and its receptor subtypes α1A-AR, α1B-AR,α1D-AR was determined by Western blot (WB) and reverse transcriptionpolymerase chain reaction (RT-PCR) to lay a the theoretical basis for thestudy and treatment of esophageal motility disorders.Methods: Thirty patients were recruited in the study. Fresh tissues ofGOJ were collected in operation theatre right after oesophagectomy, frompatients with upper oesophageal cancer. Sling fibers (S), clasp fibers (C),circular muscle strips of esophagus (E) and stomach (G) were isolatedcarefully. Total Total RNA was extracted from tissues and then wasreversetranscripted to cDNA, followed by RT-PCR with primers of a1-ARsubtypes. IOD was analyzed in amplification product. Relative concentrationof mRNA from strips was represented by the ratio of subtypes of α1-AR andendogenous control (β-actin).Total protein were extracted and adjusted to thesame concentration after measuring protein concentration by BCA. The threesubtypes were separated by electrophoresis and transferred onto apolyvinylidene difluoride (PVDF). The detection of the protein expressionwas operated using different antibody.The membrane was detected byinfrared fluorescence imaging instrument. The relative expression level ofeach protein was normalized by the value of endogenous control (GAPDH).Results:1RT-PCR shows that three subtypes of α1-adrenoceptor receptorsubtypes mRNA was expressed in all muscle strips: α1A-AR, α1B-AR,α1D-AR. α1A-AR in E,S,C,G expression levels were1.307±0.118;1.350±0.177;1.289±0.180;1.361±0.268, respectively; α1B-AR expression levelswere1.222±0.151;1.279±0.237, respectively;1.309±0.263;1.352±0.267;α1D-AR expression levels were1.258±0.297;1.323±0.237;1.310±0.295;1.282±0.288, respectively; There were no significant differences between theα1-adrenoceptor receptor subtypes in the same muscle strip (F=0.530, P=0.590), and no significant difference for each subtype across different musclestrips (F=0.037，P=0.910).2Western-blot showed that the expression of three kinds of α1- adrenoceptor receptor subtypes has its corresponding protein bands: namelyα1A-AR, α1B-AR, α1D-AR. α1A-AR in E,S,C,G,expression levels were0.431±0.118;0.474±0.177;0.503±0.180;0.485±0.268, respectively;α1B-AR expression levels were0.335±0.151;0.458±0.237;0.459±0.263;0.465±0.267, respectively; α1D-AR expression levels were0.459±0.297;0.524±0.237;0.511±0.295;0.483±0.288, respectively; There were nosignificant differences between the α1-adrenoceptor subtypes in same musclestrip (F=0.208, P=0.813), and no significant difference for a single subtypebetween the four muscle strips (F=0.188, P=0.905).Conclusion: All three α1-adrenoceptor receptor subtypes(α1A-AR,α1B-AR, α1D-AR)were expressed in sling fibers, clasp fibers, circularmuscle strips of esophagus and stomach. There were no significant differencesbetween the α1-adrenoceptor receptor subtypes in the same muscle strip, andno significant difference for each subtype across different muscle strips.Alpha1-AR expression may play a role in the function of human LESmediation.