Abstract

Methods: Between July 2015 and March 2016, muscle strips were collected from a total of 30 patients (19 males, 11 females; mean age: 62±9.9 years; range, 52 to 68 years) who underwent an esophagectomy for mid?third esophageal carcinomas. The specimens were maintained in oxygenated Krebs solution. Muscle tension measurement technique in vitro was used to examine the effects of non-selective lysophosphatidic acid receptors agonists and antagonists, as well as selective lysophosphatidic acid receptors agonists on the clasp and sling fibers of human lower esophageal sphincter.

Results: The non-selective dopamine receptor agonist lysophosphatidic acid induced the contraction of the clasp and sling fibers of the human lower esophageal sphincter. The response induced by nonselective lysophosphatidic acid receptor agonist was inhibited completely by non-selective lysophosphatidic acid receptor antagonist. The selective lysophosphatidic acid 1 and 2 receptor agonist and the selective lysophosphatidic acid 3 receptor agonist induced a concentration-dependent contractile response of the clasp and sling fibers of the human lower esophageal sphincter. There was no significant difference in contraction rates between the clasp and sling fibers (p>0.05).

Conclusion: This study indicates that lysophosphatidic acid regulates the lower esophageal sphincter is through its receptor; the lysophosphatidic acid receptors may be involved in the contractile response of the human lower esophageal sphincter.

Lysophosphatidic acid (LPA) is an intercellular phospholipid messenger with biological activity,[3,4] and it realizes its biological functions, including cell proliferation, cell survival, cell migration, the promotion of wound healing, platelet aggregation, vascular remodeling, axon retraction, inhibition/reversal differentiation, membrane depolarization, the formation of adhesion spots and stress fibers,[5-10] blood pressure regulation and smooth muscle contraction,[11-15] by binding to G protein-coupled receptors (GPCRs) on its specific cell membrane surface. To date, six GPCRs have been identified as special LPA receptors, LPA1-6,[16-22] Among them, LPA1-3 has high homology and is identified as a member of the GPCR subfamily of endothelial differentiation genes (EDGs).[23] The difference in LPA fragments implies that there are at least two different LPA receptor subtypes: smooth muscle type and platelet type.[24] All EDG family LPA receptors are more likely to be activated by acyl than alkyl-LPA, and they are smooth-muscle-type LPA receptors.[23]

We have previously shown the expression of LPA1-6 receptors in sling and clasp fibers from the human LES.[25] In the present study, we aimed to examine the effects of non-selective LPA receptor agonists and antagonists, as well as selective LPA receptor agonists, on sling and clasp fibers of the LES.

Methods

In the laboratory, fresh EGJ specimens collected in the operating room were immediately placed in 4°C Tris?buffered saline (TBS). Following washing with 37°C Krebs solution, the specimens were pinned on a wax plate containing TBS with a continuous mixed gas of 95% O₂ and 5% CO₂. The mucosa and submucosa were, then, gently removed by sharp dissection. The sling fibers and clasp fibers were separated and cut into 2x10 mm muscle strips.

The gastric 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 method described previously.[26]

Measurement of muscle strip tension
The most suitable initial length
Both ends of the muscle strips were fastened with silk, placed in a 10 mL bath containing Krebs liquid, maintained at a constant temperature of 37º and perfused with gas containing 5% CO2 and 95% O₂. The upper muscles and JZ101-type muscle tension transducer (Xinhang Electric Apparatus, Gaobeidian, China) were fastened together, and tension was recorded using MedLab 6.0 software (MedEase, Nanjing, China). Each muscle strip was stretched slightly and rapidly, until 200 mg of force was generated. This was taken as the initial length (L0). The muscle strips were, then, sequentially stretched to 200% of the L0[27] at increments of 25% of the L0 each time.[28] This was taken as the most suitable initial length.

To avoid the influence of other factors, the receptor subtypes with excitation effects on muscle strips were compared with those treated with NG-nitro-L-arginine (Sigma, Chemical Co., St. Louis, MO, USA).

Effects of non-selective LPA receptor agonists, antagonists, and selective LPA receptor agonists on human LES
The optimal initial length of the muscle was stabilized for approximately 40 min and, then, a non-selective LPA receptor agonist (LPA) (Cayman Chemical, Ann Arbor, MI, USA) was added into a thermostatic bath to activate each LPA receptor subtype in a cumulative manner from 10-9 to 1 0-4 mol/L. Each concentration of the drug was added after the reaction of the previous concentration reached a maximum. Cumulative administration concentration-response dose-response curves were established based on the above results. The maximum effect after dosing and its corresponding concentration were calculated. While observing the effects of a non-selective antagonist (tetradecyl-phosphonate) (Cayman Chemical, Ann Arbor, MI, USA), the concentration of the antagonist was found to be the same as the concentration of agonist-induced muscle maximal effect. The administration of a selective LPA1 and LPA2 receptor agonist (L-α-LPA) (Sigma, Chemical Co., St. Louis, MO, USA) and a selective LPA3 receptor agonist (OMPT) (Cayman Chemical, Ann Arbor, MI, USA) was performed using the same method. The responses in all of the experiments were quantified based upon the percentage of the baseline value of muscle strip tone relative to the nadir of the response.

Statistical analysis
Statistical analysis was performed using the IBM SPSS version 19.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Descriptive data were expressed in mean±standard error (SEM). Drug-induced muscle strip response was based on mean±SEM of contraction or relaxation percentage of muscle strips. Two-way analysis of variance (ANOVA) was used to compare the two drug concentration-response curves. A p value of <0.05 was considered statistically significant.

Results

Figure 1. Example curves of the responses of the sling and clasp muscles to LPA. The non-selective lysophosphatidic acid receptor agonist LPA induced the contraction of the human LES at concentrations of 10^-6, 10^-5, and 10^-4 mol/L.
LPA: Lysophosphatidic acid; LES: Lower esophageal sphincter.

Effect of selective LPA1 and LPA2 receptor agonist on the human LES
The selective LPA1 and LPA2 receptor agonist L-?-LPA induced a concentration-dependent contractile response of the clasp and sling fibers of the human LES at concentrations of 10-6, 10-5, and 10-4 m ol/L. T here w as n o s ignificant d ifference in contraction between the clasp and sling fibers (F=3.26, p=0.72). The optimal concentration leading to the maximum contraction percentage was 10-5 mol/L. The mean maximum contraction percentage of clasp fibers was 12.6±0.4%. The mean maximum contraction percentage of sling fibers was 13.1±0.4%. There was no significant difference in the maximum contraction percentage of clasp and sling fibers (F=0.02, p=0.90) (Figure 2a, b).

Figure 2. (a) Concentration-tension curves of the selective LPA1 and LPA2 receptor agonist. There was no significant difference in contraction between sling fibers and clasp fibers (p>0.05). The optimal concentration leading to maximum contraction was 10-5 mol/L. (b) Example curves of the responses of the two muscle strips to the selective LPA1 and LPA2 receptor agonist. The selective LPA1 and LPA2 receptor agonist induced a concentration-dependent contractile response of the human LES at concentrations of 10^-6, 10^-5, and 10^-4 mol/L.
LPA: Lysophosphatidic acid; LES: Lower esophageal sphincter.

Effect of selective LPA3 receptor agonist on the human LES
The selective LPA3 receptor agonist OMPT also induced the contraction of the human LES at concentrations of 10^-6, 10^-5, and 10^-4 mol/L in a concentration-dependent manner. There was no significant difference in contraction between the clasp and sling fibers (F=1.98, p=0.16). The optimal concentration leading to the maximum contraction percentage was 10-5 m ol/L. T he m ean m aximum contraction of clasp fibers was 6.5±0.5%, and the mean maximum relaxation of sling fibers was 6.9±0.6%, indicating no significant difference (F=0.13, p=0.72) (Figure 3a, b).

Figure 3. (a) Concentration-tension curves of the selective LPA3 agonist. There was no significant difference in contraction between the two muscle strips (p>0.05). The optimal concentration leading to maximum contraction was also 10^-5 mol/L. (b) Example curves of the responses of the two muscle strips to the selective LPA3 receptor agonist. The selective LPA3 receptor agonist induced the contraction of the human LES at concentrations of 10^-6, 10^-5, and 10^-4 mol/L in a manner that was also concentration-dependent.
LPA: Lysophosphatidic acid; LES: Lower esophageal sphincter.

Discussion

Numerous esophageal motility disorders are all associated with motor disorders of the LES. Gastroesophageal reflux is the most important cause of the development of Barrett esophagus.[29] Previous studies have shown that the regulatory mechanism of the LES involves a variety of receptors.[26,30]

Lysophosphatidic acid receptors are widely distributed in gastrointestinal smooth muscle and are closely related to gastrointestinal motility and secretion. The pharmacological effects of LPA on different parts of the gut have been confirmed.[13-15] Toews et al.[15] found that LPA both strengthened the contraction and inhibited the relaxation of tracheal smooth muscle. Xu et al.[31] confirmed that the LPA receptor induced the contraction of vascular smooth muscle and increased blood pressure. Markiewicz et al.[32] found that direct activation of LPA1, LPA2, and LPA3 receptors could enhance the contraction of the smooth muscle of pig uterus in early pregnancy, and the three receptor subtypes work together. Sriwai et al.[33] confirmed that the LPA3 receptor acted in the contraction of the smooth muscle of rabbit stomach. The use of LPA receptor agonists and antagonists has shown that the LPA1 receptor blocks the relaxation of the LES.[34] These results suggest that LPA receptors may play a key role in the contraction of smooth muscle.

Our study is based on evidence from the above studies and used in vitro muscle tension measurement technology to analyze the regulatory role of a non-selective LPA receptor agonist (LPA) and a non-selective LPA receptor antagonist (tetradecyl-phosphonate) in the contraction and relaxation responses of clasp and sling fibers in the LES. In addition, agonists of selective LPA receptors were used to further clarify the role of different subtypes of LPA receptors in the regulatory mechanism of human LES.

We found that the non-selective LPA agonist LPA could induce contraction in the clasp and sling fibers of the LES. This suggests that LPA can induce the contraction of the LES. In addition, a non-selective LPA receptor antagonist can completely inhibit the contraction of clasp and sling fibers induced by LPA. It is suggested that LPA may regulate the contraction of the human LES through the LPA receptor. The results preliminarily confirm that the LPA receptor plays an important role in enhancing the contraction of smooth muscle in the human LES. These results are consistent with those of domestic and foreign scholars.[13-15,34,35] Moreover, our study found that LPA1, LPA2, and LPA3 receptors were all involved in contraction reactions in the human LES. This result is similar to that found by Markiewicz et al.[32] However, some studies have confirmed that only the LPA3 receptor acts in the contraction reaction in the smooth muscle of rabbit stomach, and the experiment does not find the existence of LPA1 and LPA2 receptors.[33] The LPA inhibits the relaxation of the LES in cats by blocking the nitric oxide-mediated signal transduction pathway of the LPA1 receptor.[34] This result may be caused by different species and different sites of tissue action, and the exact reason is not clear and needs to be further understood.

The main limitations to the present study must be recognized. First, this is a single-center study, and we need to further carry out multicenter research. Second, this study is an in vitro study, and the results may not be in agreement with the results of in vivo studies. Third, some receptors have no specific agonists, which may have affected the results. Finally, this is the first report on the role of LPA receptors in modulating the human LES.

In conclusion, we found that all three lysophosphatidic acid receptor subtypes play a contractile role in the human lower esophageal sphincter, but the possible signal transduction pathway of the lysophosphatidic acid receptor in this process needs further investigation. In this way, the regulatory role of the lysophosphatidic acid receptor in human lower esophageal sphincter can be more clearly defined, which may play an important role in the future treatment of esophageal motor function diseases.

Acknowledgments: This project was funded by the National Natural Science Foundation of China (No. 30371413) and the Natural Science Foundation of Hebei Province (C2010000622). The authors thank Professor David Watson, Department of Surgery, Flinders University, Australia, for his supportive and helpful suggestions for this study.

Ethics Committee Approval: The project was approved by the Research Ethics Committee of the Fourth Hospital of Hebei Medical University, Shijiazhuang, China (2015mECD35). All patients gave written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Patient Consent for Publication: A written informed consent was obtained from each patient.

Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Contributions: Designed the study: Y.F., JF.L.; Analysis and interpretation of data: W.W., L.C.; Drafting the article: Y.F., W.W.; Revising it critically for important intellectual content: J.F.L., L.C.; All authors final approval of the version to be published.

Conflict of Interest: The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding: This project was funded by the National Natural Science Foundation of China (No. 30371413) and the Natural Science Foundation of Hebei Province (C2010000622).

References

1) Mittal RK, Holloway RH, Penagini R, Blackshaw LA, Dent J. Transient lower esophageal sphincter relaxation. Gastroenterology 1995;109:601-10.

2) Liebermann-Meffert D, Allgöwer M, Schmid P, Blum AL. Muscular equivalent of the lower esophageal sphincter. Gastroenterology 1979;76:31-8.

3) Eichholtz T, Jalink K, Fahrenfort I, Moolenaar WH. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J 1993;291:677-80.

4) Tigyi G, Hong L, Yakubu M, Parfenova H, Shibata M, Leffler CW. Lysophosphatidic acid alters cerebrovascular reactivity in piglets. Am J Physiol 1995;268:H2048-55.

5) Jalink K, Hordijk PL, Moolenaar WH. Growth factor-like effects of lysophosphatidic acid, a novel lipid mediator. Biochim Biophys Acta 1994;1198:185-96.

6) Moolenaar WH. Lysophosphatidic acid signalling. Curr Opin Cell Biol 1995;7:203-10.

7) Gaits F, Fourcade O, Le Balle F, Gueguen G, Gaigé B, Gassama-Diagne A, et al. Lysophosphatidic acid as a phospholipid mediator: Pathways of synthesis. FEBS Lett 1997;410:54-8.

8) Nietgen GW, Durieux ME. Intercellular signaling by lysophosphatidate. Cell Adhes Commun 1998;5:221-35.

9) Ishii I, Contos JJ, Fukushima N, Chun J. Functional comparisons of the lysophosphatidic acid receptors, LP(A1)/ VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7 in neuronal cell lines using a retrovirus expression system. Mol Pharmacol 2000;58:895-902.

10) Moolenaar WH, van Meeteren LA, Giepmans BN. The ins and outs of lysophosphatidic acid signaling. Bioessays 2004;26:870-81.

11) Tokumura A, Fukuzawa K, Tsukatani H. Effects of synthetic and natural lysophosphatidic acids on the arterial blood pressure of different animal species. Lipids 1978;13:572-4.

12) Tokumura A, Fukuzawa K, Yamada S, Tsukatani H. Stimulatory effect of lysophosphatidic acids on uterine smooth muscles of non-pregant rats. Arch Int Pharmacodyn Ther 1980;245:74-83.

13) Tokumura A, Fukuzawa K, Tsukatani H. Contractile actions of lysophosphatidic acids with a chemically-defined fatty acyl group on longitudinal muscle from guinea-pig ileum. J Pharm Pharmacol 1982;34:514-6.

14) Tokumura A, Yube N, Fujimoto H, Tsukatani H. Lysophosphatidic acids induce contraction of rat isolated colon by two different mechanisms. J Pharm Pharmacol 1991;43:774-8.

15) Toews ML, Ustinova EE, Schultz HD. Lysophosphatidic acid enhances contractility of isolated airway smooth muscle. J Appl Physiol (1985) 1997;83:1216-22.

16) Hecht JH, Weiner JA, Post SR, Chun J. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex. J Cell Biol 1996;135:1071-83.

17) An S, Bleu T, Hallmark OG, Goetzl EJ. Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid. J Biol Chem 1998;273:7906-10.

18) Bandoh K, Aoki J, Hosono H, Kobayashi S, Kobayashi T, Murakami-Murofushi K, et al. Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. J Biol Chem 1999;274:27776-85.

19) Noguchi K, Ishii S, Shimizu T. Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol Chem 2003;278:25600-6.

20) Kotarsky K, Boketoft A, Bristulf J, Nilsson NE, Norberg A, Hansson S, et al. Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes. J Pharmacol Exp Ther 2006;318:619-28.

21) Lee CW, Rivera R, Gardell S, Dubin AE, Chun J. GPR92 as a new G12/13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem 2006;281:23589-97.

22) Yanagida K, Masago K, Nakanishi H, Kihara Y, Hamano F, Tajima Y, et al. Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6. J Biol Chem 2009;284:17731-41.

23) Bandoh K, Aoki J, Taira A, Tsujimoto M, Arai H, Inoue K. Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structureactivity relationship of cloned LPA receptors. FEBS Lett 2000;478:159-65.

24) Tokumura A. A family of phospholipid autacoids: Occurrence, metabolism and bioactions. Prog Lipid Res 1995;34:151-84.

25) Feng Y, Liu JF. Expression of lysophosphatidic acid receptors in the human lower esophageal sphincter. Exp Ther Med 2014;7:423-8.

26) Liu JF, Lu HL, Wen SW, Wu RF. Effects of acetylcholine on sling and clasp fibers of the human lower esophageal sphincter. J Gastroenterol Hepatol 2011;26:1309-17.

27) Tian ZQ, Liu JF, Wang GY, Li BQ, Wang FS, Wang QZ, et al. Responses of human clasp and sling fibers to neuromimetics. J Gastroenterol Hepatol 2004;19:440-7.

28) Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physiol 2001;281:G1460-7.

29) Yeğinsu A, Ergin M, Doğan Köseoğlu R, Başsorgun Cİ. Barrett"s esophagus. Turk Gogus Kalp Dama 2009;17:221-8.

30) Liu XB, Liu JF. Expression of dopamine receptors in human lower esophageal sphincter. J Gastroenterol Hepatol 2012;27:945-50.

31) Xu YJ, Saini HK, Cheema SK, Dhalla NS. Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells. Cell Calcium 2005;38:569-79.

32) Markiewicz W, Kami?ska K, Bogacki M, Ma?lanka T, Jaroszewski J. Participation of analogues of lysophosphatidic acid (LPA): Oleoyl-sn-glycero-3-phosphate (L-alpha-LPA) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) in uterine smooth muscle contractility of the pregnant pigs. Pol J Vet Sci 2012;15:635-43.

33) Sriwai W, Zhou H, Murthy KS. G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: Reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA. Biochem J 2008;411:543-51.

34) Lee JW, Kim CH, Wang YY, Yan XM, Sohn UD. Lysophosphatidic acid presynaptically blocks NO uptake during electric field stimulation-induced relaxation via LPA(1) receptor in cat lower esophageal sphincter. Arch Pharm Res 2011;34:169-76.

35) Salmon DM, Honeyman TW. Increased phosphatidate accumulation during single contractions of isolated smoothmuscle cells. Biochem Soc Trans 1979;7:986-8.